Ligase Screening Assay

Abstract

The present invention relates to a method for identifying a MYCBP2 modulator. Suitable modulators are identified by modulation of MYCBP2 ubiquitin E3 ligase activity via covalent modification of either of two catalytic cysteines (C4520 and C4572) or by impeding the motion of a newly presented dynamic, so-called, mediator loop region where C4520 resides. The present invention also relates to the use of hydroxy group- containing small molecules and peptides as proxy substrates for measuring MYCBP2 ligase activity and their use in the method of identifying modulators.

Claims

1. A method for identifying a MYCBP2 modulator, the method comprising: a) contacting MYCBP2, an orthologue, mutant or fragment thereof with a test substance; b) providing a probe which is capable of interacting with C4520, C4572 and/or the mediator loop region (residues 4515-4531) of active MYCBP2; and c) detecting whether or not the level of interaction of the probe with MYCBP2, the orthologue, mutant or fragment thereof is modulated as compared to the level of interaction in the absence of the test substance.

2. The method according to claim 1, wherein the probe comprises a non-endogenous substrate of active MYCBP2.

3. The method according to claim 1, wherein the modulator is an inhibitor and step c) comprises detecting whether or not the level of interaction of the probe with MYCBP2, the orthologue, mutant or fragment thereof is reduced as compared to the level of interaction in the absence of the test substance.

4. The method according to claim 1, wherein the probe comprises a hydroxyl group, the probe capable of interacting with C4520 and optionally C4572 of active MYCBP2 via the hydroxyl group.

5. The method according to claim 1, wherein the probe interacts with C4572 of MYCBP2 and comprises an activated biological molecule according to the formula (I): ##STR00006## wherein X is a biological molecule and EWG is an electron withdrawing group.

6. The method according to claim 1, wherein the method does not comprise providing an endogenous MYCBP2 substrate, ATP, E1 and/or E2.

7. The method according to claim 1, wherein the probe is conjugated to a surface.

8. The method according to claim 4 wherein interaction comprises esterification by MYCBP2, the orthologue, mutant or fragment thereof of the hydroxyl group with ubiquitin.

9. The method according to claim 8, wherein the probe comprises a peptide.

10. The method according to claim 8, wherein the probe comprises threonine or serine.

11. The method according to claim 8, wherein the probe comprises a small molecule.

12. The method according to claim 9, wherein the probe comprises tris, glycerol or HEPES.

13. The method according to claim 5, wherein the activated biological molecule comprises an activated biological molecule conjugate probe which corresponds to formula (II): ##STR00007## wherein X is a first biological molecule, EWG is an electron withdrawing group and Y is a further biological molecule.

14. The method according to claim 5, wherein X comprises an E2 enzyme.

15. The method according to claim 13, wherein Y comprises ubiquitin.

16. The method according to claim 14, wherein the E2 enzyme is selected from UBE2D1, UBE2D2, UBE2D3, UBE2D4 and UBE2E1.

17. The method according to claim 1, wherein the orthologue is selected from mouse Phr1, zebrafish Esrom/phr1, Drosophila Highwire and C.elegans RPM-1.

18. The method according to claim 1, wherein the test substance comprises a peptide, a small molecule, an aptamer, an antibody or an antibody fragment.

19. The method according to claim 1, wherein the probe comprises a label.

20. The method according to claim 19, wherein the label comprises an affinity tag.

21. The method according to claim 1, wherein the MYCBP2, orthologue, mutant or fragment thereof is recombinant.

22. The method according to claim 1, wherein the method comprises contacting a fragment of MYCBP2, wherein the fragment comprises residues 4390 to 4572 of MYCBP2.

