Methods for protein ligation and uses thereof
11807671 · 2023-11-07
Assignee
Inventors
- Christopher John Squire (Auckland, NZ)
- Edward Neill Baker (Auckland, NZ)
- Paul Gary Young (Auckland, NZ)
- Yuliana Yosaatmadja (Auckland, NZ)
Cpc classification
C07K2319/24
CHEMISTRY; METALLURGY
International classification
Abstract
The invention relates to protein ligation technologies, purified or recombinant peptides, methods for making peptides and proteins with covalent bonds including reversible covalent bonds such as reversible intermolecular covalent bonds, and uses thereof. In particular, this invention relates to intermolecular ester bonds, particularly reversible ester bonds between the hydroxyl and amide groups of amino acid side chains present in recombinant chimeric peptides and proteins and the use of such peptides and proteins in protein engineering, for example in the preparation of multimeric protein complexes, including functionalised multimeric protein complexes.
Claims
1. A peptide tag and binding partner pair wherein a) the peptide tag comprises at least about 10 contiguous amino acids of an Ig-like fold of a Cpe-like domain comprising two β-sheets in a β-clasp from a β-clasp containing protein, wherein one of the amino acids is a reactive residue capable of spontaneously forming an intermolecular ester bond in an Ig-like fold of a Cpe-like domain, and wherein the peptide tag does not comprise the entire amino acid sequence of the β-clasp containing protein; and b) said binding partner comprises a separate fragment of a β-clasp containing protein wherein said fragment comprises at least about 10 contiguous amino acids of a complementary part of an Ig-like fold of a Cpe-like domain from a Cpe-like domain containing protein, wherein one of the amino acids is a reactive residue capable of forming an intermolecular ester bond with the peptide tag; and wherein either: i) the reactive residue in (a) is serine or threonine, and the reactive residue in (b) is glutamine or glutamate/glutamic acid; or ii) the reactive residue in (a) is glutamine or glutamate/glutamic acid, and the reactive residue in (b) is serine or threonine; and wherein said peptide tag and binding partner are covalently bound to each other only by an intermolecular ester bond between the reactive residues and wherein the peptide tag or the binding partner or both the peptide tag and the binding partner is covalently linked to one or more heterologous amino acid sequences.
2. The peptide tag and binding partner pair of claim 1 wherein the ester bond formed between the two reactive residues is reversibly hydrolysable.
3. The peptide tag and binding partner pair of claim 2 wherein the ester bond formed between the two reactive residues is reversibly hydrolysed when the pH is greater than 7.
4. The peptide tag and binding partner pair of claim 1 wherein said Ig-like fold domain is adhesin protein Cpe0147 from Clostridium perfringens or a protein with at least 75% identity thereto which is capable of spontaneously forming one or more ester bonds.
5. The peptide tag and binding partner pair of claim 1 wherein a) said peptide tag comprises 10 or more contiguous amino acids of the sequence set out in SEQ ID NO: 1 and corresponding to amino acids 565-587 of adhesin protein Cpe0147 from Clostridium perfringens or a sequence with at least 75% identity thereto; and/or b) said peptide tag is less than 50 amino acids in length; and/or c) said binding partner comprises 10 or more contiguous amino acids of the sequence set out in SEQ ID NO: 1 and corresponding to amino acids 439-563 of adhesin protein Cpe0147 from Clostridium perfringens or a sequence with at least 75% identity thereto.
6. The peptide tag and binding partner pair of claim 1 wherein one or both of the peptide tag and the binding partner comprises one or more amino acid residues that facilitate spontaneous intermolecular ester bond formation.
7. The peptide tag and binding partner pair of claim 6 wherein the one or more of the amino acid residues that facilitate spontaneous intermolecular ester bond formation are present in a beta-strand forming amino acid sequence together with a reactive residue.
8. The peptide tag and binding partner pair of claim 6 wherein the glutamine or glutamate/glutamic acid reactive residue is present in an amino acid sequence HXDXXDXX[Q/E] (SEQ ID NO: 30), or is present in an amino acid sequence [H/E]XDXX[D/S]XX[Q/E] (SEQ ID NO: 55), or is present in an amino acid sequence HXDXX[D/S]XX[Q/E] (SEQ ID NO: 56), or is present in an amino acid sequence HXDXXSXX[Q/E] (SEQ ID NO: 57), or is present in an amino acid sequence [H/E]XDXXXXX[Q/E], (SEQ ID NO: 58), wherein X is any amino acid.
9. The peptide tag and binding partner pair of claim 6 wherein the binding partner comprising the glutamine reactive amino acid residue or the glutamate/glutamic acid reactive amino acid residue comprises a histidine amino acid residue that facilitates spontaneous intermolecular ester bond formation, wherein the histidine is within about 6, about 5.5, about 5, about 4.5, about 4, about 3.5, about 3, about 2.5 or about 2 Angstrom of the glutamine or glutamate/glutamic acid reactive residue.
10. The peptide tag and binding partner pair of claim 9 wherein when the binding partners are contacted, the histidine that facilitates spontaneous intermolecular ester bond formation is within about 5, about 4.5, about 4, about 3.5, about 3, about 2.5 or about 2 Angstrom of the threonine and serine reactive residue.
11. The peptide tag and binding partner pair according to claim 6, wherein when contacted, the peptide tag and the binding partner form a serine protease active site-like structure, and wherein the serine protease active site-like structure comprises reactive amino acid residues present in the active site having the following relative atom locations in the Protein Data Bank conventional orthogonal coordinate system: c) Cβ (CB) Thr/Ser: 0, 0, 0; d) Cδ (CD) Gln/Glu: 0.02, 1.91, −1.61 and wherein the serine protease active site-like structure comprises accessory amino acid residues present in the active site having the following Cγ (CG) locations relative to the reactive Thr/Ser Cβ (CB) location: e) His: 1.35, 3.67, 3.34 f) Asp: −3.45, −0.89, −2.19.
12. The peptide tag and binding partner pair of claim 1 wherein at least one of the heterologous amino acid sequences comprises an enzyme, an antigen, a structural protein, an antibody, a cytokine, or a receptor.
13. The peptide tag and binding partner pair of claim 1 wherein at least one of the heterologous amino acid sequences comprises an antigen.
14. The peptide tag and binding partner pair of claim 1 wherein at least one of the heterologous amino acid sequences comprises two or more antigens.
15. The peptide tag and binding partner pair of claim 1 wherein at least one of the heterologous amino acid sequences comprises at least 8 contiguous amino acids from a heterologous protein.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The invention will now be described by way of example only and with reference to the drawings in which:
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
(25)
(26)
(27)
(28)
(29)
(30)
(31)
(32)
(33)
(34)
(35)
(36)
DETAILED DESCRIPTION OF THE INVENTION
(37) The present invention provides methods for the formation of spontaneously-formed ester bond cross-links between amino acid side chains, particularly spontaneously-formed, reversible ester bond cross-links between amino acid side chains of different proteins, thereby enabling the ligation of two or more proteins or protein-containing binding partners. Accordingly, the present invention addresses the need for protein ligation technology and particularly ligation which is reversible, by exploiting the unique characteristics of ester bonds. This technology enables complex protein assemblies to be engineered with a fine degree of control.
(38) In certain aspects the present invention relates to recombinant polypeptides comprising one or more amino acid sequences comprising an immunoglobulin (Ig) like domain, wherein the Ig-like domain is split into a truncated protein and a peptide comprising the final β-strand of the Ig-like domain. In particular, certain embodiments of the invention relate to reversible spontaneously-formed ester bond between the truncated protein, a derivative or fragment thereof and the peptide, a derivative or fragment thereof.
(39) In other aspects the invention relates to one or more truncated Ig-like domains comprising one or more heterologous amino acid sequences, wherein when two or more such truncated Ig-like domains are contacted with one another the Ig-like domains undergo self-polymerisation.
(40) In certain embodiments a heterologous amino acid sequence is referred to herein as a “cargo”, for example, as a “cargo protein”, or a “cargo enzyme”.
(41) The invention further relates to a truncated Ig-like domain comprising a plurality of attached cargo proteins, wherein the truncated Ig-like domain is reversibly self-polymerising. In certain embodiments, the self-polymerising Ig-like domains are from the same Ig-like domain-containing protein. In other embodiments, Ig-like domains from different Ig-like domain-containing proteins self-polymerise, or reversibly self-polymerise.
(42) The truncated reversibly self-polymerising Ig-like domain provides for the controllable assembly of cargo proteins. For example, one or more truncated reversibly self-polymerising Ig-like domains provide for the controllable assembly of protein ‘scaffolds’ comprising one or more cargo proteins. In particular, the truncated reversibly self-polymerising Ig-like domain is useful for the controllable assembly of a plurality of enzymes. In some embodiments, the invention is useful for emulating natural enzymatic pathways for optimising enzyme yield.
(43) Certain advantages of the invention include: a) increased peptide stability, b) highly controllable protein assembly process, c) ease and efficiency of peptide and protein manufacture, d) minimal cross-reactivity between non-adjacent or non-complementary binding pairs in multimeric protein complexes, and e) reversible self-catalysing peptide cross-links.
Certain Definitions
(44) The term “and/or” can mean “and” or “or”.
(45) The term “comprising” as used in this specification means “consisting at least in part of”. When interpreting statements in this specification which include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as “comprise” and “comprised” are to be interpreted in the same manner.
(46) As used herein “purified” does not require absolute purity; rather, it is intended as a relative term where the material in question is more pure than in the environment it was previously in. In practice the material has typically, for example, been subjected to fractionation to remove various other components, and the resultant material has substantially retained its desired biological activity or activities. The term “substantially purified” refers to material that are at least 60% free, preferably at least about 75% free, and most preferably at least about 90% free, at least about 95% free, at least about 98% free, or more, from other components with which they may be associated during manufacture.
(47) The term “α-amino acid” or “amino acid” refers to a molecule containing both an amino group and a carboxyl group bound to a carbon which is designated α-carbon. Suitable amino acids include, without limitation, both the D- and L-isomers of the naturally-occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes. Unless the context specifically indicates otherwise, the term amino acid, as used herein, is intended to include amino acid analogues.
(48) In certain embodiments a protein, polypeptide, or peptide as contemplated herein comprises only natural amino acids. The term “naturally occurring amino acid” refers to any one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V. In other embodiments, a protein, polypeptide, or peptide as contemplated herein comprises one or more amino acid analogues.
(49) The term “amino acid analogues” or “non-naturally occurring amino acid” refers to a molecule which is structurally similar to an amino acid and which can be substituted for an amino acid. Amino acid analogues include, without limitation, compounds which are structurally identical to an amino acid, as defined herein, except for the inclusion of one or more additional methylene groups between the amino and carboxyl group (e.g., α-amino β-carboxy acids), or for the substitution of the amino or carboxy group by a similarly reactive group (e.g. substitution of the primary amine with a secondary or tertiary amine, or substitution or the carboxy group with an ester).
(50) Unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry and immunology, which are within the skill of the art may be employed in practicing the methods described herein. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Handbook of Experimental Immunology (D. M. Weir & C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells G. M. Miller & M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); The Immunoassay Handbook (David Wild, ed., Stockton Press NY, 1994); Antibodies: A Laboratory Manual (Harlow et al., eds., 1987); and Methods of Immunological Analysis (R. Masseyeff, W. H. Albert, and N. A. Staines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993).
(51) The term “peptide” and the like is used herein to refer to any polymer of amino acids residues of any length. The polymer can be linear or non-linear (e.g., branched), it can comprise modified amino acids or amino acids analogues. The term also encompasses amino acid polymers that have been modified naturally or by intervention, for example, by disulphide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other modification or manipulation, for example conjugation with labelling or bioactive component.
(52) A “fragment” as used herein with reference to a specified protein typically contemplates at least about 10 contiguous amino acids of the specified protein. For example, a fragment of an Ig-like fold domain from an Ig-like fold containing protein comprises 10 or more contiguous amino acids from said Ig-like fold domain. Similarly, a fragment of an amino acid sequence presented herein as one of SEQ ID NO.s 1 to 4, 21 to 30, or 31 to 58, comprises 10 or more contiguous amino acids from the specified sequence.
(53) The term “truncated protein” is used herein to refer to protein derived from the truncation of the Ig-like domain of adhesin protein Cpe0147 (residues 439 to 563). Other uses of the term ‘truncated protein’ will be apparent from the context in which the term is used herein, for example, in reference to truncation of a different, specified protein.
(54) As used herein the terms “branch” and/or “branch domain” when used with reference to a protein-containing component herein, such as a peptide tag, binding partner, chimeric protein, protein scaffold or protein complex, contemplates a polypeptide or protein domain that provides a ligating or linking moiety or function to join one or more cargoes or functionalities to one or more other protein components of a multimeric protein complex. Examples of branch domains are provided herein, for example in Examples 18 and 19, and are depicted in, for example,
(55) As exemplified herein, a branch or branch domain will in certain embodiments ‘capture’ and/or ligate to the one or more other protein components one or more cargoes or functionalities, such as one or more cargo proteins, via a peptide tag/peptide binding partner interaction and/or spontaneous covalent bond formation as herein described.
(56) In certain specifically contemplated examples, the branch domain comprises either a) one reactive residue capable of being involved in a spontaneously-formed ester bond within a β-clasp arrangement in a β-clasp containing protein, and comprises at least 5 contiguous amino acids of said β-clasp containing protein, or b) comprises a fragment of a β-clasp containing protein wherein said fragment comprises at least about 10 contiguous amino acids of said β-clasp containing protein and comprises a reactive residue capable of being involved in the spontaneously-formed ester bond in a β-clasp containing protein. In one example, the branch domain comprises one part of the Cpe2-C2pept binding pair exemplified herein, where the complementary part of the Cpe2-C2pept binding pair is present on or comprises the cargo.
(57) As used herein the term “cargo” contemplates a functionality that ultimately is to be present in or on or otherwise incorporated into a protein as described herein, such as a chimeric protein or multimeric protein complex as described herein. In certain embodiments, a cargo is attached to or to be attached to a peptide tag or a binding partner, or comprises a part of a chimeric protein or truncated protein, which in turn may be covalently bound to one or more other protein components to form multimeric protein complexes as herein provided. Such cargoes are also referred to herein as valencies, whereby a multivalent protein or protein complex contemplates the presence of multiple cargoes, which may be the same or may be different. It will be appreciated by those skilled in the art that in certain embodiments, a heterologous amino acid sequence comprises a cargo, and may thus provide a functionality or part of a functionality.
(58) Particularly contemplated cargo proteins are enzymes, or parts of enzymes such as enzyme active sites or one or more subunits of a multimeric enzyme or enzyme complex.
(59) As used herein the terms “trunk” and/or “trunk domain” when used with reference to a protein-containing component herein, such as a peptide tag, binding partner, chimeric protein, protein scaffold or protein complex, contemplates a polypeptide or protein domain that provides a scaffold moiety or function to which one or more further trunks or trunk domains, one or more branches or branch domains, one or more cargoes or functionalities, or one or more other protein components of a multimeric protein complex as herein described is covalently bound. Accordingly, a trunk or trunk domain can be thought of as a component of a core to or around which other components, including other trunk or trunk domains, are formed. Examples of trunk domains are provided herein, for example in Examples 18 and 19, and are depicted in, for example,
(60) As exemplified herein, a trunk or trunk domain will in certain embodiments ligate to the one or more other protein components, such as one or more other trunk or trunk domains, or one or more branches or branch domains, via a peptide tag/peptide binding partner interaction and/or spontaneous covalent bond formation as herein described.
(61) In certain specifically contemplated examples, the trunk domain comprises either a) one reactive residue capable of being involved in a spontaneously-formed ester bond within a β-clasp arrangement in a β-clasp containing protein, and comprises at least 5 contiguous amino acids of said β-clasp containing protein, or b) comprises a fragment of a β-clasp containing protein wherein said fragment comprises at least about 10 contiguous amino acids of said β-clasp containing protein and comprises a reactive residue capable of being involved in the spontaneously-formed ester bond in a β-clasp containing protein. In one example, the trunk domain comprises an Ig-like domain or a part thereof, such as an Ig-like domain of a β-clasp containing protein, such as an Ig-like domain lacking its C-terminal β-strand or a part thereof. In another example, the trunk domain comprises an Ig-like domain or part thereof, in addition to a β-strand from another Ig-like domain, such as the final β-strand of another Ig-like domain, for example the final β-strand from the preceding Ig-like domain in the full length β-clasp domain of a β-clasp-containing protein. In one example, the trunk domain comprises at least a part of an Ig-like domain from Cpe0147 protein, and optionally has an additional part of another Ig-like domain from a β-clasp domain of a β-clasp-containing protein, including a part of another Ig-like domain from Cpe0147, such as the final β-strand of the preceding Ig-like domain in the full length Cpe0147 protein. In other examples, comparable trunk domains comprising one or more parts of one or more Ig-like domains such as the Mol polypeptides exemplified in Examples 6 to 16, or the Mol domains exemplified in Examples 17 to 20, are used.
(62) The term “(s)” following a noun contemplates the singular or plural form, or both.
(63) The invention consists in the foregoing and also envisages constructions of which the following gives examples only and in no way limit the scope thereof.
(64) Cpe0147 Adhesin
(65) Microbial surface components recognising adhesive matrix molecules (MSCRAMMs) are a class of bacterial surface molecules which are very long, thin and subject to large mechanical shear stresses in protease-rich environments. MSCRAMMs are typically single polypeptides folded into many domains. In particular, the present invention takes advantage of MSCRAMM adhesin proteins derived from Gram-positive bacteria. In an exemplary embodiment of the invention, adhesin derived from Clostridium perfringens is used. Other examples of adhesins or related proteins derived from bacteria other than C. perfringens and useful as described herein are contemplated, including the proteins and bacterial species exemplified herein in the Examples.
(66) Adhesins are important for mediating bacterial attachment to surfaces. Bioinformatic analysis of Clostridium perfringens adhesin (Cpe0147) predicts the structure to comprise an N-terminal adhesin domain attached to the cell wall by a shaft comprising 11 repeating domains and terminating with a C-terminal cell wall-anchoring motif (5′-LPKTG). The repeating domains have been predicted to each have an all β-strand IgG-like fold (Kwon, H.; Squire, C. J.; Young, P. G.; Baker, E. N., Autocatalytically generated Thr-Gln ester bond cross-links stabilize the repetitive Ig-domain shaft of a bacterial cell surface adhesin. P Natl Acad Sci USA 2014, 111 (4), 1367). Within each domain, the side chain of a threonine residue on the first β-strand is covalently linked to the side chain of a glutamine residue on the last β-strand by an ester bond.