Description

[0149] Embodiments of the invention will now be described by way of example and with reference to the accompanying figures, in which:

[0150] FIG. 1 shows the activity-based proteomics of E3 ligases;

[0151] FIG. 2 shows the LC-MS characterization of biotinylated ABP intermediates and biotinylated ABPs;

[0152] FIG. 3 shows the activity-based proteomic profiling of neuroblastoma SH-SY5Y cells;

[0153] FIG. 4 shows MYCBP2 is a novel class of E3 ligase and data support of a cysteine relay mechanism;

[0154] FIG. 5 shows ABPs label C4520 within MYCBP2.sub.cat with high selectivity;

[0155] FIG. 6 shows the esterification activity of MYCBP2.sub.cat and further data in support of a dual cysteine mechanism that operates in cis;

[0156] FIG. 7 shows that MYCBP2 ubiquitinates serine and threonine with selectivity for threonine;

[0157] FIG. 8 shows that MYCBP2 has serine/threonine Ub esterification activity but has preference for threonine;

[0158] FIG. 9 shows the E2 requirements of MYCBP2;

[0159] FIG. 10 is a structural comparison and representative stereo views of the MYCBP2.sub.cat crystallographic model;

[0160] FIG. 11 shows the crystal structure of MYCBP2.sub.cat;

[0161] FIG. 12 shows the structural basis for threonine selectivity, model of an E2-E3 intermediate and model of Ub relay;

[0162] FIG. 13 shows the modelling of E2-MYCBP2.sub.cat complex; and

[0163] FIG. 14 is a schematic representation for proposed model for RCR E3 ligase mechanism.

DETAILED DESCRIPTION

Introduction

[0164] Ubiquitination is initiated by ubiquitin (Ub) transfer from an E1 activating enzyme (E1) to an E2 conjugating enzyme (E2) producing a covalently linked intermediate (E2Ub).sup.1. E3 ligases (E3s) of the Really Interesting New Gene (RING) class recruit E2Ub via their RING domain and mediate direct transfer of Ub to substrates.sup.2. Homologous to E6-AP Carboxy Terminus (HECT) E3s undergo a catalytic cysteine-dependent transthiolation reaction with E2Ub forming a covalent E3Ub intermediate.sup.3,4. Additionally, RING-between-RING (RBR) E3s have a canonical RING domain that is linked to an ancillary domain. This contains a catalytic cysteine enabling a hybrid RING/HECT mechanism.sup.5. Ubiquitination is typically considered a posttranslational modification of lysine residues as human E3s endowed with non-lysine activity remain to be discovered. Herein, we carry out activity-based protein profiling of HECT/RBR-like E3s and uncover the neuron-associated E3 MYCBP2/Phr1 as a novel class of RING-linked E3 with esterification activity and intrinsic selectivity for threonine over serine. MYCBP2 contains two essential catalytic cysteine residues which relay Ub to substrate via thioester intermediates. Crystallographic characterization of this new class of E3 ligase, which we designate as an RING-Cys-Relay (RCR), reveals insights into its mechanism and threonine selectivity. These findings implicate cellular regulation of higher eukaryotes by non-lysine ubiquitination and unappreciated mechanistic diversity of E3 enzymes.

[0165] Materials and Methods

[0166] General Materials

[0167] All DNA constructs were verified by DNA sequencing, (Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee). DNA for bacterial protein expression was transformed into E. coli BL21-DE3 (Merck). All cDNA plasmids and antibodies generated for this study are available to request through our reagents website (https://mrcppureagants.dundee.ac.uk/). All solvents and reagents were purchased from Sigma-Aldrich or VWR unless otherwise stated.