(67) Spontaneous Ester Bond Formation
(68) Without wishing to be bound by any theory of mechanism, it is believed that, the ester bonds cross-links are spontaneously formed between hydroxyl and amide, or between hydroxyl and carboxylic acid/carboxylate groups on the amino acid side chains by nucleophilic attack of Thr-450 on Gln-580 (or Glu-580), proton abstraction by His-572, and bond polarisation by the Asp-480/Glu-547 pair. Comparable reactions are expected, again without wishing to be bound by any theory, in active sites comprising the reactive amino acid residues of other Ig-like domains capable of undergoing such spontaneous covalent bond formation as described herein.
(69) The term “ester bond” as used herein, refers to a covalent bond between a hydroxyl group and an amide (with the elimination of an ammonia or water molecule), or between a hydroxyl group and carboxylic acid or carboxylate groups at least one which is not derived from a protein main chain. An ester bond may form intramolecularly within a single protein or intermolecularly between two peptide/protein or protein/protein molecules.
(70) Typically, an ester bond may occur between, for example a threonine or a serine residue and a glutamine, glutamate/glutamic acid, asparagine or aspartate/aspartic acid. Each residue of the pair involved in the ester bond is referred to herein as a reactive residue. Thus, an ester bond may form between a threonine residue and a glutamine residue. Particularly, ester bonds can occur between the side chain hydroxyl of threonine and amide group of glutamine.
(71) The term “peptide binding pair” as discussed herein refers to a binding partner having one reactive residue and a second binding partner having the second reactive residue. When contacted, the reactive residues from each binding partner form an ester bond cross-link. It will be appreciated that a polypeptide, such as a chimeric polypeptide contemplated herein, comprising one reactive residue capable of forming an ester bond with one binding partner will in certain embodiments comprise one or more other reactive residues capable of forming an ester bond with another binding partner, thereby having multiple binding partners. Representative examples of such polypeptides, capable of binding more than one binding partner, are presented herein in the Examples. Accordingly, a peptide binding pair does not exclude the binding of other binding partners to the peptides present in the pair, and those of skill in the art will appreciate that further binding partners may be attached to form further binding pairs, for example as part of a multimeric protein complex.
(72) Ester bond formation between the reactive residues of the peptide binding pair may be facilitated by, for example an aspartic acid/aspartate and/or a histidine amino acid residue. Each residue facilitating the spontaneous ester bond formation is referred to herein as an accessory residue, as the residue facilitates the reaction but is unmodified by it. Thus, an aspartic acid and a histidine may facilitate the spontaneous ester bond formation between the reactive residue pair.
(73) When the reactive residues are contacted, the reactive residues form an Ig-like fold containing a serine protease active site-like geometry. The serine protease active site-like structure comprising the reactive residues and accessory residues is referred to herein as the active site (see
(74) The active site may comprise a threonine reactive residue in close proximity to the second reactive residue, for example a glutamine, glutamic acid or glutamate reactive residue, a first accessory residue, for example a histidine and a second accessory residue, for example an aspartic acid. For example, the Cβ atom of the threonine reactive residue may be within 2.40, 2.45, 2.50, 2.55, 2.60, 2.65, 2.70, 2.75, 2.80 or 2.85 Angstrom from the C6 of the reactive glutamine residue. For example, the Cβ atom of the threonine reactive residue may be within 4.50, 4.55, 4.60, 4.65, 4.70, 4.75, 4.80, 4.85, 4.90, 4.95, 5.00, 5.05, 5.15, 5.20, 5.25, 5.30, 5.35, 5.40, 5.45, 5.50, 5.55, 5.60, 5.65, or 5.70 Angstrom from the Cγ atom of the histidine accessory residue. For example, the Cβ atom of the threonine reactive residue may be within 4.00, 4.05, 4.10, 4.15, or 4.20 Angstrom from the Cγ atom of the aspartic acid accessory residue.
(75) The term “spontaneously-formed” as used herein refers to a bond e.g. an ester or covalent bond which can form in a protein or between peptides or proteins (e.g. between 2 peptides or a peptide and a protein) without any other agent (e.g. an additional enzyme catalyst) being present and/or without chemical modification of the protein or peptide. A spontaneously-formed ester bond may form almost immediately after the production of a protein or after contact between peptide or protein binding partner e.g. within 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 minutes or within 1, 2, 4, 8, 12, 16 or 20 hours. The present inventors have established an amino acid substitution in the Ig-like fold of Cpe0147, Thr450>Ser, which preserves the spontaneously-formed ester bond between amino acid side chains, but renders ester bond formation reversible. Other amino acid substitutions which enable a reversible ester bond, such as serine homologues or derivatives including non-naturally occurring derivatives, are also contemplated.
(76) The term “reversible” as used herein refers to a hydrolysable ester bond which can be hydrolysed when initiated by a trigger, for example, a pH change. Typically, a hydrolysable ester bond may form between a serine residue and a glutamine, glutamate, or glutamic acid residue. Particularly, ester bonds can occur between the side chain hydroxyl group of the serine residue and amide group of the glutamine residue.
(77) In specifically contemplated embodiments, such as those exemplified by the Ser450 substitution in the Ig-like fold of Cpe1047, maintaining the complex in which the ester bond is present at a pH of about 7 or greater leads to hydrolysis of the serine-containing ester bond. Notably, other ester bonds which may be present in the complex and which are not reversible, for example, do not involve a serine-glutamine or a serine-glutamate/glutamic acid ester bond, are not hydrolysed. Those skilled in the art will recognise this specificity contributes to the directed construction of the multimeric protein complexes described and exemplified herein.
(78) A reversible ester bond as contemplated herein will in certain embodiments be almost immediately hydrolysed after the protein complex, protein, polypeptide, or peptide in or between which the reversible ester bond is present is introduced into suitable conditions. For example, the bond is hydrolysed within 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 minutes or within 1, 2, 4, 8, 12, 16 or 20 hours. As exemplified in the Examples presented herein, pH is a significant determinant of reversibility of the serine-glutamine or a serine-glutamate/glutamic acid ester bonds described herein, where increasing the pH to 7 or above leads to hydrolysis. As outlined in the Examples, other factors, such as buffer conditions and buffering agents, the presence or absence of divalent cations, and/or the presence or absence of molecular crowding agents such as glycerol, can influence the kinetics and equilibrium of the hydrolysis reaction, and those skilled in the art can, using the description provided herein, identify reaction conditions to provide desired and/or optimal hydrolysis of reversible ester bonds present in the protein complexes described herein.
(79) A “conservative amino acid substitution” is one in which an amino acid residue is replaced with another residue having a chemically similar or derivatised side chain. Families of amino acid residues having similar side chains, for example, have been defined in the art. These families include, for example, amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartate/aspartic acid, glutamate/glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Amino acid analogues (e.g., phosphorylated or glycosylated amino acids) are also contemplated in the present invention, as are peptides substituted with non-naturally occurring amino acids, including but not limited to N-alkylated amino acids (e.g. N-methyl amino acids), D-amino acids, β-amino acids, and γ-amino acids.
(80) Peptide Motif
(81) In addition to the Cpe0147, the inventors have identified other peptide/structures, including multiple adhesin protein domains from Mobiluncus mulieris [SEQ ID Nos. 21-29, 31], comprising reactive and accessory amino acid residues that are capable of spontaneous ester bond formation. A HxDxxDxxQ peptide sequence motif was identified containing the glutamine reactive residue and histidine accessory residue. The HxDxxDxxQ peptide sequence motif may, for example form one peptide of a peptide binding pair, a part of a chimeric protein as herein described, or a part of a protein component for or of a multimeric protein complex as herein described. A representative list of peptide/structures containing the HxDxxDxxQ peptide motif capable of spontaneous ester bond formation is shown in
(82) Numerous representative engineered peptide binding pairs comprising reactive and accessory amino acid residues as above are exemplified herein in the Examples, wherein one or more of the reactive and accessory amino acid residues are present in one peptide (see, for example, the peptide sequences presented in Tables 42 and 44, and one or more of the other reactive and accessory amino acid residues comprising a binding pair are present in another peptide (see, for example, the peptide sequences presented in Tables 41 and 43). Furthermore, the Examples herein exemplify peptide binding pairs wherein one peptide comprises more than one set of reactive and accessory amino acid residues, to enable ligation to multiple binding partners. For example, certain peptide constructs presented in, for example, Example 18 and 19, comprise one set of reactive and accessory amino acids to enable ligation to a complementary ‘trunk’ domain (such as the Mol trunk domain depicted in
(83) Applications of the Technology
(84) It will be appreciated that the present invention is useful in the fields of molecular biology, immunology, synthetic biology, nanotechnology and other related fields. For example, the present invention is useful in purification, detection and identification of peptides and/or proteins of interest, protein scaffolding for enhancing resilience and efficacy of enzymes.
(85) Those skilled in the art will appreciate that the polypeptides of the invention are in certain embodiments suited for engineering self-assembling enzymatic complexes that emulate the natural efficiency of clustered multienzyme complexes. It will be appreciated that current protein scaffolds are restricted to small, two or three enzyme complexes with limited control of enzyme stoichiometry (Lee, H.; DeLoache, W. C.; Dueber, J. E., Spatial organization of enzymes for metabolic engineering. Metab Eng 2012, 14(3) 242-251, Horn, A. H. C.; Sticht, H., Synthetic protein scaffolds based on peptide motifs and cognate adaptor domains for improving metabolic productivity. Frontiers in Bioengineering and Biotechnology, 2015, 3(191), 1-7 and Chen, R. Chen, Q.; Kim, H.; Siu, K. H.; Sun, Q.; Tsai, S. L.; Chen, W., Biomolecular scaffolds for enhanced signalling and catalytic efficiency. Curr Opin Biotech 2014, 28, 59-68). All enzymes whether or not presently characterised, that form part of enzymatic pathways are contemplated.
(86) In certain embodiments, the present invention are modular building blocks which co-localizes an enzymatic pathway in a specific arrangement. By selecting appropriate peptide tag/binding partner pairs, for example within trunk domains, multimeric protein complexes can be assembled in a directed manner, such that the cargo proteins or other functionalities can be positioned in a predetermined arrangement. In certain embodiments, such as is shown in the Examples, in particular in Examples 17-19, the appropriate selection of 5 different trunk domains enabled, unlike current technology, the complex assembly of each building block in a specific order of at least 5 domains, in the case of the trunk domains employed in Example 17 in a single reaction.
(87) The constructs and methods described herein enable those skilled in the art to select and construct trunk domains that self-arrange in a specific order, whereby the trunk domains comprise at least part of one or more Ig-like folds, such that specific complementation between a first trunk domain comprising a first part of an Ig-like fold and a second trunk domain comprising a complementary part of the Ig-like fold leads to the specific binding of and formation of an ester bond between the two trunk domains. As those skilled in the art will appreciate, particularly in light of the examples provided herein such as those presented in Examples 17-19, the appropriate construction of one or more trunk domains having more than one part of an Ig-like fold enables such trunk domains to bind more than one other trunk domain, in turn enabling the directed binding of trunk domains in a predetermined order. For example, in certain embodiments, one trunk domain comprises, for example, at least a part of the first or last β-strand of a β-sheet usually present in a β-clasp arrangement, and a second trunk domain comprises at least a complementary part of the β-strand or the β-sheet thereby to recapitulate the β-clasp arrangement on binding of the first and second trunk domains.
(88) In certain embodiments, multimeric protein complexes contemplated herein comprise a single trunk or branch domain to which a cargo or functionality is attached. In other embodiments, multimeric protein complexes contemplated herein comprise multiple trunk and/or branch domains, to any one or more of which one or more cargoes or functionalities are attached. Accordingly, in certain embodiments, multimeric protein complexes contemplated herein comprise at least 2, at least 3, at least 4, at least 5, or more than 5 protein components, such as 2, 3, 4, 5 or more than 5 trunk domains, branch domains, or cargo proteins. For example, multimeric protein complexes comprising 2, 3, 4, 5, or more than 5 trunk domains are specifically contemplated, as exemplified herein. Such multimeric protein complexes, including those exemplified herein, comprise additional protein components, for example 2, 3, 4, 5, or more than 5 branch domains, and/or 2, 3, 4, 5, or more than 5 cargo proteins.
(89) As outlined above, it will be appreciated that unlike current technology, the present invention enables complex assembly of each building block in a specific order of at least 5 domains, commonly in a single reaction. In certain embodiments of the invention, a nanochain of five or more modular building blocks may be created. A nanochain of 10 or more building blocks are contemplated. It will also be appreciated that unlike current technology, the present invention enables complex assemblies to be made in a single reaction mixture.
(90) In certain embodiments, the present invention is useful for purification, detection and identification applications, for example in the isolation of rare cells, including circulating tumour cells (CTC) (magnetic bead-affibody capture). Current technologies are typically limited by difficulty of capturing CTCs expressing low levels of tumour markers. It will be appreciated that the strong spontaneously-formed covalent bonds enabled by the present invention increases the sensitivity of diagnostics methods.
(91) In certain embodiments, the invention is useful for investigating mechanical/physical characteristics of proteins, for example, by immobilisation onto atomic force microscopy (AFM) tips. It will be appreciated that the binding specificity of the present invention is suitable for studying mechanical properties of proteins compared with current approaches based on less specific disulphide bonds.
(92) In certain embodiments, the invention is useful for enhancing the resilience and stability of enzymes, for example by circularisation of enzymes. It will be appreciated that increased enzyme resilience and stability enabled by the present invention is desired in many important applications such as biotransformation, biofuel production and molecular diagnostics.
(93) In certain embodiments, the invention is useful for synthetic vaccine generation, for example in covalently attaching antigens to antibodies. Current technologies for synthetic vaccine generation are typically time consuming, costly and often limited by the size of the molecules expressed. The present invention enables generation of different protein subunits which can be assembled with high specificity at a lower cost and shorter time.
(94) In certain embodiments, the invention is useful for therapeutic delivery of enzyme or proteins in outer membrane vesicles. It will be appreciated that using current technology, recombinant products are typically produced using a single microbial culture and purified for use. Accumulation of recombinant products within host expressing the product often leads to toxicity and limited yield. The present invention enables production of recombinant products in outer membrane vesicles reducing cellular toxicity and increases yield.
(95) In certain embodiments, the invention is useful for constructing catalytic biofilms. The present invention enables engineering of biofilms to display functional peptides such as catalytic enzymes.
(96) In certain embodiments, the invention is useful for protein expression and solubility analysis. It will be appreciated that current methods for expression and solubility analysis are typically very time consuming requiring many purification steps. The present invention enables rapid expression and solubility analysis by attachment of fluorescent labels to proteins of interest.
(97) In certain embodiments, the invention is useful for producing protein based hydrogels. It will be appreciated that the present invention enables the synthesis of protein scaffolds that form spontaneously under physiological conditions.
(98) In certain embodiments, the invention is useful for producing synthetic nanofibers. The present invention enables production of self-polymerising protein monomers.
(99) It will be appreciated on reading this specification that multivalent protein structures can be assembled using various combinations of peptide binding pairs, where one binding partner comprises components of a spontaneously-reacting active site as described herein, and the other binding partner comprises the remaining components of a spontaneously-reacting active site, sufficient to recapitulate the active site and enable spontaneous bond formation. By appropriate selection of binding partners and active site components, binding specificity and selectivity can be achieved so as to enable the ordered construction of protein scaffolds carrying multiple functionalities. One representation of such a scaffold is shown herein in
(100) A protein scaffold carrying multiple functionalities, such as that depicted in
(101) Homovalent multimeric protein complexes, including multi-homovalent multimeric protein complexes, and heterovalent multimeric protein complexes, including multi-heterovalent multimeric protein complexes, including such complexes comprising one or more of the protein components specifically described and/or exemplified herein, including one or more protein components comprising 10 or more contiguous amino acids of the amino acid sequence of any one of SEQ ID NO.s 1-4 or 21-30, and/or comprising 10 or more contiguous amino acids of the amino acid sequence of any one of SEQ ID NO.s 31-58, such as 10 or more contiguous amino acids of the amino acid sequence of any one of SEQ ID NO.s 31-58, and comprises at least one amino acid from two or more of the domains present in said amino acid sequence as identified in one of Tables 37 to 41 herein, are specifically contemplated herein.
(102) The following examples are intended to illustrate but not to limit the invention in any manner, shape, or form, either explicitly or implicitly. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.
EXAMPLES
(103) Methodology
(104) Bacterial Strains, Plasmid and Oligonucleotides
(105) E. coli strain DH5a was used for all DNA manipulation and the BL21 (ΔDE3) (Stratagene) strain was used for protein expression. Cultures were grown at 37° C. in 2×YT medium supplemented with ampicillin (100 μg/ml). The oligonucleotide primers used are listed in Table 1.
(106) Cloning C2 Cpe0147 Constructs
(107) DNA encoding the Cpe0147 amino acid sequence 439-563 (for full sequence see Uniprot entry B1R775) was PCR amplified from the C2 construct previously reported in Kwon et al. (2014) using primers PYC2NtermFwd [SEQ ID No. 5] and PYC2NtermRev [SEQ ID No. 6]. Amplified PCR fragments were digested with EcoRI and KasI restriction endonucleases, and cloned into the expression vector pMBP-ProExHta (Invitrogen). pMBP-ProExHta, previously reported in Ting, Y. T.; Batot, G.; Baker, E. N.; Young, P. G. Acta crystallographica. Section F, Structural biology communications 2015, 71, 61, was generated by inserting the maltose binding protein (MBP) gene between the His.sub.6-tag and the rTEV (recombinant Tobacco Etch Virus protease) cleavage site of pProExHta. The resulting vector, pMBP-Cpe0147.sup.439-563, produces an N-terminal His.sub.6-tagged MBP fusion protein followed by an rTEV cleavage site and the Cpe0147.sup.439-563 truncated protein domain.