[0168] Biotin Functionalized ABP Preparation

[0169] Ub with a GCSSG N-terminal extension was expressed from plasmid pTXB1-Ub74-76-T3C plasmid. An equivalent plasmid encoding Ub residues 1-74 (pTXB1-Ub75-76-T3C) was also created. Ub thioesters were obtained as described previously generating cysteine tagged Cys-Ub.sub.1-73-SR and Cys-Ub.sub.1-74-SR, respectively.sup.7. The extended Ub.sub.1-74 was included as this retains Arg74 which forms a favourable electrostatic interaction with the RBR E3 HOIP.sup.31. Cys-Ub.sub.1-73-SR (30 mg) was reconstituted by the addition of DMSO (116 L) followed by H.sub.2O (456 L). An aqueous stock solution (48 mM) of EZ-link lodoacetyl-PEG2-biotin (Thermofisher) was prepared and 200 L was added to the Cys-Ub.sub.1-73-SR solution (580 l) followed by the addition of 900 l degased buffer (50 mM Na.sub.2HPO.sub.4 pH 7.5, 150 mM NaCl). The reaction was incubated at 23 C. for 1 hour and monitored by LC-MS. The protein (Biotin-Ub.sub.1-73-SR) was then further purified by semi-preparative RP-HPLC (Column: BioBasic-4; Part number: 72305-259270). A gradient of 20% buffer A to 50% buffer B was applied at a flow rate of 10 mL min.sup.1 over 60 min (buffer A=0.1% TFA in H.sub.2O, buffer B=0.1% TFA in acetonitrile). The above procedure was repeated to generate Biotin-Ub.sub.1-74-SR. HPLC fractions containing Biotin-Ub.sub.1-7X-SR were pooled and lyophilized (Yield: Biotin-Ub.sub.1-73-SR 75-85%, Biotin-Ub.sub.1-74-SR 40-50%) (FIGS. 3a and d). Biotin-tagged ABPs containing thioacrylamide warheads were then prepared as previously described.sup.7 employing the E2 recognition elements UBE2D2*, UBE2D2* F62A, UBE2L3* and UBE2L3* F63A furnishing ABPs 1, 3, 2 and 4 (FIGS. 3b, c, e and f). *denotes E2 where non-catalytic Cys residues were mutated to Ser. ABPs based on UBE2D2* and UBE2D3* bearing hexahistidine reporter tags and thioacrylamide warheads (FIG. 5a), were also prepared yielding ABPs 5 and 6, respectively.

[0170] Cell Culture and Lysis Protocol

[0171] SH-SY5Y cells were cultures as previously described.sup.7. HEK293 were cultured (37 C., 5% CO.sub.2) in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) Fetal Bovine Serum (FBS), 2.0 mM L-glutamine, and antibiotics (100 units mL.sup.1 penicillin, 0.1 mg mL.sup.1 streptomycin). Cell transfections were performed using polyethylenimine (Polysciences) according to the manufacturer's instruction. MG-132 (50 M) was added to cells two hours prior to lysis. Cells were rinsed with ice-cold PBS and extracted in lysis buffer (1% NP-40, 50 mM Tris-HCl pH 7.5, 1.0 mM EGTA, 1.0 mM EDTA, 0.27 M sucrose, 10 mM sodium 2-glycerophosphate, 0.2 mM phenylmethane sulfonyl fluoride (PMSF), 1.0 mM benzamidine, 1.0 mM sodium ortho-vanadate, 50 mM sodium fluoride and 5.0 mM sodium pyrophosphate, 50 mM iodoacetamide and cOmplete, EDTA-free protease inhibitor cocktail (Roche)). Lysates were then clarified by centrifugation at 4 C. for 30 min at 14,800 rpm. Supernatants were collected (total cell extracts) and protein concentration determined by Bradford assay. For the base-lability test, indicated cell lysates were further incubated with 0.5M hydroxylamine, pH 9.0 at 37 C. for 30 minutes.

[0172] Immunoblotting

[0173] Samples were mixed with NuPAGE LDS sample buffer (Thermofisher) without boiling, and resolved by SDS-PAGE (4-12% NuPage gel, Thermofisher) with MOPS or MES running buffer and transferred on to 0.45 m nitrocellulose membranes (GE Life Sciences). Membranes were blocked with PBS-T buffer (PBS+0.1% Tween-20) containing 5% (w/v) non-fat dried skimmed milk powder (PBS-TM) at room temperature for 1 h. Membranes were subsequently probed with the indicated antibodies in PBS-T containing 5% (w/v) Bovine Serum Albumin (BSA) overnight at 4 C. Detection was performed using HRP-conjugated secondary antibodies in PBS-TM for 1 h at 23 C. ECL western blotting detecting reagent (GE Life Sciences) was used for visualization in accordance with the manufacturers protocol.