(108) A second construct that lacks the cleavable rTEV recognition sequence was created by sub-cloning Cpe0147.sup.439-563 into the vector pMBP3, previously described in Ting et al. 2015. The resulting vector, pMBP3L-Cpe0147.sup.439-563, produces an N-terminal His.sub.6-tagged MBP fusion protein followed by an -AGA- three residue linker and the Cpe0147.sup.439-563 truncated protein domain.
(109) A third, self-polymerising construct, was produced by the PCR amplification of Cpe0147 amino acid sequence 416-563 from the C2 construct using primers Fwdcomp1 [SEQ ID No. 7] and PYC2NtermRev [SEQ ID No. 6]. Amplified PCR fragments were digested with EcoRI and KasI restriction endonucleases, and were cloned into the expression vector pMBP-ProExHta to create the construct pMBP-Cpe0147.sup.416-563Poly.
(110) A construct comprising enhanced green fluorescent protein (eGFP) engineered with an N-terminal peptide tag derived from residues 565-587 of Cpe0147, was produced as follows. Customized, complementary 76 bp synthetic oligonucleotides CtermpeptF2 [SEQ ID No. 8] and CtermpeptR2 [SEQ ID No. 9]; Integrated DNA Technologies) encoding residues 565-587 of Cpe0147 were annealed by applying a temperature gradient from 100° C. to 20° C. The annealed product contained single-strand overhangs complementary to KasI and NcoI restriction endonuclease sites, and was inserted at the N-terminus of eGFP in the construct SP-GFP (Ting et al., 2015) between KasI and NcoI sites to create the construct pC2pept-GFP. This construct contains an N-terminal His.sub.6-tag sequence followed by an rTEV cleavage site and the Cpe0147.sup.565-587 peptide sequence fused to eGFP. All constructs were sequence verified at the DNA sequencing facility, School of Biological Sciences, University of Auckland.
(111) TABLE-US-00001 TABLE 1 List of primers used. Primer name Sequence SEQ ID PYC2NtermFwd AAA GGC GCC AAT CTG CCT GAA GTG AAA GAT GG 5 PYC2NtermRev TTT GAA TTC TCA GTT GTA ATC TTT ATC CGT ATC GAT 6 Fwdcomp1 AAA GGC GCC GAT ACC AAA CAG GTG GTG AAA C 7 PYC2T13SFwd {circle around (P)}-AGC ACC GTT ATT GCA GAT GGC G 10 PYC2T13CRev {circle around (P)}-ACG CAG TGT ACC ATC TTT CAC 11 CtermpeptF2 {circle around (P)}-GCG CCG ACA CAA AAC AGG TTG TCA AAC ATG AGG ACA AAA ACG ACA AAG CAC 8 AGA CAC TGG TGG TTG AAA AAC CGA C CtermpeptR2 {circle around (P)}-CAT GGT CGG TTT TTC AAC CAC CAG TGT CTG TGC TTT GTC GTT TTT GTC CTC ATG 9 TTT GAC AAC CTG TTT TGT GTC G {circle around (P)}= 5′ Phosphate
Site-Directed Mutagenesis of Cpe0147
(112) A T450S variant of pMBP3L-Cpe0147.sup.439-563 was made by inverse PCR site-directed mutagenesis using the phosphorylated primers PYC2T13SFwd [SEQ ID No. 10] and PYC2T13SRev [SEQ ID No. 11] with pMBP3L-Cpe0147.sup.439-563 as the template. Briefly, a high-fidelity DNA polymerase (iProof, Bio-Rad) was used for the PCR amplification of the pMBP3L-Cpe0147.sup.439-563 plasmid to produce a linearized PCR product with the desired mutation at the 5′ end of the sense primer. The methylated parental template without the T450S mutation was then removed from the non-methylated linear PCR product by DpnI digestion. Finally, the PCR product was re-circularized by intermolecular ligation. The resulting plasmid pMBP3L-Cpe0147-T450S.sup.439-563 was transformed into E. coli DH5α cells, amplified, extracted and purified for sequence verification. A fully intact domain Cpe0147-T450S.sup.439-587 was also engineered.
(113) Protein Expression and Purification
(114) The E. coli BL21 (20E3) cells harboring recombinant expression constructs were grown in 2×YT medium supplemented with ampicillin (100 μg/ml) at 37° C. in an orbital shaker (@180 rpm) to an optical density of OD.sub.600=0.5-0.6. Protein expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.3 mM and cultures were left to incubate for an additional 16 h at 18° C. Cells were pelleted at 4000 g at 4° C. for 20 minutes, snap-frozen, and stored at −20° C.
(115) Recombinant protein was purified from frozen cells, which were thawed and resuspended in lysis buffer [50 mM HEPES pH 7.0, 300 mM NaCl, 5% (v/v) glycerol, 10 mM imidazole] with the addition of Complete EDTA-free Protease Inhibitor Cocktail tablets (Roche) and lysed using a cell disruptor at 18,000 psi (Constant Systems). The insoluble protein fraction was removed by centrifugation (55,000 g at 4° C. for 30 minutes) and the soluble recombinant protein fraction loaded onto a 5 mL Protino NiNTA 5 column (Macherey-Nagel) for purification by immobilized metal affinity chromatography (IMAC). Recombinant protein was washed with Wash Buffer [50 mM HEPES pH 7, 300 mM NaCl, 20 mM imidazole] and eluted in a linear gradient with Elution Buffer (Wash Buffer with 500 mM imidazole).
(116) For constructs with removable His- or His/MBP affinity tags, fractions from IMAC containing recombinant protein were dialyzed overnight against a >100×volume of dialysis buffer [20 mM HEPES pH7, 100 mM NaCl, 1 mM beta-mercaptoethanol] and the His6-tag or His-MBP concomitantly removed using recombinant TEV protease at a 1:50 molar ratio of rTEV to recombinant protein. Undigested protein and rTEV protease were removed by a second round of IMAC. Proteins with cleaved His-MBP tags were subjected to an additional purification by passage through an amylose resin (NEB) to remove contaminating cleaved MBP protein. Purified protein was concentrated and subjected to size-exclusion chromatography (SEC) on a Superdex 200 10/300 column (GE Healthcare) equilibrated with 10 mM HEPES pH7 and 100 mM NaCl. SEC-purified protein was concentrated to ˜20 mg/ml and flash cooling in liquid nitrogen for subsequent storage at −80° C.
(117) Peptide Synthesis
(118) A synthetic peptide comprising Cpe0147.sup.565-587, was prepared using the Fmoc/tBu solid phase methodology on a Tribute (Tucson, Az.) automated synthesizer on a 0.1 mmol scale using appropriately functionalized aminomethyl polystyrene resin. Briefly, the N-Fmoc group was removed with 20% piperidine in DMF (v/v) for 2×5 mins and the incoming Fmoc amino acid (0.5 mmol) was coupled with HATU (0.45 mmol) and DIPEA (1 mmol) in DMF for 20 mins. The peptide was released from the resin with 95% TFA, 2.5% TIPS and 2.5% water (v/v/v) for 3 h, precipitated with ether and recovered by centrifugation. Crude peptide was purified by reverse phase HPLC using an appropriate gradient based on its analytical profile and the mass confirmed by LC-MS.
(119) Mass Spectrometry
(120) Protein masses for Cpe0147.sup.439-563-Cpe0147.sup.565-587 products were confirmed by LC-MS using an Agilent 1120 Compact LC system with a Hewlett Packard Series 1100 MSD mass spectrometer using ESI in the positive mode. LC-MS was performed using a Zorbax SB-300 C3 (5 μm; 3.0×150 mm) column (Agilent) and a linear gradient of 5% to 65% B over 21 mins (˜3% B per minute) at a flow rate of 0.3 ml/min. The solvent system used was A (0.1% formic acid in H.sub.2O) and B (0.1% formic acid in acetonitrile). Date was acquired in the m/z range of 400-2000 and the m/z values were deconvoluted to yield the monoisotopic mass. All other mass spectrometry experiments were performed by the Mass Spectrometry Centre, The University of Auckland, Auckland, New Zealand, using an LC-MS/MS, Q-Star XL Quadrupole-Time-of-Flight system.
(121) Ester Bond Ligation Reactions
(122) Initial protein purification of Cpe0147.sup.439-563 was performed in a TRIS.Math.HCl pH 8.0 buffering system. The initial experiments exploring the effect on pH and buffering systems contained residual TRIS.Math.HCl (˜2-5 mM) and NaCl (5 mM) from the diluted protein. For subsequent experiments protein was purified with a HEPES buffering system. Reactions for determining ester bond formation were performed with a protein concentration of 10 μM. Concentrated protein stored at −80° C. was thawed and diluted ˜20 fold to 10 μM in the reaction buffer while the concentration of the other components was varied. All reactions were incubated at 20° C. unless otherwise stated. For time course experiments, samples were collected from a larger volume in the reaction tube and were stopped by adding SDS loading buffer and heating at 99° C. for ˜3 min.
(123) NMR Spectroscopy
(124) NMR experiments were conducted using a Bruker 500 MHz instrument equipped with a BBFO probe. Conventional 5 mm NMR tubes (Norell) were used. Samples typically contained 90% H.sub.2O and 10% D20. Unless otherwise stated, all experiments were conducted at 300 K. Standard .sup.1H proton pulse sequence was used and water suppression was achieved by the excitation sculpting method with a 2 ms Squa100.1000 pulse. The pulse tip-angle calibration using the single-pulse nutation method (Bruker pulsecal routine)(Wu, P. S. C.; Otting, G. J. Magn. Reson. 2005, 176, 115) was undertaken for each sample.
(125) Small-Angle X-Ray Scattering
(126) Samples for small angle X-ray scattering were buffer exchanged into 10 mM HEPES pH 7.0, 100 mM NaCl with size exclusion chromatography (SEC). Data were collected at the Australian Synchrotron SAXS/WAXS beamline at a wavelength of 1.03 Å with a camera length of 1.6 m covering a momentum transfer range of 0.006<q<0.6 Å.sup.−1 (q=4π sin(θ)/λ). Data were collected by SEC-SAXS and images were processed using scatterBrain.sup.4 and PRIMUS.sup.5. SAXS data were further analyzed using programs in the ASTAS package including ab initio modeling produced in GASBOR and DAMMIF and with consensus models generated with DAMAVER (Petoukhov, M. V.; Franke, D.; Shkumatov, A. V.; Tria, G.; Kikhney, A. G.; Gajda, M.; Gorba, C.; Mertens, H. D.; Konarev, P. V.; and Svergun, D. I. J Appl Crystallogr. 2012, 45, 342).
(127) Small angle X-ray scattering of the ligated MBP-Cpe-GFP assembly was undertaken to determine a low-resolution envelope of the structure. Data were collected every 2 seconds by SEC-SAXS from 25 μl of 12 mg/ml protein injected onto a Superdex S200 increase 5/150 GL column (GE Healthcare Life Sciences). Images representing the central peak of the SEC elution profile (images 120-130) were used for analysis, as shown in the scattering curve (
(128) TABLE-US-00002 TABLE 2 Small Angle X-ray scattering parameters and statistics. Data collection parameters Beamline.sup.a AS SAX/WAX Wavelength (Å) 1.03320 Detector 1M Pilatus detector Camera length (mm) 1575 SEC column S200 increase 5/150 GL q range (Å.sup.−1) 0.006-0.6 Sample capillary flow rate (ml/min) 0.5 Exposure time/images (s) 2 Number of images used 10 Sample concentration (mg/ml) 12 Sample volume (μl) 25 Temperature (K) 283 Structural parameters I(0) (cm.sup.−1) (from P(r)) 0.05 Rg (Å) (from P(r)) 47.2 I(0) (cm.sup.−1) (from Guinier) 0.05 Rg (Å) (from Guinier) 45.4 D.sub.max (Å) 176.7 Porod volume estimate (Å.sup.3) 134714 MW calc from sequence (kDa) 84.7 MW calc from Porod volume (kDa) 84.2 Software Primary data collection ScatterBrain Data processing ScatterBrain Data analysis Primus, ATSAS .sup.aFull details of the beamline specifications are available at the Australian Synchrotron website.
X-Ray Crystallography of Three Domain Constructs of Mobiluncus mulieris Adhesin
(129) Cloning of three-domain ester bond constructs of Mobiluncus mulieris strain BV 64-5 [ATCC® 35240™] was achieved by PCR amplification from genomic DNA and restriction cloning. Four overlapping three-domain ester bond constructs were PCR amplified from M. mulieris genomic DNA (ATCC® 35240™) using the gene specific primer pairs listed in Table 3. Briefly, a high-fidelity DNA polymerase (iProof, Bio-Rad) with GC-rich buffer was used for the PCR amplification of the 3-domain constructs from 0.5 ng genomic DNA. Amplified PCR fragments were digested with the KasI and XhoI restriction endonucleases and cloned into the expression vector pProExHta (Invitrogen) to create the constructs; Mol3-5, Mol5-7, Mol7-9, and Mol9-11. The resulting plasmids were sequence-verified and transformed into E. coli BL21 (DE3) cells for protein expression.
(130) The E. coli BL21 (20E3) cells harboring recombinant expression constructs were grown in 2×YT medium supplemented with ampicillin (100 μg/ml) at 37° C. in an orbital shaker (@180 rpm) to an optical density of OD.sub.600=0.5-0.6. Protein expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.3 mM and cultures were left to incubate for an additional 16 h at 18° C. Cells were pelleted at 4000 g at 4° C. for 20 minutes, snap-frozen, and stored at −20° C.
(131) Recombinant protein was purified from frozen cells, which were thawed and resuspended in lysis buffer [50 mM HEPES pH 7.0, 300 mM NaCl, 5% (v/v) glycerol, 10 mM imidazole] with the addition of Complete EDTA-free Protease Inhibitor Cocktail tablets (Roche) and lysed using a cell disruptor at 18,000 psi (Constant Systems). The insoluble protein fraction was removed by centrifugation (55,000 g at 4° C. for 30 minutes) and the soluble recombinant protein fraction loaded onto a 5 mL Protino NiNTA 5 column (Macherey-Nagel) for purification by immobilized metal affinity chromatography (IMAC). Recombinant protein was washed with Wash Buffer [50 mM HEPES pH 7, 300 mM NaCl, 20 mM imidazole] and eluted in a linear gradient with Elution Buffer (Wash Buffer with 500 mM imidazole).
(132) Fractions from IMAC containing recombinant protein were dialyzed overnight against a >100×volume of dialysis buffer [20 mM HEPES pH7, 100 mM NaCl, 1 mM beta-mercaptoethanol] and the His6-tag concomitantly removed using recombinant TEV protease at a 1:50 molar ratio of rTEV to recombinant protein. Undigested protein and rTEV protease were removed by a second round of IMAC. Purified protein was concentrated and subjected to size-exclusion chromatography (SEC) on a Superdex 200 10/300 column (GE Healthcare) equilibrated with 10 mM HEPES pH7 and 100 mM NaCl. SEC-purified protein was concentrated to ˜200-400 mg/ml, and in the case of Mol5-7 concentrated to 750 mg/ml, and flash cooling in liquid nitrogen for subsequent storage at −80° C.
(133) Selenomethionine-labelled Mol7-9 protein was produced using a modified protocol based on the inhibition of methionine biosynthesis (Doublie S, Carter C. (1992) Preparation of Selenomethionyl Protein Crystals. Oxford University Press. New York). Briefly, 2×YT media was substituted with M9 minimal media and the cells were grown as in the above protocol described for the expression of native protein. Once OD600 reached 1.5, 100 mg/l each of lysine, phenylalanine and threonine and 50 mg/leach of isoleucine, leucine and valine were added to the cultures. An abundance of L-selenomethionine (60 mg/1) was then added and the cells were grown for an additional 15 min at 37° C. prior to induction with 0.1 mM IPTG at 18° C. for 16 h.
(134) Purified recombinant proteins were subjected to sitting-drop vapour diffusion crystallization screening trials at 290 K using a locally compiled crystallization screen. Initial crystallization conditions were optimised by hanging-drop vapour diffusion format with 1 μl protein solution mixed with an equal volume of well solution. The crystallization conditions, protein concentration and cryoprotection solution for each of the constructs is listed in Tables 4 and 5.
(135) X-ray diffraction data were collected at the Australian Synchrotron (MX1 and MX2). Data were processed and scaled with XDS (Kabsch, W. (2010). XDS. Acta Cryst. D66, 125-132) and POINTLESS/AIMLESS (Evans, P. R. & Murshudov, G. N. (2013) Acta Cryst. D69, 1204-1214). The structure of Smet-Mol7-9 was solved by SAD phasing. Phase determination, density modification and model building used SHELX-CDE (A short history of SHELX. Sheldrick, G. M. (2008). Acta Cryst. A64, 112-122) Model building was completed with COOT (Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486-501). The SeMet-Mol7-9 structure was refined with REFMAC (Murshudov G. N., Skubák P., Lebedev A. A., Pannu N. S., Steiner R. A., Nicholls R. A., Winn M. D., Long, F. & Vagin, A. A. (2011). Acta Cryst. D67, 355-367). The Mol7-9 native structure and the Mol9-11, Mol5-7 and Mol3-5 structures were solved by molecular replacement using the overlapping domain of each previously solved structure and refined using REFMAC. Final validation used MOLPROBITY (Chen V. B., Arendall W. B. 3.sup.rd, Headd J. J., Keedy D. A., Immormino R. M., Kapral, G. J., Murray, L. W., Richardson, J. S. & Richardson, D. C. (2010). Acta Cryst. D66, 12-21.).
(136) TABLE-US-00003 TABLE 3 List of primers used to amplify Mobiluncus mulieris strain BV 64-5 [ATCC ® 35240 ™]. Primer name Sequence SEQ ID MOB 3-5 FWD AAA GGCGCC CCCGGTGTCACCACGGATGCCACCG 12 MOB 3-5 REV AAA CTCGAG TCA CAGGCTCGGGTGGTACACGACCTGGG 13 MOB 5-7 FWD AAA GGCGCC GTGCAAGAGGTAGAGATTACCACCACGGCC 14 MOB 5-7 REV AAA CTCGAG TCA ACTCGGGGTGTAAACCGTCTGGGCGTCATC 15 MOB 7-9 FWD AAA GGCGCC GTTGGTTCTCTGGATACCACCGCTACCGATG 16 MOB 7-9 RRV AAA CTCGAG TCA ATGCTCCGACACCACCGTTTGGTTCGGATC 17 MOB 9-11 FWD AAA GGCGCC GGCACGAGCCCGTCTCTAAAGACCGTG 18 MOB 9-11 REV AAA CTCGAG TCA AGGCTTCTTGGACGTAACCGTCTGGTTTTCATCC 19
(137) TABLE-US-00004 TABLE 4 The crystallization conditions and protein concentration for each of the constructs. Protein Construct concentration Crystallization condition MOB 3-5 140 mg/ml 20% PEG 3350, 0.2M ammonium formate MOB 5-7 750 mg/ml 6% PEG 20K, 22% PEG 550, 0.03M MgCl.sub.2, 0.03M CaCl.sub.2, O.1M MES/imidazole pII 6.5 MOB 7-9 270 mg/ml 4% PEG 8K, 8% PEG 1K, 0.2M MgCl.sub.2 MOB 9-11 360 mg/ml 8% MPEG 5K, 0.2M citric acid pH 5.1 MOB 7-9 250 mg/ml 12% PEG 8K, 6% PEG 1K, 0.2M MgCl.sub.2 Smet
(138) TABLE-US-00005 TABLE 5 Cryoprotection solution for each of the constructs. Construct Crystallization Cryo MOB 3-5 20% PEG 3350, 0.2M ammonium formate, 20% glycerol MOB 5-7 6% PEG 20K, 22% PEG 550, 0.03M MgCl.sub.2, 0.03M CaCl.sub.2, O.1M MES/imidazole pII 6.5 MOB 7-9 4% PEG 8K, 8% PEG 1K, 0.2M MgCl.sub.2, 20% glycerol MOB 9-11 8% MPEG 5K, 0.2M citric acid pH 5.1, 20% glycerol MOB 7-9 Smet 12% PEG 8K, 6% PEG 1K, 0.2M MgCl.sub.2, 20% glycerol
Example 1
(139) This example demonstrates the spontaneous formation of ester bond between Cpe0147.sup.439-563 truncated protein domain and Cpe0147.sup.565-587 peptide.
(140) Method
(141) The Ig-like domain, encompassing Cpe0147 residues 439-587, is split into two parts; a truncated protein comprising the sequence 439-563 and a peptide comprising the final β-strand of the domain, residues 565-587 (DTKQVVKHEDKNDKAQTLVVEKP [SEQ ID No. 3]). The truncated protein was produced recombinantly in E. coli as a maltose binding protein (MBP) construct, with the MBP tag subsequently removed, while the complementary C-terminal peptide was chemically synthesized.
(142) Results
(143) When mixed together, the N-terminal truncation and the peptide spontaneously form a covalent ester bond linkage that is evident in SDS-PAGE analysis (
(144) Interestingly, a pH/buffer screen suggests that the particular buffer molecule used has a greater impact on bond formation than the pH of the solution itself (
Example 2
(145) This example demonstrates covalent cross-linking between two proteins.
(146) Method
(147) The N-terminally MBP-tagged Cpe0147 truncated protein was paired with an enhanced green fluorescent protein (eGFP) engineered with an N-terminal peptide tag derived from residues 565-587 of the Cpe0147 adhesin protein.
(148) Results
(149) Incubation of Cpe0147.sup.439-563 truncated protein domain with Cpe0147.sup.565-587 peptide sequence fused to eGFP in the previously optimized buffer system produces a dimeric, irreversibly cross-linked assembly with a mass of 84,580 Da. The MBP-Cpe0147-eGFP ligation product was visualized by small-angle X-ray scattering (SAXS). A constructed ab initio envelope (
Example 3
(150) This example demonstrates the in vivo self-polymerisation of Cpe0147.sup.439-563 truncated protein domain.
(151) Method
(152) The Ig-like domain of Cpe0147 was engineered as a self-polymerizing construct to form nanochains comprising a central Cpe0147-derived stalk displaying MBP-cargo protein along the entire length (
(153) Results
(154) SDS-PAGE analysis shows a diagnostic laddering pattern indicative of polymerization, with a mixture of species ranging from ˜56 kDa (monomer) to >500 kDa molecular mass (
Example 4
(155) This example demonstrates a reversible ester bond.
(156) Method
(157) The cross-linking Thr-Gln pair was replaced with a Ser-Gln pair (T450S variant). The formation of a Ser-Gln crosslink under the previously optimized buffer system using the T450S variant of the Ig-like domain 2 of Cpe0147, was first confirmed by tryptic digest and mass spectrometry (
(158) Results
(159) Under low pH conditions and in the presence of CaCl.sub.2) and glycerol, this construct forms ester bonds that are stable and that do not hydrolyze (
(160) The time courses of ester bond formation and hydrolysis, illustrated in
(161) Following hydrolysis, the separated MBP and eGFP constructs can be re-ligated simply by switching the buffer to our low pH optimized condition which initiates ester bond formation. The implication here is that the wild type Gln can be replaced with a Glu in the active site and still form an ester bond. Intriguingly, the process of bond making and bond breaking can be completed through at least three cycles on the same sample (
Example 5
(162) This example demonstrates the peptide sequence, relative atom locations and interatomic distances of the essential residues in the active site of Cpe0147-domain 2.
(163) Method
(164) The peptide sequence for Cpe0147-domain 2 (residues 439-587) was obtained from Uniprot (entry B1R775).
(165) The coordinate data of the atoms for each of the four essential residues for spontaneous intermolecular ester bond formation of Cpe0147-domain 2 was obtained from Protein Data Bank file 4MKM. The Protein Data Bank (PDB) conventional orthogonal coordinate system was used. The Cβ (CB) atom of the threonine reactive residue was chosen as the reference coordinate (0, 0, 0).
(166) The interatomic distances were obtained by using the distance measurement tools within the software programme Pymol.
(167) Cpe0147-Domain 2
(168) Table 6 shows the peptide sequence of Cpe0147-domain 2. The essential reactive and accessory amino acids are in bold. The HxDxxDxxQ is underlined.
(169) TABLE-US-00006 TABLE 6 Peptide sequence of Cpe0147 - domain 2. Peptide and SEQ position Sequence ID Cpe0147 - LPEVKDGTLRTTVIADGVNGSSEKEALVSFENSKDG 1 domain 2 VDVKDTINYEGLVANQNYTLTGTLMHVKADGSLEE 439-587 IATKTTNVTAGENGNGTWGLDFGNQKLQVGEKYV VFENAESVENLIDTDKDYNLDTKQVVKHEDKNDK AQTLVVEKP
(170) The coordinates of the atoms of threonine, aspartic acid, histidine and glutamine residues are listed in Table 7 below.
(171) TABLE-US-00007 TABLE 7 Relative atom locations of essential residues for spontaneous intermolecular ester bond formation. Residue Residue X Y Z Temperature Atom Atom type Chain number coordinate coordinate coordinate Occupancy factor type Cpe0147- 1193 N THR A 160 2.211 −0.667 0.879 1.00 32.76 N domain 1194 CA THR A 160 1.188 −0.952 −0.122 1.00 32.66 C 2 1195 C THR A 160 1.706 −0.934 −1.570 1.00 35.12 C 1196 O THR A 160 2.670 −0.230 −1.893 1.00 33.46 O 1197 CB THR A 160 0.000 0.000 0.000 1.00 37.84 C 1198 OG1 THR A 160 0.408 1.307 −0.446 1.00 41.05 O 1199 CG2 THR A 160 −0.621 0.056 1.388 1.00 32.20 C 1409 N ASP A 190 −5.567 −3.182 −4.385 1.00 36.17 N 1410 CA ASP A 190 −4.390 −2.782 −3.609 1.00 32.95 C 1411 C ASP A 190 −3.907 −3.947 −2.748 1.00 36.79 C 1412 O ASP A 190 −4.726 −4.618 −2.146 1.00 35.26 O 1413 CB ASP A 190 −4.706 −1.574 −2.713 1.00 33.42 C 1414 CG ASP A 190 −3.453 −0.882 −2.188 1.00 36.02 C 1415 OD1 ASP A 190 −2.605 −0.474 −3.022 1.00 37.62 O 1416 OD2 ASP A 190 −3.354 −0.685 −0.962 1.00 37.50 O 2126 N HIS A 282 −1.293 4.493 5.905 1.00 31.69 N 2127 CA HIS A 282 −0.308 4.745 4.862 1.00 32.37 C 2128 C HIS A 282 −1.010 5.680 3.890 1.00 36.40 C 2129 O HIS A 282 −1.850 5.234 3.099 1.00 36.28 O 2130 CB HIS A 282 0.162 3.454 4.201 1.00 33.33 C 2131 CG HIS A 282 1.353 3.676 3.343 1.00 38.04 C 2132 ND1 HIS A 282 2.583 3.981 3.888 1.00 40.88 N 2133 CD2 HIS A 282 1.437 3.770 2.001 1.00 39.83 C 2134 CE1 HIS A 282 3.397 4.159 2.861 1.00 40.97 C 2135 NE2 HIS A 282 2.750 4.020 1.708 1.00 40.66 N 2209 N GLN A 290 1.986 2.894 −4.694 1.00 34.45 N 2210 CA GLN A 290 0.536 3.078 −4.538 1.00 32.16 C 2211 C GLN A 290 −0.125 3.900 −5.636 1.00 35.86 C 2212 O GLN A 290 −1.346 4.120 −5.565 1.00 33.51 O 2213 CB GLN A 290 0.260 3.821 −3.204 1.00 31.70 C 2214 CG GLN A 290 0.818 3.147 −1.975 1.00 31.37 C 2215 CD GLN A 290 0.023 1.910 −1.605 1.00 39.23 C 2216 OE1 GLN A 290 −0.719 1.304 −2.384 1.00 32.11 O
(172) The interatomic distance (in Angstrom) of Cβ (CB) of threonine to Cδ (CD) of glutamine, Cγ (CG) of histidine and Cγ (CG) of aspartic acid is listed in table 8 below.
(173) TABLE-US-00008 TABLE 8 Interatomic distances. Cpe0147 - domain 2 Thr CB to Gln CD 2.49 Thr CB to His CG 5.15 Thr CB to Asp CG 4.18
Example 6
(174) This example demonstrates the peptide sequence and the essential residues in the active site of T450S-Cpe0147-domain 2.
(175) Method
(176) The peptide sequence for Cpe0147-domain 2 (residues 439-587) was obtained from Uniprot (entry B1R775) and the threonine at amino acid position 450 replaced with a serine amino acid.
(177) T450S-Cpe0147-Domain 2
(178) Table 9 shows the peptide sequence of T450S-Cpe0147-domain 2. The essential reactive and accessory amino acids are in bold. The HxDxxDxxQ peptide sequence motif is underlined.
(179) TABLE-US-00009 TABLE 9 Peptide sequence of T450S-Cpe0147 - domain 2. Peptide and SEQ position Sequence ID T450S- LPEVKDGTLRSTVIADGVNGSSEKEALVSFENSKDG 20 Cpe0147 - VDVKDTINYEGLVANQNYTLTGTLMHVKADGSLEE domain 2 IATKTTNVTAGENGNGTWGLDFGNQKLQVGEKYV 439-587 VFENAESVENLIDTDKDYNLDTKQVVKHEDKNDK AQTLVVEKP
Example 7
(180) This example demonstrates the peptide sequence, relative atom locations and interatomic distances of the essential residues in the active site of Mol3.
(181) Method
(182) The peptide sequence for Mol3 was obtained from Uniprot (entry E0QN07).
(183) The coordinate data of the atoms for each of the four essential residues for spontaneous intermolecular ester bond formation of Mol3 was obtained from an unpublished X-ray crystal structure of a Mol3-Mol4-Mol5 construct (E0QN07 sequence 5430-5825). The Protein Data Bank (PDB) conventional orthogonal coordinate system was used. The Cβ (CB) atom of the threonine reactive residue was chosen as the reference coordinate (0, 0, 0).
(184) The interatomic distances were obtained by using the distance measurement tools within the software programme Pymol.
(185) Mol3
(186) Table 10 shows the peptide sequence of Mol3. The essential reactive and accessory amino acids are in bold. The HxDxxDxxQ peptide sequence motif is underlined.
(187) TABLE-US-00010 TABLE 10 Peptide sequence of Mol3. Peptide and SEQ position Sequence ID Mol3 P G V T T D 21 from A T D G D G D K Y V D S S Q N F T I K D E0QN07 T V T A T G L I P G K T Y D V S G E L M 5430- V D N G T P Q G A T T G I K Q T G T I T 5553 A K A D G T G E T V L E F P V T A Q Q A Q D L G L V G K P I V V F E D L S L D G K K V A V H H D I K D E K Q T V Y N
(188) The coordinates of the atoms of threonine, aspartic acid, histidine and glutamine residues are listed in Table 11 below.
(189) TABLE-US-00011 TABLE 11 Relative atom locations of essential residues for spontaneous intermolecular ester bond formation. Residue Residue X Y Z Temperature Atom Atom type Chain number coordinate coordinate coordinate Occupancy factor type Mol3 1 N THR A 7 0.237 1.677 1.706 1.00 33.54 N 2 CA THR A 7 0.222 1.462 0.290 1.00 37.70 C 3 C THR A 7 −0.863 2.270 −0.399 1.00 41.11 C 4 O THR A 7 −1.707 2.846 0.282 1.00 41.48 O 5 CB THR A 7 0.000 0.000 0.000 1.00 40.97 C 6 CG2 THR A 7 0.797 −0.938 0.865 1.00 42.06 C 7 OG1 THR A 7 −1.353 −0.237 0.276 1.00 42.72 O 9 N ASP A 28 0.505 −0.819 −7.497 1.00 35.39 N 10 CA ASP A 28 0.485 −0.550 −6.062 1.00 35.04 C 11 C ASP A 28 1.736 0.171 −5.646 1.00 33.23 C 12 O ASP A 28 2.792 −0.375 −5.791 1.00 31.66 O 13 CB ASP A 28 0.420 −1.857 −5.261 1.00 34.95 C 14 CG ASP A 28 0.019 −1.618 −3.836 1.00 34.09 C 15 OD1 ASP A 28 −0.902 −0.840 −3.634 1.00 30.49 O 16 OD2 ASP A 28 0.563 −2.244 −2.918 1.00 43.42 O 18 N HIS A 114 1.572 −5.364 5.517 1.00 41.36 N 19 CA HIS A 114 0.373 −4.532 5.404 1.00 32.60 C 20 C HIS A 114 −0.668 −5.379 4.744 1.00 33.25 C 21 O HIS A 114 −0.743 −5.439 3.497 1.00 27.81 O 22 CB HIS A 114 0.678 −3.274 4.644 1.00 34.16 C 23 CG HIS A 114 −0.357 −2.221 4.815 1.00 32.78 C 24 CD2 HIS A 114 −1.447 −1.902 4.077 1.00 39.05 C 25 ND1 HIS A 114 −0.386 −1.404 5.909 1.00 35.38 N 26 CE1 HIS A 114 −1.431 −0.595 5.835 1.00 39.36 C 27 NE2 HIS A 114 −2.087 −0.870 4.723 1.00 36.33 N 29 N GLN A 122 −5.755 0.651 −0.506 1.00 22.69 N 30 CA GLN A 122 −5.228 −0.488 −1.249 1.00 27.25 C 31 C GLN A 122 −6.282 −1.151 −2.160 1.00 30.92 C 32 O GLN A 122 −6.027 −2.263 −2.652 1.00 37.38 O 33 CB GLN A 122 −4.702 −1.532 −0.275 1.00 27.08 C 34 CG GLN A 122 −3.566 −1.003 0.597 1.00 26.96 C 35 CD GLN A 122 −2.274 −1.241 −0.107 1.00 25.59 C 36 OE1 GLN A 122 −2.165 −1.045 −1.339 1.00 25.28 O
(190) The interatomic distance (in Angstrom) of Cβ (CB) of threonine to Cδ (CD) of glutamine, Cγ (CG) of histidine and Cγ (CG) of aspartic acid is listed in table 12 below.
(191) TABLE-US-00012 TABLE 12 Interatomic distances. Mol3 Thr CB to Gln CD 2.59 Thr CB to His CG 5.31 Thr CB to Asp CG 4.16
Example 8
(192) This example demonstrates the peptide sequence, relative atom locations and interatomic distances of the essential residues in the active site of Mol4.
(193) Method
(194) The peptide sequence for Mol4 was obtained from Uniprot (entry E0QN07).
(195) The coordinate data of the atoms for each of the four essential residues for spontaneous intermolecular ester bond formation of Mol4 was obtained from an unpublished X-ray crystal structure of a Mol3-Mol4-Mol5 construct (E0QN07 sequence 5430-5825). The Protein Data Bank (PDB) conventional orthogonal coordinate system was used. The Cβ (CB) atom of the threonine reactive residue was chosen as the reference coordinate (0, 0, 0).
(196) The interatomic distances were obtained by using the distance measurement tools within the software programme Pymol.
(197) Mol4
(198) Table 13 shows the peptide sequence of Mol4. The essential reactive and accessory amino acids are in bold. The HxDxxDxxQ peptide sequence motif is underlined.
(199) TABLE-US-00013 TABLE 13 Peptide sequence of Mol4. Peptide and SEQ position Sequence ID Mol4 G G 22 from L K T K A V D A A D E N Q A M V P G Q K E0QN07 S A A V V D T V T F N G R F E K S H S Y 5554- T L V G E L H Y V N G T V V P G T K T E 5680 T K T F Q S D Q D G A I A A Q K M T F T V P A E Y I K A G Q N M V V F E K L F D A K K K D G T P V A S H E D P N D P D Q T I T V Q
(200) The coordinates of the atoms of threonine, aspartic acid, histidine and glutamine residues are listed in Table 14 below.