[0174] Antibodies

[0175] His-tagged species were probed with 1:10000 anti-His primary antibody (Clontech, #631212). Alpha tubulin (1E4C11) mouse mAb (Proteintech) was used at 1:10000 dilution. The MYCBP2 antibody was used at 0.5 g mL.sup.1 and raised by in sheep by MRC PPU Reagents and Services and affinity-purified against the indicated antigen: anti-MYCBP2 (2nd bleed of SA357, residues 4378-4640 of human MYCBP2). Mouse monoclonal NMNAT2 antibody (clone 2E4; Sigma Aldrich) was used at 0.5 g mL.sup.1.

[0176] Activity-Based Proteomic Profiling of SH-SY5Y Cells

[0177] SH-SY5Y total cell lysate (4.5 mg, 550 L) was mixed with ABPs 1, 2, 3 and 4 (3 M) and incubated at 30 C. for 4 hours. To induce Parkin activation cells were administered with oligomycin (5 M) and antimycin A (10 M) (OA) for 3 hours. Control enrichments were also performed where probe was withheld. Extracts were mixed with 100 L of Pierce Streptavidin Plus UltraLink Resin (ThermoFisher Scientific) and diluted with 6% SDS solution (20 L) to a final concentration of 0.2% in phosphate buffer. Samples were incubated for 4 hours at 4 C. and resin washed (2 ml 0.2% SDS/PBS, 2 ml PBS, 1 ml 4 M urea/PBS, 2 ml PBS) and then resuspended in 190 l Tris buffer (50 mM Tris pH 8, 1.5 M urea). Resin-bound proteins were reduced with TCEP (5 mM) for 30 minutes at 37 C. and then alkylated with iodoacetamide (10 mM) at 23 C. for 20 minutes. DTT (10 mM) was then added followed by washing with buffer (50 mM Tris pH 8, 1.5 M urea) to a final volume of 300 L. Trypsin (2 g) was then added and further incubated at 37 C. for 14 hours. Trifluoroacetic acid was added to a final concentration of 0.1% and samples were desalted with a C.sup.18 MacroSpin column (The Nest Group Inc). LC-MS/MS analysis was performed on an LTQ Orbitrap Velos instrument (Thermo Scientific) coupled to an Ultimate nanoflow HPLC system (Dionex). A gradient running from 3% solvent B to 99% solvent B over 345 min was applied (solvent A=0.1% formic acid and 3% DMSO in H.sub.2O; solvent B=0.08% formic acid and 3% DMSO in 80% MeCN).

[0178] Data Processing

[0179] Raw files were searched against the Swissprot database and a decoy database using the MASCOT server (Matrix Science). Trypsin specificity with up to three missed cleavages was applied. Cysteine carbamylation was set as a fixed modification and variable modifications were methione oxidation/dioxidation. A PERL script was used to extract the number of rank 1 peptides for each protein from the MASCOT search results and this figure was used as the number of spectral counts. A second PERL script filtered the data by searching the human swisspfam_v30 database using the E3 domain terms RING, HECT, IBR and zf-UBR. Manual curation was also carried out which involved the addition of E1 enzymes. Any proteins with less than 3 spectral counts and less than 14-fold spectral count enrichment, relative to control experiments where ABP was withheld, were omitted from the list. Pairwise datasets were then plotted as column charts in Prism (Graphpad Software).

[0180] Cloning of MYCBP2.sub.cat

[0181] Human MYCBP2 (NM_015057.4) sequences were amplified from full-length Addgene plasmid #2570. Wild type and mutant fragments were subcloned as BamHI/Not1 inserts into pGEX6P-1 (GE Life Sciences) for bacterial expression, or a modified version of pcDNA TM5/FRT/TO (ThermoFisher) containing an N-terminal Myc tag for mammalian expression.

[0182] UBE1 and E2 Expression and Purification

[0183] 6His-UBE1 was expressed in Sf21 cells and purified via its tag as previously described.sup.32. Phosphate Buffered Saline was used throughout the purification and hydroxy containing compounds avoided. UBE2D3 was expressed as an N-terminally 6His-tagged protein in BL21 cells and purified over Ni-NTA-agarose and finally dialysed into 50 mM Na.sub.2HPO.sub.4 pH 7.5, 150 mM NaCl, 0.5 mM TCEP. UBE2A was expressed as a GST-fusion in E. coli and the GST tag was proteolytically removed. The remaining E2s were expressed as recombinant bacterial proteins and purified via their His-tags and buffer exchanged by size exclusion chromatography into running buffer (50 mM Na.sub.2HPO.sub.4 pH 7.5, 150 mM NaCl, 0.5 mM TCEP, 0.015% Brij-35) using a Superdex 75 column (GE Life Sciences).