(201) TABLE-US-00014 TABLE 14 Relative atom locations of essential residues for spontaneous intermolecular ester bond formation. Residue Residue X Y Z Temperature Atom Atom type Chain number coordinate coordinate coordinate Occupancy factor type Mol4 1 N THR A 131 0.221 1.930 1.461 1.00 33.74 N 2 CA THR A 131 0.104 1.497 0.088 1.00 36.50 C 3 C THR A 131 −1.092 2.062 −0.639 1.00 34.44 C 4 O THR A 131 −2.144 2.305 −0.055 1.00 32.42 O 5 CB THR A 131 0.000 0.000 0.000 1.00 40.07 C 6 CG2 THR A 131 1.120 −0.722 0.781 1.00 42.85 C 7 OG1 THR A 131 −1.265 −0.339 0.535 1.00 34.89 O 9 N ASP A 154 0.752 −0.619 −7.662 1.00 38.88 N 10 CA ASP A 154 0.806 −0.284 −6.238 1.00 40.90 C 11 C ASP A 154 2.032 0.563 −5.905 1.00 41.39 C 12 O ASP A 154 3.156 0.247 −6.311 1.00 39.05 O 13 CB ASP A 154 0.742 −1.547 −5.382 1.00 44.36 C 14 CG ASP A 154 0.235 −1.284 −3.951 1.00 48.17 C 15 OD1 ASP A 154 −0.946 −0.907 −3.780 1.00 55.41 O 16 OD2 ASP A 154 1.002 −1.500 −2.972 1.00 50.04 O 18 N HIS A 240 1.991 −4.931 5.169 1.00 36.54 N 19 CA HIS A 240 0.658 −4.345 5.017 1.00 34.75 C 20 C HIS A 240 −0.220 −5.373 4.289 1.00 33.79 C 21 O HIS A 240 0.072 −5.785 3.188 1.00 41.11 O 22 CB HIS A 240 0.741 −2.995 4.325 1.00 39.22 C 23 CG HIS A 240 −0.457 −2.109 4.547 1.00 42.36 C 24 CD2 HIS A 240 −0.551 −0.821 4.945 1.00 44.98 C 25 ND1 HIS A 240 −1.744 −2.513 4.278 1.00 42.94 N 26 CE1 HIS A 240 −2.582 −1.533 4.558 1.00 43.45 C 27 NE2 HIS A 240 −1.883 −0.485 4.938 1.00 45.04 N 29 N GLN A 248 −5.688 0.389 −0.797 1.00 25.84 N 30 CA GLN A 248 −5.213 −0.808 −1.500 1.00 28.94 C 31 C GLN A 248 −6.290 −1.524 −2.361 1.00 31.66 C 32 O GLN A 248 −6.060 −2.615 −2.925 1.00 31.79 O 33 CB GLN A 248 −4.818 −1.824 −0.443 1.00 31.09 C 34 CG GLN A 248 −3.705 −1.339 0.437 1.00 31.22 C 35 CD GLN A 248 −2.467 −1.176 −0.326 1.00 25.89 C 36 OE1 GLN A 248 −2.236 −1.875 −1.294 1.00 30.46 O
(202) The interatomic distance (in Angstrom) of Cβ (CB) of threonine to Cδ (CD) of glutamine, Cγ (CG) of histidine and Cγ (CG) of aspartic acid is listed in table 15 below.
(203) TABLE-US-00015 TABLE 15 Interatomic distances. Mol4 Thr CB to Gln CD 2.75 Thr CB to His CG 5.03 Thr CB to Asp CG 4.16
Example 9
(204) This example demonstrates the peptide sequence, relative atom locations and interatomic distances of the essential residues in the active site of Mol5.
(205) Method
(206) The peptide sequence for Mol5 was obtained from Uniprot (entry E0QN07).
(207) The coordinate data of the atoms for each of the four essential residues for spontaneous intermolecular ester bond formation of Mol5 was obtained from an unpublished X-ray crystal structure of a Mol3-Mol4-Mol5 construct (E0QN07 sequence 5430-5825). The Protein Data Bank (PDB) conventional orthogonal coordinate system was used. The Cβ (CB) atom of the threonine reactive residue was chosen as the reference coordinate (0, 0, 0).
(208) The interatomic distances were obtained by using the distance measurement tools within the software programme Pymol.
(209) Mol5
(210) Table 16 shows the peptide sequence of Mol5. The essential reactive and accessory amino acids are in bold. The HxDxxDxxQ peptide sequence motif is underlined.
(211) TABLE-US-00016 TABLE 16 Peptide sequence of Mol5. Peptide and SEQ position Sequence ID Mol5 E V E I T T T A Y D G A A G D 23 from K S D P K D K N L D A S K E T V T I Y D E0QN07 Q V D Y K G L N V G E E Y T I T G T L H 5681- Y Q A D A T L A D G T Q V K R G D E V P 5825 A Q Y V N V T P V K I T A N K A S S D E S G A V K A I V K F E V Q K T A L A T A P V V V F E T L Y Q G T V E V A T H Q D I D D G S Q V V Y H
(212) The coordinates of the atoms of threonine, aspartic acid, histidine and glutamine residues are listed in Table 17 below.
(213) TABLE-US-00017 TABLE 17 Relative atom locations of essential residues for spontaneous intermolecular ester bond formation. Residue Residue X Y Z Temperature Atom Atom type Chain number coordinate coordinate coordinate Occupancy factor type Mol5 1 N THR B 10 0.325 1.794 1.605 1.00 17.87 N 2 CA THR B 10 0.212 1.510 0.184 1.00 17.43 C 3 C THR B 10 −0.912 2.303 −0.518 1.00 16.69 C 4 O THR B 10 −1.880 2.726 0.113 1.00 15.96 O 5 CB THR B 10 0.000 0.000 0.000 1.00 17.71 C 6 CG2 THR B 10 1.096 −0.823 0.609 1.00 18.64 C 7 OG1 THR B 10 −1.226 −0.373 0.641 1.00 17.12 O 9 N ASP B 39 1.125 −0.307 −7.429 1.00 14.50 N 10 CA ASP B 39 0.949 −0.093 −6.021 1.00 14.78 C 11 C ASP B 39 2.150 0.627 −5.443 1.00 15.66 C 12 O ASP B 39 3.232 0.042 −5.305 1.00 15.45 O 13 CB ASP B 39 0.740 −1.411 −5.293 1.00 14.69 C 14 CG ASP B 39 0.098 −1.223 −3.967 1.00 14.62 C 15 OD1 ASP B 39 −0.966 −0.539 −3.903 1.00 14.86 O 16 OD2 ASP B 39 0.667 −1.736 −2.987 1.00 15.02 O 18 N HIS B 137 1.832 −5.036 5.020 1.00 23.31 N 19 CA HIS B 137 0.574 −4.310 5.040 1.00 22.65 C 20 C HIS B 137 −0.451 −5.176 4.343 1.00 23.54 C 21 O HIS B 137 −0.476 −5.285 3.110 1.00 22.59 O 22 CB HIS B 137 0.704 −2.933 4.399 1.00 23.38 C 23 CG HIS B 137 −0.506 −2.071 4.581 1.00 24.12 C 24 CD2 HIS B 137 −1.630 −1.943 3.838 1.00 23.11 C 25 ND1 HIS B 137 −0.645 −1.198 5.640 1.00 25.07 N 26 CE1 HIS B 137 −1.805 −0.573 5.546 1.00 24.90 C 27 NE2 HIS B 137 −2.425 −1.010 4.461 1.00 24.22 N 29 N GLN B 145 −5.944 0.750 −0.789 1.00 14.31 N 30 CA GLN B 145 −5.372 −0.407 −1.478 1.00 14.19 C 31 C GLN B 145 −6.329 −1.122 −2.395 1.00 12.90 C 32 O GLN B 145 −5.997 −2.181 −2.878 1.00 12.73 O 33 CB GLN B 145 −4.872 −1.385 −0.442 1.00 14.29 C 34 CG GLN B 145 −3.815 −0.776 0.429 1.00 15.31 C 35 CD GLN B 145 −2.473 −0.725 −0.265 1.00 15.46 C 36 OE1 GLN B 145 −2.324 −0.790 −1.497 1.00 15.59 O
(214) The interatomic distance (in Angstrom) of Cβ (CB) of threonine to Cδ (CD) of glutamine, Cγ (CG) of histidine and Cγ (CG) of aspartic acid is listed in table 18 below.
(215) TABLE-US-00018 TABLE 18 Interatomic distances. Mol5 Thr CB to Gln CD 2.59 Thr CB to His CG 5.05 Thr CB to Asp CG 4.15
Example 10
(216) This example demonstrates the peptide sequence, relative atom locations and interatomic distances of the essential residues in the active site of Mol6.
(217) Method
(218) The peptide sequence for Mol6 was obtained from Uniprot (entry E0QN07).
(219) The coordinate data of the atoms for each of the four essential residues for spontaneous intermolecular ester bond formation of Mol6 was obtained from an unpublished X-ray crystal structure of a Mol5-Mol6-Mol7 construct (E0QN07 sequence 5681-6100). The Protein Data Bank (PDB) conventional orthogonal coordinate system was used. The Cβ (CB) atom of the threonine reactive residue was chosen as the reference coordinate (0, 0, 0).
(220) The interatomic distances were obtained by using the distance measurement tools within the software programme Pymol.
(221) Mol6
(222) Table 19 shows the peptide sequence of Mol6. The essential reactive and accessory amino acids are in bold. The HxDxxDxxQ peptide sequence motif is underlined.
(223) TABLE-US-00019 TABLE 19 Peptide sequence of Mol6. Peptide and SEQ position Sequence ID Mol6 P S L R T L A T V N 24 from G A K V I Q M K K D S K E N L T V T D Q E0QN07 I T W A N L A P G T Y T L E G S L M E V 5826- K D G Q L V S N T P V A K G Q T Q K V E 5957 V A A G K A G A T T S T G E A Q M T F K L P V D K V K S G S Q F V V Y Q I L K D K S G Q V V A T H A D P K S D D Q T V T V G
(224) The coordinates of the atoms of threonine, aspartic acid, histidine and glutamine residues are listed in Table 20 below.
(225) TABLE-US-00020 TABLE 20 Relative atom locations of essential residues for spontaneous intermolecular ester bond formation. Residue Residue X Y Z Temperature Atom Atom type Chain number coordinate coordinate coordinate Occupancy factor type Mol6 1 N THR B 154 0.011 1.987 1.418 1.00 16.51 N 2 CA THR B 154 −0.086 1.529 0.059 1.00 16.14 C 3 C THR B 154 −1.356 2.073 −0.573 1.00 15.93 C 4 O THR B 154 −2.313 2.380 0.132 1.00 16.35 O 5 CB THR B 154 0.000 0.000 0.000 1.00 16.49 C 6 CG2 THR B 154 1.212 −0.573 0.724 1.00 16.12 C 7 OG1 THR B 154 −1.159 −0.519 0.645 1.00 17.71 O 9 N ASP B 178 0.041 −0.697 −7.584 1.00 14.03 N 10 CA ASP B 178 0.114 −0.395 −6.168 1.00 14.72 C 11 C ASP B 178 1.320 0.505 −5.913 1.00 15.00 C 12 O ASP B 178 2.442 0.110 −6.220 1.00 13.65 O 13 CB ASP B 178 0.232 −1.694 −5.341 1.00 14.32 C 14 CG ASP B 178 −0.123 −1.487 −3.868 1.00 14.43 C 15 OD1 ASP B 178 −1.206 −0.920 −3.581 1.00 13.91 O 16 OD2 ASP B 178 0.681 −1.872 −2.998 1.00 13.88 O 18 N HIS B 268 1.696 −5.379 5.533 1.00 17.63 N 19 CA HIS B 268 0.498 −4.573 5.294 1.00 17.43 C 20 C HIS B 268 −0.553 −5.479 4.671 1.00 16.51 C 21 O HIS B 268 −0.493 −5.789 3.478 1.00 16.41 O 22 CB HIS B 268 0.780 −3.361 4.406 1.00 17.89 C 23 CG HIS B 268 −0.314 −2.348 4.429 1.00 19.44 C 24 CD2 HIS B 268 −1.524 −2.319 3.825 1.00 20.99 C 25 ND1 HIS B 268 −0.237 −1.203 5.178 1.00 21.14 N 26 CE1 HIS B 268 −1.341 −0.496 5.018 1.00 21.60 C 27 NE2 HIS B 268 −2.144 −1.160 4.214 1.00 21.28 N 29 N GLN B 276 −5.974 0.084 −0.325 1.00 12.48 N 30 CA GLN B 276 −5.398 −1.090 −0.969 1.00 12.31 C 31 C GLN B 276 −6.382 −1.884 −1.822 1.00 12.24 C 32 O GLN B 276 −5.985 −2.895 −2.414 1.00 11.33 O 33 CB GLN B 276 −4.776 −2.041 0.058 1.00 12.64 C 34 CG GLN B 276 −3.660 −1.477 0.885 1.00 12.59 C 35 CD GLN B 276 −2.563 −0.842 0.051 1.00 13.42 C 36 OE1 GLN B 276 −2.310 −1.130 −1.135 1.00 13.64 O
(226) The interatomic distance (in Angstrom) of Cβ (CB) of threonine to Cδ (CD) of glutamine, Cγ (CG) of histidine and Cγ (CG) of aspartic acid is listed in table 21 below.
(227) TABLE-US-00021 TABLE 21 Interatomic distances. Mol6 Thr CB to Gln CD 2.7 Thr CB to His CG 5.02 Thr CB to Asp CG 4.15
Example 11
(228) This example demonstrates the peptide sequence, relative atom locations and interatomic distances of the essential residues in the active site of Mol7.
(229) Method
(230) The peptide sequence for Mol7 was obtained from Uniprot (entry E0QN07).
(231) The coordinate data of the atoms for each of the four essential residues for spontaneous intermolecular ester bond formation of Mol7 was obtained from an unpublished X-ray crystal structure of a Mol5-Mol6-Mol7 construct (E0QN07 sequence 5681-6100). The Protein Data Bank (PDB) conventional orthogonal coordinate system was used. The Cβ (CB) atom of the threonine reactive residue was chosen as the reference coordinate (0, 0, 0).
(232) The interatomic distances were obtained by using the distance measurement tools within the software programme Pymol.
(233) Mol7
(234) Table 22 shows the peptide sequence of Mol7. The essential reactive and accessory amino acids are in bold. The HxDxxDxxQ peptide sequence motif is underlined.
(235) TABLE-US-00022 TABLE 22 Peptide sequence of Mol7. Peptide and SEQ position Sequence ID Mol7 S L D T T A T D A A D G N K H A D N 25 from A A A V T I N D K V D Y S G L N L A A T E0QN07 Y P D G T L K A Y L V R G E L M D K A T 5958- G K P V A G V A P V E R V I G A A N S V 6100 Y R V G D Q N R P V E E E I T S G A G S V V L S F Q V P A K L T Q G K V L V A F E T V Y E E G R E F L I H H D I N D D A Q T V Y T
(236) The coordinates of the atoms of threonine, aspartic acid, histidine and glutamine residues are listed in Table 23 below.
(237) TABLE-US-00023 TABLE 23 Relative atom locations of essential residues for spontaneous intermolecular ester bond formation. Residue Residue X Y Z Temperature Atom Atom type Chain number coordinate coordinate coordinate Occupancy factor type Mol7 1 N THR B 285 0.064 1.998 1.432 1.00 16.63 N 2 CA THR B 285 −0.064 1.534 0.060 1.00 16.84 C 3 C THR B 285 −1.335 2.100 −0.590 1.00 16.83 C 4 O THR B 285 −2.390 2.249 0.052 1.00 16.72 O 5 CB THR B 285 0.000 0.000 0.000 1.00 17.18 C 6 CG2 THR B 285 1.138 −0.593 0.781 1.00 16.65 C 7 OG1 THR B 285 −1.170 −0.511 0.614 1.00 17.83 O 9 N ASP B 307 0.335 −0.151 −7.481 1.00 16.17 N 10 CA ASP B 307 0.297 −0.010 −6.038 1.00 16.74 C 11 C ASP B 307 1.514 0.835 −5.648 1.00 16.96 C 12 O ASP B 307 2.649 0.394 −5.769 1.00 18.66 O 13 CB ASP B 307 0.344 −1.371 −5.344 1.00 16.38 C 14 CG ASP B 307 −0.119 −1.315 −3.909 1.00 16.82 C 15 OD1 ASP B 307 −1.021 −0.511 −3.577 1.00 17.21 O 16 OD2 ASP B 307 0.413 −2.093 −3.097 1.00 16.46 O 18 N HIS B 412 1.936 −4.634 5.460 1.00 15.81 N 19 CA HIS B 412 0.666 −3.987 5.318 1.00 16.00 C 20 C HIS B 412 −0.306 −5.014 4.771 1.00 16.18 C 21 O HIS B 412 −0.364 −5.252 3.547 1.00 15.52 O 22 CB HIS B 412 0.783 −2.755 4.424 1.00 16.62 C 23 CG HIS B 412 −0.427 −1.883 4.465 1.00 17.57 C 24 CD2 HIS B 412 −1.545 −1.871 3.700 1.00 18.43 C 25 ND1 HIS B 412 −0.593 −0.891 5.400 1.00 17.73 N 26 CE1 HIS B 412 −1.754 −0.293 5.206 1.00 18.09 C 27 NE2 HIS B 412 −2.351 −0.868 4.179 1.00 18.86 N 29 N GLN B 420 −6.070 0.585 −0.588 1.00 16.56 N 30 CA GLN B 420 −5.509 −0.587 −1.264 1.00 17.21 C 31 C GLN B 420 −6.491 −1.391 −2.116 1.00 17.36 C 32 O GLN B 420 −6.088 −2.405 −2.714 1.00 17.13 O 33 CB GLN B 420 −4.931 −1.528 −0.209 1.00 16.74 C 34 CG GLN B 420 −3.796 −0.931 0.578 1.00 17.41 C 35 CD GLN B 420 −2.510 −0.919 −0.231 1.00 16.90 C 36 OE1 GLN B 420 −2.459 −0.813 −1.463 1.00 16.71 O
(238) The interatomic distance (in Angstrom) of Cβ (CB) of threonine to Cδ (CD) of glutamine, Cγ (CG) of histidine and Cγ (CG) of aspartic acid is listed in table 24 below.