[0184] Expression and Purification of MYCBP2 and GST-MYCBP2

[0185] GST-tagged MYCBP2.sub.cat (Ser4378-Phe4640), wt and mutants, were expressed at 16 C. overnight and purified against glutathione resin (Expedeon) using standard procedures. GST-tagged constructs were eluted with glutathione and untagged constructs were obtained by on-resin cleavage with Rhinovirus 3C protease. Proteins were buffer exchanged into 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1.0 mM TCEP buffer and flash frozen for storage at 80 C.

[0186] Expression and Purification of NMNAT2

[0187] NMNAT2 was expressed with a 6His-SUMO tag in BL21(DE3) cells, induced with 0.1 mM IPTG and incubated for expression at 16 C. The cells were collected and lysed in buffer (50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 0.2 mM EGTA, 20 mM imidazole, 20 mM L-arginine, 0.015% Brij 35, 1 mM Leupeptin, 1 mM Pefabloc, 1 mM DTT using standard protocols and the protein was purified over Ni-NTA-agarose. The eluted protein was incubated with His-SENP1 protease during dialysis against PBS, 20 mM L-arginine, 1 mM DTT. The tag and protease were depeleted against Ni-NTA-agarose and NMNAT2 was concentrated and subjected to chromatography on a Superdex 75 HR 10/30 into buffer (PBS, 20 mM L-arginine).

[0188] Activity-Based Protein Profiling of MYCBP2 Cysteine Mutants

[0189] The indicated MYCBP2 mutant was diluted into Tris buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl) to a final concentration of 3 M. Probe 6 was added (12 M) and incubated with E3 ligase at 30 C. for four hours. Reactions were quenched by the addition of 4LDS loading buffer (supplemented with 680 mM 2-mercaptoethanol) and samples were resolved by SDS-PAGE (4-12% NuPage gel) followed by Coomassie staining or anti-His immunoblotting.

[0190] Tryptic MS/MS Sequencing of Probe-Labelled MYCBP2

[0191] Crosslinking MS using ABP 6 was carried out as previously described.sup.7. In summary Coomassie stained SDS-PAGE band corresponding to ABP-labelled WT MYCBP2 was analyzed by LC-MS/MS using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific) coupled to an Ultimate nanoflow HPLC system (Dionex). A gradient running from 0% solvent A to 60% solvent B over 120 min was applied (solvent A=0.1% formic acid in H.sub.2O; solvent B=0.08% formic acid in 80% MeCN). Fragment Ions were generated by HCD and 1.sup.+, 2.sup.+ and 3.sup.+ precursor ions excluded. Raw data was searched using the pLink software.sup.33 against UBE2D3* and MYCBP2 sequences with trypsin specificity (up to 2 missed cleavages). The error window for MS/MS fragment ion mass values was set to the software default of 20 ppm. A crosslinker monoisotopic mass of 306.1805 Da was manually added which accounted for the theoretical mass difference associated with formation of a bisthioether between 2 Cys residues derived from probe 6, which was based on UBE2D3* and contained a thioacrylamide AVS warhead.sup.7.

[0192] Tris/Glycerol-Mediated E2 Discharge Assay

[0193] Assays were carried out in buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM TCEP, 5 mM MgCl.sub.2) containing the indicated MYCBP2 mutant (15 M), UBE1 (1.5 M), UBE2D3 (15 M), Ub (37 M) and ATP (10 mM). The reactions were incubated at 37 C. for 30 minutes. Reactions were terminated by the addition of 4LDS loading buffer (with and without 680 mM 2-mercaptoethanol). A C4572S sample was further incubated with 0.14 N NaOH at 37 C. for 20 minutes and samples were resolved by SDS-PAGE (4-12% NuPage gel) and visualized by Coomassie staining.

[0194] LC-MS Analysis of Nucleophile Discharge Assays

[0195] Reactions were prepared as described for discharge assay. After 30 minutes, the reaction was analyzed using an Agilent 1200/6130 LC-MS system (Agilent Technologies) using a 10-75% gradient over 20 minutes (buffer A=H.sub.2O+0.05% TFA, buffer B=acetonitrile+0.04% TFA).

[0196] Preparation of Cy3b-Ub

[0197] Ub bearing a TEV protease-cleavable N-terminal hexahistidine tag followed by a ACG motif was expressed in bacteria from a pET plasmid (kindly provide Ronald Hay, University of Dundee). Protein was purified by Ni affinity chromatography, cleaved from the tag with TEV protease then buffer exchanged into reaction buffer (50 mM HEPES, pH 7.5, 0.5 mM TCEP). Protein was concentrated to 2 mg mL.sup.1 and 221 L (50 nmol) was mixed with Cy3b-maleimide (150 nmol, GE Life Sciences) in a final volume of 300 L and agitated for 2 h at 25 C. Labelled protein was then further purified with a P2 Centri-Pure desalting column (EMP Biotech) with degassed buffer (50 mM Na.sub.2HPO.sub.4, 150 mM NaCl).

[0198] MYCBP2 Thioester/Ester Trapping Assay

[0199] UBE1 (2 M) was mixed with Cy3b-Ub (1 M) in buffer (40 mM Na.sub.2HPO.sub.4HCl pH 7.5, 150 mM NaCl, 0.5 mM TCEP, 5 mM MgCl.sub.2) (FIG. 2e, Lane 1, 2). The reaction was then initiated by the addition of ATP (5 mM) and incubated for 10 minutes at 25 C. Samples (lane 3, 4) were taken and combined with UBE2D3 (10 M). After a further 10 minutes at 25 C., samples (lane 5, 6) were taken and combined with GST-MYCBP2.sub.cat (WT, C4520S, C4520A, C4572S, C4572A, C4520A/C4572S or C4520S/C4572A) (15 M). The reactions were incubated at 25 C. for 30 seconds and terminated by the addition of 4LDS loading buffer (either non-reducing or reducing). For Ub-GST-MYCBP2.sub.cat C4572S ester bond cleavage, 0.14 N NaOH was added after E3 reaction with E1/E2 mixture for 30 seconds and then further incubated at 37 C. for 20 minutes. The gel was then scanned with a Chemidoc Gel Imaging System (BioRad).

[0200] Multiple Turnover Amino Acid and Peptide Panel Discharge Assays

[0201] Stock solutions (0.5 M) of amino acids were dissolved in MQ water and pH was adjusted to pH 8. Peptides of the sequence Ac-EGXGN-NH.sub.2 (X=K, S or T) were obtained from Bio-Synthesis Inc. Stock peptide solutions (200 mM) were dissolved in MQ water and pH was adjusted to pH 8. An E2 (UBE2D3) charging reaction was carried out in buffer (40 mM Na.sub.2HPO.sub.4HCl pH 8.0, 150 mM NaCl, 0.5 mM TCEP) containing UBE1 (250-500 nM), UBE2D3 (20 M), Ub (50 M), or Cy3B-Ub (25 M), MgCl.sub.2 (5 mM) and ATP 10 (mM). The reaction was incubated at 37 C. for 15 minutes and then equilibrated to 23 C. for 3 minutes. An equivalent volume of nucleophile sample containing small molecule/peptide nucleophile (100 mM) and GST-MYCBP2 (10 M) was then added and incubated at 23 C. Samples were taken at the specified time points and analyzed as described for Tris/glycerol-mediated E2 discharge assay.

[0202] Cy3B-Ub was visualized using a Chemidoc Gel Imaging System (Biorad). LC-MS was carried out as described for Tris/glycerol discharge but amino acid substrate samples were quenched by the addition of 2:1 parts quenching solution (75% acetonitrile, 2% TFA) and peptide substrate samples were quenched by addition of 1:1 parts quenching solution.