(239) TABLE-US-00024 TABLE 24 Interatomic distances. Mol7 Thr CB to Gln CD 2.68 Thr CB to His CG 4.86 Thr CB to Asp CG 4.13
Example 12
(240) This example demonstrates the peptide sequence, relative atom locations and interatomic distances of the essential residues in the active site of Mol8.
(241) Method
(242) The peptide sequence for Mol8 was obtained from Uniprot (entry E0QN07).
(243) The coordinate data of the atoms for each of the four essential residues for spontaneous intermolecular ester bond formation of Mol8 was obtained from an unpublished X-ray crystal structure of a Mol7-Mol8-Mol9 construct (E0QN07 sequence 5958-6383). The Protein Data Bank (PDB) conventional orthogonal coordinate system was used. The Cβ (CB) atom of the threonine reactive residue was chosen as the reference coordinate (0, 0, 0).
(244) The interatomic distances were obtained by using the distance measurement tools within the software programme Pymol.
(245) Mol8
(246) Table 25 shows the peptide sequence of Mol8. The essential reactive and accessory amino acids are in bold. The HxDxxDxxQ peptide sequence motif is underlined.
(247) TABLE-US-00025 TABLE 25 Peptide sequence of Mol8. Peptide and SEQ position Sequence ID Mol8 P S V K T Q A R V D S E R N L 26 from L L A D K D S T I K D T V T L S G L K T E0QN07 G E T Y V L S G V L M D K A T G Q P V L 6101- G K D M Q A I T A V S E P L K A E S G A 6246 F V K T D A V S F T V P A G T V K A D T E L V V F E K L W V A N E V T V D T K T K T V T P K D T K T G K S Q P A A S H E D I T D E N Q T V K S
(248) The coordinates of the atoms of threonine, aspartic acid, histidine and glutamine residues are listed in Table 26 below.
(249) TABLE-US-00026 TABLE 26 Relative atom locations of essential residues for spontaneous intermolecular ester bond formation. Residue Residue X Y Z Temperature Atom Atom type Chain number coordinate coordinate coordinate Occupancy factor type Mol8 1 N THR A 152 0.293 1.796 1.630 1.00 11.45 N 2 CA THR A 152 0.146 1.509 0.213 1.00 11.34 C 3 C THR A 152 −1.050 2.221 −0.435 1.00 11.97 C 4 O THR A 152 −2.087 2.462 0.210 1.00 11.54 O 5 CB THR A 152 0.000 0.000 0.000 1.00 10.89 C 6 CG2 THR A 152 1.076 −0.805 0.650 1.00 11.06 C 7 OG1 THR A 152 −1.226 −0.419 0.616 1.00 11.03 O 9 N ASP A 173 0.739 −0.153 −7.599 1.00 13.80 N 10 CA ASP A 173 0.712 0.121 −6.166 1.00 13.43 C 11 C ASP A 173 1.924 0.938 −5.750 1.00 12.91 C 12 O ASP A 173 3.065 0.494 −5.934 1.00 13.50 O 13 CB ASP A 173 0.660 −1.178 −5.372 1.00 13.48 C 14 CG ASP A 173 0.141 −0.971 −3.979 1.00 13.31 C 15 OD1 ASP A 173 0.921 −0.599 −3.092 1.00 13.65 O 16 OD2 ASP A 173 −1.067 −1.167 −3.750 1.00 12.94 O 18 N HIS A 281 1.692 −5.346 5.351 1.00 11.74 N 19 CA HIS A 281 0.440 −4.632 5.291 1.00 12.22 C 20 C HIS A 281 −0.568 −5.524 4.571 1.00 11.72 C 21 O HIS A 281 −0.593 −5.614 3.338 1.00 11.49 O 22 CB HIS A 281 0.592 −3.269 4.618 1.00 12.58 C 23 CG HIS A 281 −0.628 −2.427 4.760 1.00 13.08 C 24 CD2 HIS A 281 −1.511 −1.972 3.842 1.00 13.45 C 25 ND1 HIS A 281 −1.119 −2.047 5.989 1.00 13.82 N 26 CE1 HIS A 281 −2.232 −1.358 5.820 1.00 13.53 C 27 NE2 HIS A 281 −2.495 −1.303 4.527 1.00 12.99 N 29 N GLN A 289 −5.843 0.359 −0.505 1.00 11.93 N 30 CA GLN A 289 −5.317 −0.813 −1.211 1.00 11.99 C 31 C GLN A 289 −6.315 −1.535 −2.086 1.00 11.76 C 32 O GLN A 289 −6.023 −2.625 −2.572 1.00 11.05 O 33 CB GLN A 289 −4.750 −1.821 −0.212 1.00 11.97 C 34 CG GLN A 289 −3.630 −1.274 0.625 1.00 11.74 C 35 CD GLN A 289 −2.394 −0.942 −0.175 1.00 12.21 C 36 OE1 GLN A 289 −2.245 −1.153 −1.376 1.00 11.40 O
(250) The interatomic distance (in Angstrom) of Cβ (CB) of threonine to Cδ (CD) of glutamine, Cγ (CG) of histidine and Cγ (CG) of aspartic acid is listed in table 27 below.
(251) TABLE-US-00027 TABLE 27 Interatomic distances. Mol8 Thr CB to Gln CD 2.58 Thr CB to His CG 4.58 Thr CB to Asp CG 4.1
Example 13
(252) This example demonstrates the peptide sequence, relative atom locations and interatomic distances of the essential residues in the active site of T6105S-Mol8.
(253) Method
(254) The peptide sequence for Mol8 was obtained from Uniprot (entry E0QN07) and the threonine at amino acid position 6105 replaced with a serine amino acid.
(255) The coordinate data of the atoms for each of the four essential residues for spontaneous intermolecular ester bond formation of T6105S-Mol8 was obtained from an unpublished X-ray crystal structure of a Mol7-T6105S-Mol8-Mol9 construct (E0QN07 sequence 5958-6383). The wild type DNA sequence of Mol7-Mol8-Mol9 (E0QN07 sequence 5958-6383) was subjected to site-directed mutagenesis to produce a T6105S variant of Mol8. Mol7 and Mol9 domain sequences comprised wild type sequence. The Protein Data Banks (PDB) conventional orthogonal coordinate system was used. The Cβ (CB) atom of the serine reactive residue was chosen as the reference coordinate (0, 0, 0).
(256) The interatomic distances were obtained by using the distance measurement tools within the software programme Pymol.
(257) T6105S-Mol8
(258) Table 28 shows the peptide sequence of T6105S-Mol8. The essential reactive and accessory amino acids are in bold. The HxDxxDxxQ peptide sequence motif is underlined.
(259) TABLE-US-00028 TABLE 28 Peptide sequence of T6105S-Mol8. Peptide and SEQ position Sequence ID T6105S- P S V K S Q A R V D S E R N L 27 Mol8 L L A D K D S T I K D T V T L S G L K T from G E T Y V L S G V L M D K A T G Q P V L E0QN07 G K D M Q A I T A V S E P L K A E S G A 6101- F V K T D A V S F T V P A G T V K A D T 6246 E L V V F E K L W V A N E V T V D T K T K T V T P K D T K T G K S Q P A A S H E D I T D E N Q T V K S
(260) The coordinates of the atoms of serine, aspartic acid, histidine and glutamine residues are listed in Table 29 below.
(261) TABLE-US-00029 TABLE 29 Relative atom locations of essential residues for spontaneous intermolecular ester bond formation. Residue Residue X Y Z Temperature Atom Atom type Chain number coordinate coordinate coordinate Occupancy factor type T6105S- 1 N SER A 152 −0.316 −2.311 −0.669 1.00 24.09 N 2 CA SER A 152 0.068 −1.470 0.419 1.00 24.03 C 3 C SER A 152 −0.784 −1.686 1.653 1.00 23.81 C 4 O SER A 152 −2.008 −1.994 1.565 1.00 21.38 O 5 CB SER A 152 0.000 0.000 0.000 1.00 20.00 C 6 OG SER A 152 −1.331 0.385 −0.296 1.00 20.00 O 7 N ASP A 173 3.082 3.229 6.073 1.00 22.28 N 8 CA ASP A 173 2.618 2.398 4.992 1.00 20.70 C 9 CB ASP A 173 2.183 3.261 3.833 1.00 21.48 C 10 CG ASP A 173 1.266 2.547 2.910 1.00 22.16 C 11 OD2 ASP A 173 1.776 2.009 1.932 1.00 21.07 O 12 OD1 ASP A 173 0.045 2.458 3.193 1.00 22.48 O 13 C ASP A 173 3.711 1.419 4.555 1.00 21.71 C 14 O ASP A 173 4.816 1.808 4.150 1.00 22.34 O 15 N HIS A 281 −1.034 2.264 −7.596 1.00 27.78 N 16 CA HIS A 281 −2.104 1.752 −6.766 1.00 29.22 C 17 CB HIS A 281 −1.571 0.843 −5.691 1.00 30.29 C 18 CG HIS A 281 −2.645 0.139 −4.932 1.00 29.81 C 19 ND1 HIS A 281 −3.173 −1.061 −5.343 1.00 30.82 N 20 CE1 HIS A 281 −4.109 −1.446 −4.488 1.00 32.00 C 21 NE2 HIS A 281 −4.187 −0.544 −3.526 1.00 30.73 N 22 CD2 HIS A 281 −3.300 0.470 −3.794 1.00 30.01 C 23 C HIS A 281 −2.864 2.925 −6.154 1.00 31.03 C 24 O HIS A 281 −2.445 3.546 −5.134 1.00 27.11 O 25 N GLN A 289 −5.392 0.299 2.528 1.00 21.99 N 26 CA GLN A 289 −4.815 1.638 2.409 1.00 23.21 C 27 CB GLN A 289 −4.674 2.057 0.931 1.00 21.32 C 28 CG GLN A 289 −3.886 1.101 0.067 1.00 21.10 C 29 CD GLN A 289 −2.403 1.005 0.483 1.00 21.15 C 30 OE1 GLN A 289 −1.851 1.720 1.340 1.00 20.69 O 31 C GLN A 289 −5.553 2.737 3.153 1.00 23.46 C 32 O GLN A 289 −5.191 3.906 2.987 1.00 24.32 O
(262) The interatomic distance (in Angstrom) of Cβ (CB) of serine to Cδ (CD) of glutamine, Cγ (CG) of histidine and Cγ (CG) of aspartic acid is listed in table 30 below.
(263) TABLE-US-00030 TABLE 30 Interatomic distances. T6105S-Mol8 Ser CB to Gln CD 2.65 Ser CB to His CG 5.60 Ser CB to Asp CG 4.07
Example 14
(264) This example demonstrates the peptide sequence, relative atom locations and interatomic distances of the essential residues in the active site of Mol10.
(265) Method
(266) The peptide sequence for Mol10 was obtained from Uniprot (entry E0QN07).
(267) The coordinate data of the atoms for each of the four essential residues for spontaneous intermolecular ester bond formation of Mol10 was obtained from an unpublished X-ray crystal structure of a Mol9-Mol10-Mol11 construct (E0QN07 sequence 6247-6669). The Protein Data Bank (PDB) conventional orthogonal coordinate system was used. The Cβ (CB) atom of the threonine reactive residue was chosen as the reference coordinate (0, 0, 0).
(268) The interatomic distances were obtained by using the distance measurement tools within the software programme Pymol.
(269) Mol10
(270) Table 31 shows the peptide sequence of Mol10. The essential reactive and accessory amino acids are in bold. The HxDxxDxxQ peptide sequence motif is underlined.
(271) TABLE-US-00031 TABLE 31 Peptide sequence of Mol10. Peptide and SEQ position Sequence ID Mol10 H N P G I T T T L T D A 28 from Q A A K G T D G K V I S L T R D A Q L K E0QN07 D V V R V T Q T G L I E G A K Y H V F S 6384- K L V N Q A N P D Q V V S A G M Q E F T 6541 A T G D Q L R S V T V K F T V P K E T L Q E L A G S D P S A E F K L V A Y E Y L A L D S D T D I V N K E A T S E I E A V G F K T G K T W A A T H A D P N D A G Q T V T V V K
(272) The coordinates of the atoms of threonine, aspartic acid, histidine and glutamine residues are listed in Table 32 below.
(273) TABLE-US-00032 TABLE 32 Relative atom locations of essential residues for spontaneous intermolecular ester bond formation. Residue Residue X Y Z Temperature Atom Atom type Chain number coordinate coordinate coordinate Occupancy factor type Mol 1 N THR A 146 0.097 1.989 1.463 1.00 21.29 N 2 CA THR A 146 0.094 1.517 0.054 1.00 21.32 C 3 C THR A 146 −1.091 2.123 −0.682 1.00 22.18 C 4 O THR A 146 −2.118 2.392 −0.084 1.00 21.01 O 5 CB THR A 146 0.000 0.000 0.000 1.00 21.82 C 6 CG2 THR A 146 1.096 −0.715 0.715 1.00 23.80 C 7 OG1 THR A 146 −1.242 −0.384 0.577 1.00 25.23 O 9 N ASP A 172 0.904 −0.293 −7.607 1.00 20.59 N 10 CA ASP A 172 0.832 −0.045 −6.194 1.00 18.21 C 11 C ASP A 172 2.093 0.709 −5.754 1.00 21.29 C 12 O ASP A 172 3.231 0.178 −5.921 1.00 18.93 O 13 CB ASP A 172 0.754 −1.353 −5.455 1.00 18.89 C 14 CG ASP A 172 0.225 −1.176 −4.029 1.00 23.67 C 15 OD1 ASP A 172 −1.021 −1.272 −3.793 1.00 20.52 O 16 OD2 ASP A 172 1.025 −0.893 −3.138 1.00 24.02 O 18 N HIS A 283 1.713 −4.889 4.998 1.00 22.82 N 19 CA HIS A 283 0.453 −4.170 4.988 1.00 22.33 C 20 C HIS A 283 −0.527 −5.014 4.254 1.00 22.61 C 21 O HIS A 283 −0.378 −5.241 3.058 1.00 20.75 O 22 CB HIS A 283 0.500 −2.795 4.387 1.00 23.27 C 23 CG HIS A 283 −0.774 −2.015 4.579 1.00 26.16 C 24 CD2 HIS A 283 −1.731 −1.663 3.703 1.00 25.08 C 25 ND1 HIS A 283 −1.165 −1.472 5.798 1.00 25.84 N 26 CE1 HIS A 283 −2.330 −0.848 5.656 1.00 24.78 C 27 NE2 HIS A 283 −2.665 −0.912 4.381 1.00 25.17 N 29 N GLN A 291 −5.834 0.440 −0.914 1.00 21.93 N 30 CA GLN A 291 −5.193 −0.738 −1.483 1.00 24.16 C 31 C GLN A 291 −6.123 −1.545 −2.382 1.00 21.62 C 32 O GLN A 291 −5.736 −2.609 −2.829 1.00 19.95 O 33 CB GLN A 291 −4.770 −1.730 −0.404 1.00 23.96 C 34 CG GLN A 291 −3.656 −1.272 0.538 1.00 23.15 C 35 CD GLN A 291 −2.393 −0.851 −0.236 1.00 22.36 C 36 OE1 GLN A 291 −2.205 −1.013 −1.457 1.00 19.46 O
(274) The interatomic distance (in Angstrom) of Cβ (CB) of threonine to Cδ (CD) of glutamine, Cγ (CG) of histidine and Cγ (CG) of aspartic acid is listed in table 33 below.
(275) TABLE-US-00033 TABLE 33 Interatomic distances. Mol10 Thr CB to Gln CD 2.55 Thr CB to His CG 5.06 Thr CB to Asp CG 4.2
Example 15
(276) This example demonstrates the peptide sequence, relative atom locations and interatomic distances of the essential residues in the active site of Mol11.
(277) Method
(278) The peptide sequence for Mol11 was obtained from Uniprot (entry E0QN07).
(279) The coordinate data of the atoms for each of the four essential residues for spontaneous intermolecular ester bond formation of Mol11 was obtained from an unpublished X-ray crystal structure of a Mol9-Mol10-Mol11 construct (E0QN07 sequence 6247-6669). The Protein Data Banks (PDB) conventional orthogonal coordinate system was used. The Cβ (CB) atom of the threonine reactive residue was chosen as the reference coordinate (0, 0, 0).
(280) The interatomic distances were obtained by using the distance measurement tools within the software programme Pymol.
(281) Mol11
(282) Table 34 shows the peptide sequence of Mol11. The essential reactive and accessory amino acids are in bold. The HxDxxDxxQ peptide sequence motif is underlined.
(283) TABLE-US-00034 TABLE 34 Peptide sequence of Mol11. Peptide and SEQ position Sequence ID Mol11 A P K I G T T L K Y G Q S K 29 from T V W V A D K V E L T D T V E Y F N L Q E0QN07 P K T K Y T L S G N L M G G T S A E S L 6542- S D T G V K A T T E F T T P A A A N G A 6669 Q T V S G T A V V K F T V P R E V L E R N E K L V A Y E Y L T I D G N P V A S H E D P K D E N Q T V T S K K
(284) The coordinates of the atoms of threonine, aspartic acid, histidine and glutamine residues are listed in Table 35 below.