[0203] Multiple Turnover E2 Discharge Panel

[0204] E2s were screened for threonine discharge activity with GST-MYCBP2.sub.cat as described for the amino acid panel. E2s were also incubated in the presence of threonine but in the absence of GST-MYCBP2.sub.cat. These samples provided a reference to distinguish between intrinsic E2Ub instability and E3-dependent discharge.

[0205] Single Turnover E2 Mutant Discharge by in-Gel Fluorescence

[0206] E2 mutants.sup.16,17,34-36 (10 M) were charged with Cy3b-labelled Ub (12.5 M) in a final volume of 12 L at 37 C. for 20 minutes then cooled at 23 C. for 3 minutes. E2 recharging was then blocked by the addition of MLN4924 derivative, Compound 1 (25 M).sup.37, which inhibits E1, and then incubated for a further 15 minutes. The mixture was then mixed with 12 L of GST-MYCBP2.sub.cat (5 M) and threonine (100 mM) and incubated at 23 C. for the specified time. Analysis was carried out as for multiple turnover assays. To account for intrinsic E2Ub instability the mean % discharge (n=2) calculated against a parallel incubation where E3 was withheld. Data were plotted using Prism (Graphpad).

[0207] Expression and Purification of ARIH1 and UBE3C

[0208] ARIH1 residues 1-394 (Dundee clone DU24260) was expressed as an N-terminally GST-tagged fusion protein in BL21 cells. UBE3C residues 641-1083 (Dundee Clone DU45301) was expressed as a N-terminally GST-tagged fusion protein in Sf21 cells using the baculovirus infection system.

[0209] Calculation of Observed Rate Constants for E3-Substrate Dependent Single Turnover E2Ub Discharge

[0210] UBE2D3 or UBE2L3 (5 M) were charged with Cy3b-labelled Ub (8 M) in a final volume of 30 L at 37 C. for 25 minutes then incubated at 23 C. for 3 minutes. Single turnover conditions for E2Ub discharge were achieved by E1 inhibition with MLN4924 derivative, Compound 1 (25 M) and then incubated for a further 15 minutes. The mixture was then mixed with 30 L of MYCBP2.sub.cat or ARIH1.sub.1-394 (HHARI) or UBE3C.sub.641-1083 (1 M) and threonine (100 mM) and incubated at 23 C. for the specified time. Samples were quenched with non-reducing 4LDS loading buffer and resolved by SDS-PAGE (Bis-Tris 4-12%). The gel was then scanned with a Chemidoc Gel Imaging System (BioRad) and subsequently Coomassie stained. E2Ub signals were quantified using the Fiji software. Observed rate constants were obtained by fitting reaction progressive curves to a single exponential function using Prism (Graphpad Software).

[0211] MYCBP2 Crystallization

[0212] MYCBP2 was expressed as described for untagged protein. After protease cleavage of the tag the protein was further purified by size exclusion chromatography using an KTA FPLC system and a HiLoad 26/600 Superdex 75 g column (GE Life Sciences). The running buffer consisted of 20 mM HEPES pH 7.4, 150 mM NaCl, 4 mM DTT. Combined fractions were concentrated to 10.4 mg mL.sup.1. Sparse matrix screening was carried out and Bipyrimidal crystals were obtained from the Morpheus screen condition C1 (Molecular Dimensions). A subsequent optimization screen yielded multiple crystals (Buffer system 1 (MES/imidazole) pH 6.7, 23.3 mM Na.sub.2HPO.sub.4, 23.3 mM (NH.sub.4).sub.2SO.sub.4, 23.3 mM NaNO.sub.3, 18% PEG500 MME, 9% PEG20000). A single crystal was soaked in mother liquor and further cryoprotected by supplementation with 5% PEG400 and frozen in liquid N.sub.2. Data were collected to 1.75 at the European Synchrotron Radiation Facility at Beamline ID23-1. Energy was set to the peak value of 9.669 keV (1.2823 ), as determined by an absorption edge energy scan. A total of 360 were collected with an oscillation range of =0.1. The phase problem was solved by locating 6 Zn.sup.2+ sites in the anomalous signal and solvent flattening with the SHELX suite. An initial model was built by ARP/wARP.sup.38 and subsequently optimized by manual building in COOT.sup.39 and refinement with REFMAC5.sup.40 resulting in the final model with statistics as shown in FIG. 10. Final Ramachandran statistics were favored: 95.55%, allowed: 3.24%, outliers: 1.21%.