(285) TABLE-US-00035 TABLE 35 Relative atom locations of essential residues for spontaneous intermolecular ester bond formation-Mol11 Residue Residue X Y Z Temperature Atom Atom type Chain number coordinate coordinate coordinate Occupancy factor type 1 N THR A 303 0.229 2.000 1.478 1.00 18.55 N 2 CA THR A 303 0.106 1.594 0.079 1.00 17.69 C 3 C THR A 303 −1.062 2.266 −0.650 1.00 17.48 C 4 O THR A 303 −2.160 2.461 −0.080 1.00 18.08 O 5 CB THR A 303 0.000 0.000 0.000 1.00 18.35 C 6 CG2 THR A 303 1.012 −0.769 0.782 1.00 18.46 C 7 OG1 THR A 303 −1.280 −0.404 0.486 1.00 20.39 O 9 N ASP A 323 0.767 −0.080 −7.474 1.00 17.21 N 10 CA ASP A 323 0.742 −0.035 −6.018 1.00 17.43 C 11 C ASP A 323 1.939 0.770 −5.579 1.00 16.91 C 12 O ASP A 323 3.075 0.280 −5.543 1.00 20.98 O 13 CB ASP A 323 0.678 −1.444 −5.359 1.00 18.89 C 14 CG ASP A 323 0.129 −1.386 −3.936 1.00 19.81 C 15 OD1 ASP A 323 −0.799 −0.585 −3.758 1.00 18.90 O 16 OD2 ASP A 323 0.606 −2.132 −3.007 1.00 19.63 O 18 N HIS A 411 1.693 −4.552 5.258 1.00 17.21 N 19 CA HIS A 411 0.419 −3.803 5.249 1.00 17.57 C 20 C HIS A 411 −0.585 −4.743 4.576 1.00 21.51 C 21 O HIS A 411 −0.518 −5.019 3.350 1.00 19.05 O 22 CB HIS A 411 0.528 −2.494 4.505 1.00 18.90 C 23 CG HIS A 411 −0.704 −1.623 4.637 1.00 18.52 C 24 CD2 HIS A 411 −1.680 −1.262 3.770 1.00 18.46 C 25 ND1 HIS A 411 −1.000 −1.017 5.822 1.00 20.56 N 26 CE1 HIS A 411 −2.092 −0.285 5.673 1.00 23.69 C 27 NE2 HIS A 411 −2.540 −0.438 4.443 1.00 20.98 N 29 N GLN A 419 −5.798 1.052 −0.795 1.00 18.52 N 30 CA GLN A 419 −5.271 −0.153 −1.427 1.00 19.82 C 31 C GLN A 419 −6.287 −0.875 −2.286 1.00 18.39 C 32 O GLN A 419 −5.983 −1.926 −2.861 1.00 20.28 O 33 CB GLN A 419 −4.824 −1.127 −0.317 1.00 20.02 C 34 CG GLN A 419 −3.700 −0.681 0.530 1.00 19.80 C 35 CD GLN A 419 −2.393 −0.647 −0.226 1.00 18.36 C 36 OE1 GLN A 419 −2.296 −0.598 −1.470 1.00 18.86 O
(286) The interatomic distance (in Angstrom) of Cβ (CB) of threonine to Cδ (CD) of glutamine, Cγ (CG) of histidine and Cγ (CG) of aspartic acid is listed in table 36 below.
(287) TABLE-US-00036 TABLE 36 Interatomic distances. Mol11 Thr CB to Gln CD 2.49 Thr CB to His CG 4.96 Thr CB to Asp CG 4.17
Example 16
(288) This example demonstrates the peptide sequence, relative atom locations and interatomic distances of the essential residues in the active site of Mol9.
(289) Method
(290) The peptide sequence for Mol9 was obtained from Uniprot (entry E0QN07).
(291) The coordinate data of the atoms for each of the four essential residues for spontaneous intermolecular ester bond formation of Mol9 was obtained from an unpublished X-ray crystal structure of a Mol9-Mol10-Mol11 construct (E0QN07 sequence 6247-6669). The Protein Data Banks (PDB) conventional orthogonal coordinate system was used. The Cβ (CB) atom of the threonine reactive residue was chosen as the reference coordinate (0, 0, 0).
(292) The interatomic distances were obtained by using the distance measurement tools within the software programme Pymol.
(293) Mol9
(294) Table 37 shows the peptide sequence of Mol9. The essential reactive and accessory amino acids are in bold. The HxDxxDxxQ peptide sequence motif is underlined.
(295) TABLE-US-00037 TABLE 37 Peptide sequence of Mol9. Pep- tide and posi- SEQ tion Sequence ID Mol9 G T S P S L K T V L S A D G K R E W V E N 31 from N T N I P T V P H A S D S L I D T V L Y T E0QN07 G L T E G V S Y R L D A K L M E I N P V T 6247- G K V S E T P V A T G Y T E F T A K T S D 6383 G T A Q V T F N G I T G K L K A G Y K Y V A Y E K M T R P G Q P D K P V P P P H E D P K D P N Q T V V S E
(296) The coordinates of the atoms of serine, aspartic acid, histidine and glutamine residues are listed in Table 38 below.
(297) TABLE-US-00038 TABLE 38 Relative atom locations of essential residues for spontaneous intermolecular ester bond formation-Mol9. Residue Residue X Y Z Temperature Atom Atom type Chain number coordinate coordinate coordinate Occupancy factor type 1 N THR A 6254 −1.474 −1.086 1.715 1.00 26.99 N 2 CA THR A 6254 −1.306 0.052 0.793 1.00 25.35 C 3 CB THR A 6254 0.000 0.000 0.000 1.00 24.17 C 4 OG1 THR A 6254 −0.082 −1.137 −0.900 1.00 24.60 O 5 CG2 THR A 6254 1.226 −0.197 0.833 1.00 22.81 C 6 C THR A 6254 −2.532 0.106 −0.137 1.00 24.59 C 7 O THR A 6254 −3.113 −0.928 −0.507 1.00 22.88 O 8 N ASP A 6283 −0.671 6.909 −3.265 1.00 21.90 N 9 CA ASP A 6283 −0.502 5.717 −2.458 1.00 21.36 C 10 CB ASP A 6283 0.774 4.891 −2.781 1.00 21.23 C 11 CG ASP A 6283 0.593 3.445 −2.406 1.00 19.70 C 12 OD1 ASP A 6283 −0.495 2.858 −2.666 1.00 20.24 O 13 OD2 ASP A 6283 1.458 2.870 −1.711 1.00 20.64 O 14 C ASP A 6283 −0.554 6.079 −0.964 1.00 20.57 C 15 O ASP A 6283 0.330 6.766 −0.448 1.00 24.37 O 16 N HIS A 6370 6.585 −3.885 0.695 1.00 26.09 C 17 CA HIS A 6370 5.388 −4.415 0.195 1.00 24.44 C 18 CB HIS A 6370 4.199 −3.727 0.751 1.00 26.18 C 19 CG HIS A 6370 2.943 −4.373 0.347 1.00 26.11 C 20 ND1 HIS A 6370 2.589 −5.620 0.812 1.00 32.01 N 21 CE1 HIS A 6370 1.426 −5.956 0.300 1.00 32.92 C 22 NE2 HIS A 6370 1.025 −4.981 −0.497 1.00 29.37 N 23 CD2 HIS A 6370 1.985 −4.009 −0.524 1.00 25.39 C 24 C HIS A 6370 5.453 −4.228 −1.313 1.00 25.81 C 25 O HIS A 6370 5.232 −3.145 −1.836 1.00 23.66 O 26 N GLN A 6378 −3.321 −2.170 −4.282 1.00 21.15 N 27 CA GLN A 6378 −2.221 −1.412 −4.725 1.00 19.36 C 28 CB GLN A 6378 −0.909 −2.103 −4.452 1.00 21.66 C 29 CG GLN A 6378 −0.616 −2.262 −2.987 1.00 21.70 C 30 CD GLN A 6378 −0.538 −0.926 −2.256 1.00 22.43 C 31 OE1 GLN A 6378 −0.505 0.227 −2.773 1.00 21.93 O 32 C GLN A 6378 −2.317 −1.123 −6.273 1.00 18.51 C 33 O GLN A 6378 −1.430 −0.516 −6.820 1.00 18.86 O
(298) The interatomic distance (in Angstrom) of Cβ (CB) of threonine to Cδ (CD) of glutamine, Cγ (CG) of histidine and Cγ (CG) of aspartic acid is listed in table 39 below.
(299) TABLE-US-00039 TABLE 39 Interatomic distances. Mol9 Thr CB to Gln CD 2.50 Thr CB to His CG 5.28 Thr CB to Asp CG 4.24
Example 17
(300) This example demonstrates preparation of a covalently linked multimeric protein complex having a ‘trunk’ structure through the spontaneous formation of ester bonds between engineered Mobiluncus mulieris (Mol) domains that ligate together in a specific order to form a stalk or trunk-like multimeric structure.
(301) Method
(302) The Ig-like domains of Mol7, Mol8, Mol9, Mol10 and Mol11 were modified whereby the domain boundaries of each individual construct were shifted such that the Mol7 to Mol10 constructs lacked their own final beta-strand, and each Mol construct was extended at the N-terminus to include the final beta-strand of the preceding Mol domain, also referred to herein as the strand complementation sequence. A His.sub.6-tag and rTEV cleavage motif was fused to each N-terminal extension, and each protein was expressed and isolated from the crude bacterial lysate using immobilized metal affinity chromatography (IMAC) resin, and the His.sub.6-tag was then removed with rTEV protease.
(303) The amino acid sequences of the modified Mol constructs Mol7a-Mol11 used in this example are shown in Table 40 below and in
(304) TABLE-US-00040 TABLE 40 Peptide sequence of Mol constructs. Con- SEQ struct Sequence ID Mol7a MSYYHHHHHHDYDIPTTENLYFQGAVGSLDTTATDAADG 32 NKHADNAAAVTINDKVDYSGLNLAATYPDGTLKAYLVRG ELMDKATGKPVAGVAPVERVIGAANSVYRVGDQNRPVEE EITSGAGSVVLSFQVPAKLTQGKVLVAFETVYEE* Mol8 MSYYHHHHHHDYDIPTTENLYFQGAGREFLIHHDINDDA 33 QTVYTPSVKTQARVDSERNLLLADKDSTIKDTVTLSGLK TGETYVLSGVLMDKATGQPVLGKDMQAITAVSEPLKAES GAFVKTDAVSFTVPAGTVKADTELVVFEKLWVANEVTVD AKTKTVTPKDTK* Mol9 MSYYHHHHHHDYDIPTTENLYFQGAGKSQPAASHEDITD 34 ENQTVKSGTSPSLKTVLSADGKREWVENNTNIPTVPHAS DSLIDTVLYTGLTEGVSYRLDAKLMEINPVTGKVSETPV ATGYTEFTAKTSDGTAQVTFNGITGKLKAGYKYVAYEKM TRPG* Mol10 MSYYHHHHHHDYDIPTTENLYFQGAPDKPVPPPHEDPKD 35 PNQTVVSEHNPGITTTLTDAQAAKGTDGKVISLTRDAQL KDVVRVTQTGLIEGAKYHVFSKLVNQANPDQVVSAGMQE FTATGDQLRSVTVKFTVPKETLQELAGSDPSAEFKLVAY EYLALDSDTDIVNKEATSEIEAVGFK* Mol11 MSYYHHHHHHDYDIPTTENLYFQGAGKTWAATHADPNDA 36 GQTVTVVKAPKIGTTLKYGQSKTVWVADKVELTDTVEYF NLQPKTKYTLSGNLMGGTSAESLSDTGVKATTEFTTPAA ANGAQTVSGTAVVKFTVPREVLERNEKLVAYEYLTIDGN PVASHEDPKDENQTVTSKKP*
(305) When mixed together each construct ligates to others in a specific order through strand complementation and ester bond formation (see
(306) The specificity of each construct was tested by adding equimolar amounts of the Mol domains in an optimized reaction buffer (50 mM HEPES pH 7.0, 10 mM NaCl, 100 μM CaCl.sub.2) and 20% glycerol) for 24 h. Bond formation was analysed by SDS-PAGE.
(307) Results
(308) SDS-PAGE analysis shows that an ester bond only forms between adjacent pairs (
(309) Discussion
(310) This example demonstrates that multimeric protein complexes having a desired defined structure can be prepared via selection of appropriate complementarity between individual component constructs. Here, the selection of complementary amino acid sequences from the β-clasp domains of Mol Ig-like proteins enables the directed ligation and formation of a multimeric protein complex providing a trunk-like scaffold. Furthermore, the desired defined structure can be achieved even when all individual components are present in a single reaction.
Example 18
(311) This example demonstrates the formation of a multivalent multimeric protein complex having a ‘tree-like’ structure with functional activities (‘valencies’) positioned in desired relationships to one another.
(312) Here, the Mol trunk domains were engineered to carry a Cpe0147 Ig-like domain 2 (Cpe2) branch domain that captures a cargo protein with a specific ester bond peptide tag. Spontaneous formation of ester bonds between the engineered, chimeric Mol-Cpe2 (i.e., Mobiluncus mulieris-Clostridium perfringens Cpe0147 Ig-like domain 2) constructs and cargo protein ligate each component together in a specific order to form a multimeric protein complex having a tree-like structure.
(313) Method
(314) The Ig-like domains of Mol7, Mol8, Mol9, Mol10 and Mol11 from Example 17 were engineered to be combined with a Cpe2 domain, previously described in Example 1. Here, the Cpe2 domains were fused to a helical linker (HL), and the helical linker to the N-terminus of the N-terminal strand complementation peptide of each domain (Cpe2-HL-Mol). This forms a construct in which the Mol trunk domain and the Cpe2 branch domain are separated by an alpha-helical linker (
(315) The amino acid sequences of the Cpe2-HL-Mol constructs prepared in this example are shown in Table 41 below and in
(316) TABLE-US-00041 TABLE 41 Peptide sequence of Cpe2-HL-Mol constructs. Con- SEQ struct Sequence ID Cpe2-HL- MSYYHHHHHHDYDIPTTENLYFQGANLPEVKDGTLRTTV 37 Mol7b IADGVNGSSEKEALVSFENSKDGVDVKDTINYEGLVANQ NYTLTGTLMHVKADGSLEEIATKTTNVTAGENGNGTWGL DFGNQKLQVGEKYVVFENAESVENLIDTDKDYNSHGKAE AAAKEAAAKEAAAKEAAAKEAAAKAAAPTSAGQVVATHA DPKSDDQTVTVGSLDTTATDAADGNKHADNAAAVTINDK VDYSGLNLAATYPDGTLKAYLVRGELMDKATGKPVAGVA PVERVIGAANSVYRVGDQNRPVEEEITSGAGSVVLSFQV PAKLTQGKVLVAFETVYEE Cpe2-HL- MSYYHHHHHHDYDIPTTENLYFQGANLPEVKDGTLRTTV 38 Mol8 IADGVNGSSEKEALVSFENSKDGVDVKDTINYEGLVANQ NYTLTGTLMHVKADGSLEEIATKTTNVTAGENGNGTWGL DFGNQKLQVGEKYVVFENAESVENLIDTDKDYNSHGKAE AAAKEAAAKEAAAKEAAAKEAAAKAAAPTSAGREFLIHH DINDDAQTVYTPSVKTQARVDSERNLLLADKDSTIKDTV TLSGLKTGETYVLSGVLMDKATGQPVLGKDMQAITAVSE PLKAESGAFVKTDAVSFTVPAGTVKADTELVVFEKLWVA NEVTVDAKTKTVTPKDTK Cpe2-HL- MSYYHHHHHHDYDIPTTENLYFQGANLPEVKDGTLRTTV 39 Mol9 IADGVNGSSEKEALVSFENSKDGVDVKDTINYEGLVANQ NYTLTGTLMHVKADGSLEEIATKTTNVTAGENGNGTWGL DFGNQKLQVGEKYVVFENAESVENLIDTDKDYNSHGKAE AAAKEAAAKEAAAKEAAAKEAAAKAAAPTSAGKSQPAAS HEDITDENQTVKSGTSPSLKTVLSADGKREWVENNTNIP TVPHASDSLIDTVLYTGLTEGVSYRLDAKLMEINPVTGK VSETPVATGYTEFTAKTSDGTAQVTENGITGKLKAGYKY VAYEKMTRPG Cpe2-HL- MSYYHHHHHHDYDIPTTENLYFQGANLPEVKDGTLRTTV 40 Mol10 IADGVNGSSEKEALVSFENSKDGVDVKDTINYEGLVANQ NYTLTGTLMHVKADGSLEEIATKTTNVTAGENGNGTWGL DFGNQKLQVGEKYVVFENAESVENLIDTDKDYNSHGKAE AAAKEAAAKEAAAKEAAAKEAAAKAAAPTSAPDKPVPPP HEDPKDPNQTVVSEHNPGITTTLTDAQAAKGTDGKVISL TRDAQLKDVVRVTQTGLIEGAKYHVFSKLVNQANPDQVV SAGMQEFTATGDQLRSVTVKFTVPKETLQELAGSDPSAE FKLVAYEYLALDSDTDIVNKEATSEIEAVGFK JT-Mol11 MSYYHHHHHHDYDIPTTENLYFQGAGKTWAATHADPNDA 41 GQTVTVVKAPKIGTTLKYGQSKTVWVADKVELTDTVEYF NLQPKTKYTLSGNLMGGTSAESLSDTGVKATTEFTTPAA ANGAQTVSGTAVVKFTVPREVLERNEKLVAYEYLTIDGN PVASHEDPKDENQTVTSKKP
(317) T antigen cargo proteins (C2pept-T protein) were engineered with an N-terminal C2pept tag. Four different T-antigens were used, each expressed naturally by different strains of S. pyogenes, to yield four different C2pept-T protein constructs.
(318) The amino acid sequences of these C2pept-T constructs are shown in Table 42 below and in
(319) TABLE-US-00042 TABLE 42 Peptide sequence of C2pept-T constructs. Con- SEQ struct Sequence ID C2pept- MSYYHHHHHHDYDIPTTENLYFQGADTKQVVKHEDKNDK 42 T1 AQTLVVEKPTGSGSGAETVVNGAKLTVTKNLDLVNSNAL IPNTDFTFKIEPDTTVNEDGNKFKGVALNIPMTKVTYTN SDKGGSNTKTAEFDFSEVTFEKPGVYYYKVTEEKIDKVP GVSYDTTSYTVQVHVLWNEEQQKPVATYIVGYKEGSKVP IQFKNSLDSTTLTVKKKVSGTGGDRSKDFNFGLTLKANQ YYKASEKVMIEKTTKGGQAPVQTEASIDQLYHFTLKDGE SIKVTNLPVGVDYVVTEDDYKSEKYTTNVEVSPQDGAVK NIAGNSTEQETSTDKDMTITFTNKKFE C2pept- MSYYHHHHHHDYDIPTTENLYFQGADTKQVVKHEDKNDK 43 T3.2 AQTLVVEKPTGSGSGAETAGVSENAKLIVKKTFDSYTDN EVLMPKADYTFKVEADSTASGKTKDGLEIKPGIVNGLTE QIISYTNTDKPDSKVKSTEFDFSKVVFPGIGVYRYIVSE KQGDVEGITYDTKKWTVDVYVGNKEGGGFEPKFIVSKEQ GTDVKKPVNFNNSFATTSLKVKKNVSGNTGELQKEFDFT LTLNESTNFKKDQIVSLQKGNEKFEVKIGTPYKFKLKNG ESIQLDKLPVGITYKVNEMEANKDGYKTTASLKEGDGQS KMYQLDMEQKTDESADEIVVTNKRD C2pept- MSYYHHHHHHDYDIPTTENLYFQGADTKQVVKHEDKNDK 44 T13 AQTLVVEKPTGSGSGAETAGVVTGKTLPITKSMIYTDNE ILMPKTTFTFTIEPDTTASGKTKDGLEIKSGETTGLTTK AIVSYDNTDKESAKNKTSNFNFETVTFSGIGIYRYTVSE QNDGIEGIQYDGKKWTVDVYVGNKEGGGFEPKYVVSKEV NSDVKKPIRFENSFKTTSLKIEKQVTGNTGELQKDFNFT LILEASALYEKGQVVKIIQDGQTKDVVIGQEYKFTLHDH QSIMLAKLPIGISYKLTEDKADGYTTTATLKEGEIDAKE YVLGNLQKTDESADEIVVTNKRD C2pept- MSYYHHHHHHDYDIPTTENLYFQGADTKQVVKHEDKNDK 45 T18 AQTLVVEKPTGSGSGAETAGVIDGSTLVVKKTFPSYTDD KVLMPKADYTFKVEADDNAKGKTKDGLDIKPGVIDGLEN TKTIHYGNSDKTTAKEKSVNFDFANVKFPGVGVYRYTVS EVNGNKAGIAYDSQQWTVDVYVVNREDGGFEAKYIVSTE GGQSDKKPVLFKNFFDTTSLKVIKKVIGNTGEHQRSFSF TLLLTPNECFEKGQVVNILQGGETKKVVIGEEYSFTLKD KESVTLSQLPVGIEYKVTEEDVTKDGYKTSATLKDGDVT DGYNLGDSKTTDKSTDEIVVTNKRD
(320) The protein constructs were expressed and purified individually. Each component was expressed with a His6-tag and rTEV cleavage motif fused to the N-terminus of the construct (i.e., His6-rTEV-Cpe-like-HL-Mol and His6-rTEV-pept-T18.1). Recombinant proteins were isolated from the crude bacterial lysate using immobilized metal affinity chromatography, and the His6-tag subsequently removed with rTEV protease from all protein constructs with the exception of T18.1.
(321) By way of outline, the multimeric protein complex was prepared as follows. Each Cpe2-HL-Mol construct was first ligated to the paired C2pept-T protein individually as depicted in
(322) Equimolar amounts of each Cpe2-HL-Mol construct was first ligated to the paired C2pept-T protein in separate reactions. Aliquots were removed for verification of bond formation by SDS-PAGE.
(323) In the next step, all individually ligated T-protein-Cpe2-HL-Mol constructs were mixed together. After a 24 h incubation the multimeric protein complex was purified by IMAC to remove any partially formed scaffolds and any monomeric proteins. Only the T18.1 protein retained a His-tag and because all His-affinity tags on the other constructs were removed with rTEV protease, thus only complexes containing T18.1 were retained on the affinity column.
(324) Results
(325) SDS-PAGE analysis (
(326) Representative results of the second ligation step, where the four Cpe2-HL-Mol-C2pept-T proteins were mixed together, incubated, then purified by IMAC, are shown in
(327) High molecular weight species were observed in the incubated sample prior to IMAC (
(328) These two species were separated by size exclusion chromatography (SEC), as shown in
(329) Discussion
(330) This example demonstrates that multimeric protein complexes having a defined, desired structure and carrying different cargo proteins at pre-determined positions can be prepared via appropriate complementarity between individual component constructs. Here, the selection of complementary amino acid sequences from the β-clasp domains of Mol Ig-like proteins enables the directed ligation and formation of a multimeric protein complex providing a trunk-like scaffold, where each different ‘trunk’ component carries a specific functional protein cargo via further covalent linkage between specific Cpe2-C2pept binding partners.
(331) In this instance, individual monovalent multimeric protein constructs (“trunk-branch-cargo” protein constructs), where each monovalent construct had a single, different antigen, were first prepared in separate reactions. This was followed by the formation of a desired multivalent multimeric protein complex having a defined structure by the combination of multiple monovalent multimeric protein constructs in a single second reaction. The structural relationship between the different functional activities present in the multivalent multimeric protein can readily be adapted by appropriate selection of complementarity between ligation partners, and by appropriate sequencing of ligation reactions.
Example 19
(332) This example demonstrates the formation of a multimeric protein complex having a ‘tree-like’ structure with functional activities positioned in desired relationships to one another.
(333) Here, Mol trunk domains were engineered to carry diverse Cpe-like branch domains derived from bacterial adhesins from species other than Clostridium perfringens, where each Cpe-like branch domain has a covalently linked peptide tag. Spontaneous formation of ester bonds between the Cpe-like branch domains and their specific peptide tagged cargo (here, enhanced green fluorescent protein, eGFP) enabled each component to be ligated together in a specific order to form a multivalent multimeric protein complex having a tree-like structure.
(334) Method
(335) Cpe-like domains were cloned from the following sources:
(336) Geberg1—Gemella bergeriae ATCC 700627, ACCESSION AWVP01000087
(337) Gberg2—Gemella bergeriae ATCC 700627, ACCESSION ERK56535
(338) Corio—Coriobacteriaceae bacterium 68-1-3, ACCESSION NZ_CP009302
(339) Ig-like domains of Mol7, Mol8, Mol9, Mol10 and Mol11 (as described in Example 18 above) were engineered with the Cpe-like domains fused to the N-terminus of the N-terminal strand complementation peptide of each domain via a helical linker (eg. Corio-HL-Mol,
(340) TABLE-US-00043 TABLE 43 Peptide sequence of Cpe-like-HL-Mol constructs. Con- SEQ struct Sequence ID Corio- MSYYHHHHHHDYDIPTTENLYFQGAGGEEPFVPGNGDTP 46 HL- SLKTTVKAASSTASSEAAAKLTASEAAKGASVVDTIDYA Mol7b NLYGGKQYEVTARLMPVKDGVVTGDPLVTVTVRRTADLS GSGSWTVPLGTVEGLEKDTSYVVFEKAVSIDNLVDRDGD GNSHGKAEAAAKEAAAKEAAAKEAAAKEAAAKAAAPTSA GQVVATHADPKSDDQTVTVGSLDTTATDAADGNKHADNA AAVTINDKVDYSGLNLAATYPDGTLKAYLVRGELMDKAT GKPVAGVAPVERVIGAANSVYRVGDQNRPVEEEITSGAG SVVLSFQVPAKLTQGKVLVAFETVYEE Gberg1- MSYYHHHHHHDYDIPTTENLYFQGATVTDQDKYVNPKGE 47 HL- LKTTVEADGQSSTTEKSVEVTENKDGVKVVDTIKYKGLV Mol8 EGKDYTVTGQLYEVKDGKIVGEAKATKTETKKADKDEGN WNLDFGTVKGLEAGKSYVVYETATSLENLVDTDNDNKSH GKAEAAAKEAAAKEAAAKEAAAKEAAAKAAAPTSAGREF LIHHDINDDAQTVYTPSVKTQARVDSERNLLLADKDSTI KDTVTLSGLKTGETYVLSGVLMDKATGQPVLGKDMQAIT AVSEPLKAESGAFVKTDAVSFTVPAGTVKADTELVVFEK LWVANEVTVDAKTKTVTPKDTK Gberg2- MSYYHHHHHHDYDIPTTENLYFQGARVTNKKIVSSLQTT 48 HL- VEADGQSSTAEKSAEVTENKDGVNVVDTIHYKGLIPKQK Mol9 YEVVGILYEVKDGKLVDPNKPITISNGTGEYTVSDSGEG EWKLNFGKIDGVEARKSYVVYEEVTSVENLVDTDNDGNS HGKAEAAAKEAAAKEAAAKEAAAKEAAAKAAAPTSAGKS QPAASHEDITDENQTVKSGTSPSLKTVLSADGKREWVEN NTNIPTVPHASDSLIDTVLYTGLTEGVSYRLDAKLMEIN PVTGKVSETPVATGYTEFTAKTSDGTAQVTFNGITGKLK AGYKYVAYEKMTRPG CpeC2- MSYYHHHHHHDYDIPTTENLYFQGANLPEVKDGTLRTTV 49 HL- IADGVNGSSEKEALVSFENSKDGVDVKDTINYEGLVANQ Mol10 NYTLTGTLMHVKADGSLEEIATKTTNVTAGENGNGTWGL DFGNQKLQVGEKYVVFENAESVENLIDTDKDYNSHGKAE AAAKEAAAKEAAAKEAAAKEAAAKAAAPTSAPDKPVPPP HEDPKDPNQTVVSEHNPGITTTLTDAQAAKGTDGKVISL TRDAQLKDVVRVTQTGLIEGAKYHVFSKLVNQANPDQVV SAGMQEFTATGDQLRSVTVKFTVPKETLQELAGSDPSAE FKLVAYEYLALDSDTDIVNKEATSEIEAVGFK JT-Mol11 MSYYHHHHHHDYDIPTTENLYFQGAGKTWAATHADPNDA 50 GQTVTVVKAPKIGTTLKYGQSKTVWVADKVELTDTVEYF NLQPKTKYTLSGNLMGGTSAESLSDTGVKATTEFTTPAA ANGAQTVSGTAVVKFTVPREVLERNEKLVAYEYLTIDGN PVASHEDPKDENQTVTSKKP
(341) GFP cargo proteins were engineered, with each having a specific Cpe-like pept tag complementary to a Cpe-like domain as outlined above at its N-terminus, to yield four different Cpe-like pept-GFP protein constructs. The amino acid sequence of the four different Cpe-like pept-GFP constructs are shown in Table 44 below and in
(342) TABLE-US-00044 TABLE 44 Peptide sequence of Cpe-like pept-GFP constructs. Construct Sequence SEQ ID Coriopept-GFP MSYYHHHHHHDYDIPTTENLYFQGGGDELQTGSHEDPRD 51 SSQTVTVASDPGSGSGAMVSKGEELFTGVVPILVELDGD VNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL VTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIF FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILG HKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQ LADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRD HMVLLEFVTAAGITLGMDELYK Gberg1pept-GFP MSYYHHHHHHDYDIPTTENLYFQGGGDKKQEVEHKDPKD 52 KSQTFVVKPKTPGSGSGAMVSKGEELFTGVVPILVELDG DVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPT LVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNIL GHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSV QLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKR DHMVLLEFVTAAGITLGMDELYK Gberg2pept-GFP MSYYHHHHHHDYDIPTTENLYFQGGGDKKHEVEHKDPKD 53 KSQTFVVKPKTPGSGSAMVSKGEELFTGVVPILVELDGD VNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL VTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIF FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILG HKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQ LADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRD HMVLLEFVTAAGITLGMDELYK C2pept-GFP MSYYHHHHHHDYDIPTTENLYFQGADTKQVVKHEDKNDK 54 AQTLVVEKPTGSGSGAMVSKGEELFTGVVPILVELDGDV NGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLV TTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFF KDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGH KLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQL ADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDH MVLLEFVTAAGITLGMDELYK
(343) The constructs were purified individually before mixing and assembly into multimeric protein scaffold complexes having a ‘tree-like’ structure. Each component was expressed with a His6-tag and rTEV cleavage motif fused to the N-terminus of the construct (ie. His6-rTEV-Cpe-like-HL-Mol and His6-rTEV-pept-GFP). Recombinant proteins were isolated from the crude bacterial lysate using immobilized metal affinity chromatography with the His6-tag subsequently removed with rTEV protease.
(344) By way of outline, the multimeric protein complex was prepared as follows. Each Cpe-like-HL-Mol construct was first ligated to the paired Cpe-like pept-GFP protein individually as depicted in
(345) Equimolar amounts of each Cpe-like-HL-Mol construct was first ligated to the paired Cpe-like pept-GFP protein in separate reactions. After a 24 h incubation, the four individual GFP-Cpe-like-HL-Mol ligation assemblies were combined along with the capping Mol11 domain. Aliquots were removed for verification of bond formation by SDS-PAGE.
(346) In the next step, individual reactions were mixed together to explore ligation product formation.
(347) Results
(348) SDS-PAGE analysis (
(349) After a 24 h incubation the four individual GFP-Cpe-like-HL-Mol ligation assemblies were combined along with the capping Mol11 domain. The Mol trunk domains associated through strand complementation to form a multivalent multimeric protein complex having a ‘tree-like’ structure that is then covalently linked through ester bond formation to yield the covalently linked multimeric protein.
(350) As can readily be seen in
(351) Discussion
(352) This example demonstrates that multivalent multimeric protein complexes having a desired defined structure and carrying multiple cargo proteins at pre-determined positions can be prepared via appropriate complementarity between individual component constructs. Here, the selection of complementary amino acid sequences from the β-clasp domains of Ig-like proteins from different bacterial species enables the directed ligation and formation of a multimeric protein complex providing a trunk-like scaffold, where each different ‘trunk’ component carries a functional protein cargo via further covalent linkage between specific Cpe-like-Cpe-like pept binding partners.
(353) In this instance, individual monovalent multimeric protein constructs (“trunk-branch-cargo” protein constructs), where each monovalent construct had a protein functionality, were first prepared in separate reactions. This was followed by the formation of a desired multivalent multimeric protein complex having a defined structure by the combination of multiple monovalent multimeric protein constructs in a multiple, stepwise reactions. The structural relationship between the functional activities present in the multivalent multimeric protein can readily be adapted by appropriate selection of complementarity between ligation partners, and by appropriate sequencing of ligation reactions.
Example 20
(354) This example demonstrates the functional activity of multiple protein cargoes and the co-location of these protein functionalities via the formation of a multivalent multimeric protein complex having a ‘tree-like’ structure with functional activities positioned in desired relationships to one another.
(355) Method
(356) The immunogenicity of the multivalent T antigen-comprising multimeric protein complexes prepared as described in Example 18 were analysed by Western blot and ELISA.
(357) Aliquots of multimeric protein complexes were electrophoresed by SDS-PAGE (as outlined above in Example 18 and as depicted in
(358) Results
(359) Western blot analysis (data not shown) established that the multivalent multimeric protein complex exhibited immunogenicity with T antisera, confirming that T antigens co-located with the multivalent multimeric protein complex.
(360) The results of ELISA are shown in
(361) Discussion
(362) These results clearly demonstrate that the function of protein cargoes is maintained when present in a multivalent multimeric protein complex as herein described. The Western blot analysis reported above establishes the presence of the linear epitopes comprising the component T antigens in the protein complex. Furthermore, the ELISA, being performed under non-denaturing conditions, establishes that the antigens present in the multivalent multimeric complex retain their native conformation and immunogenic functionality.
(363) This example demonstrates that the function of multiple protein cargoes—in this case the immunogenic function of each of the T antigen ‘valencies’—is maintained and presented by the multivalent multimeric protein complexes as described herein. Hence, the directed ligation and formation of a multimeric protein complex providing a trunk-like scaffold, where each different ‘trunk’ component carries a functional protein cargo enables the presentation and co-location of multiple functionalities in a defined, structured manner.
INDUSTRIAL APPLICATION
(364) The present invention provides peptide and protein ligation techniques to allow for the controlled assembly and disassembly of multimeric complexes, particularly covalently linked multimeric protein complexes. The present invention thus has application in a wide range of industries including the biomedical, pharmaceutical, diagnostic, engineering, agricultural, and horticultural sectors.
(365) Where in the foregoing description reference has been made to elements or integers having known equivalents, then such equivalents are included as if they were individually set forth.
(366) Although the invention has been described by way of example and with reference to particular embodiments, it is to be understood that modifications and/or improvements may be made without departing from the scope or spirit of the invention.
(367) In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognise that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
PUBLICATIONS
(368) Kwon, H.; Squire, C. J.; Young, P. G.; Baker, E. N., Autocatalytically generated Thr-Gln ester bond cross-links stabilize the repetitive Ig-domain shaft of a bacterial cell surface adhesin. P Natl Acad Sci USA 2014, 111 (4), 1367. Lee, H.; DeLoache, W. C.; Dueber, J. E., Spatial organization of enzymes for metabolic engineering. Metab Eng 2012, 14(3) 242-251 Horn, A. H. C.; Sticht, H., Synthetic protein scaffolds based on peptide motifs and cognate adaptor domains for improving metabolic productivity. Frontiers in Bioengineering and Biotechnology, 2015, 3(191), 1-7 Chen, R. Chen, Q.; Kim, H.; Siu, K. H.; Sun, Q.; Tsai, S. L.; Chen, W., Biomolecular scaffolds for enhanced signalling and catalytic efficiency. Curr Opin Biotech 2014, 28, 59-68 Ting, Y. T.; Batot, G.; Baker, E. N.; Young, P. G. Acta crystallographica. Section F, Structural biology communications 2015, 71, 61 Wu, P. S. C.; Otting, G. J. Magn. Reson. 2005, 176, 115 Petoukhov, M. V.; Franke, D.; Shkumatov, A. V.; Tria, G.; Kikhney, A. G.; Gajda, M.; Gorba, C.; Mertens, H. D.; Konarev, P. V.; and Svergun, D. I. J Appl Crystallogr. 2012, 45, 342. Doublie S, Carter C. (1992) Preparation of Selenomethionyl Protein Crystals. Oxford University Press. New York. Kabsch, W. (2010). XDS. Acta Cryst. D66, 125-132. Evans, P. R. & Murshudov, G. N. (2013) Acta Cryst. D69, 1204-1214 A short history of SHELX”. Sheldrick, G. M. (2008). Acta Cryst. A64, 112-122 Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486-501. Murshudov G. N., Skubak P., Lebedev A. A., Pannu N. S., Steiner R. A., Nicholls R. A., Winn M. D., Long, F. & Vagin, A. A. (2011). Acta Cryst. D67, 355-367. Chen V. B., Arendall W. B. 3rd, Headd J. J., Keedy D. A., Immormino R. M., Kapral, G. J., Murray, L. W., Richardson, J. S. & Richardson, D. C. (2010). Acta Cryst. D66, 12-21.