[0213] Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS)

[0214] SEC-MALS experiments were performed on a Ultimate 3000 HPLC system (Dionex) with an in-line miniDAWN TREOS MALS detector and Optilab T-rEX refractive index detector (Wyatt). In addition, the elution profile of the protein was also monitored by UV absorbance at 280 nm. A Superdex 75 10/300 GL column (GE Life Sciences) was used. Buffer conditions were 50 mM Na.sub.2HPO.sub.4 pH 7.5, NaCl 150 mM, 1.0 mM TCEP and a flow rate of 0.3 mL min.sup.1 was applied. Sample (50 L, 5.5 mg mL.sup.1) was loaded onto the column with a Dionex autosampler. Molar masses spanning elution peaks were calculated using ASTRA software v6.0.0.108 (Wyatt).

[0215] Mediator Loop Modelling

[0216] Mediator loop residues where built and geometry optimized within the Bioluminate Software (Schrodinger). Side chains were modified within COOT.sup.39 and figures were generated with Pymol (Schrodinger). Ramachandran analysis was carried with the RAMPAGE server.sup.41.

[0217] NMNAT2 Ubiquitination Assay

[0218] NMNAT2 (5 M) was mixed with E1 (500 nM), UBE2D3 (10 M), MYCBP2.sub.cat (10 M), Ub (50 M), ATP (10 mM) and made up with 10pH 7.5 buffer (40 mM Na.sub.2H.sub.2PO.sub.4 pH 7.5, 150 mM NaCl, 5 mM MgCl.sub.2, 0.5 mM TCEP). The reactions were incubated at 37 C. for 1 hour and terminated by the addition of 4LDS loading buffer (either non-reducing or reducing). For base lability test, reactions were supplemented with 0.14 N NaOH and then further incubated at 37 C. for 20 minutes.

[0219] Bioinformatic Analysis

[0220] Proteins belonging to the RCR family were identified by generalized profile searches. Overall 671 such sequences were identified. The sequences were aligned by profile-guided alignment using the pftools package. For identifying representative sequences from various taxa, the Belvu program (Sanger Institute) was used to remove sequences with >80% identity to other sequences. Truncated and misassembled proteins were removed manually, resulting in 130 representative TC domain sequences.

[0221] Data Availability Statement

[0222] Coordinates have been deposited with the Protein Data Bank (PDB ID 5O6C).

[0223] Results

[0224] We prepared biotinylated variants of our recently developed activity-based probes (ABPs).sup.6 which profile the hallmark transthiolation activity of HECT/RBR E3s (FIG. 1a and FIG. 2).sup.7. Interfacing the ABP technology with mass spectrometry enabled parallelized profiling of E3 activity in neuroblastoma SH-SY5Y cell extracts 80% (FIG. 1b). E3s were filtered using criteria ensuring signals for at least a subset of detected E3s correlated with E3 activity and/or abundance (FIG. 3). Profiling of 80% of the 50 known HECT/RBR E3s was achieved but unexpectedly, 33 RING E3s, devoid of HECT or RBR ancillary domains, were also enriched (FIG. 1c-e). To explore the possibility that hitherto undiscovered RING-linked E3s were being labelled we focused on MYCBP2/Phr1 (Myc-binding protein 2; PAM/Highwire/Rpm-1) (FIG. 1c). MYCBP2 is a large 0.5 MDa neuron-associated protein which contains a C-terminal RING domain (FIG. 4a), and is involved in a range of cellular processes including regulation of nervous system development and axon degeneration.sup.10-12.

[0225] A recombinant C-terminal version of MYCBP2 encompassing the RING domain (residues 4378-4640; MYCBP2.sub.cat; FIG. 4a) and an uncharacterized C-terminal cysteine-rich region underwent robust ABP labelling with an efficiency comparable to that of E3s known to demonstrate transthiolation activity.sup.7,13 (FIG. 5a). To map the putative catalytic cysteine we used a combination of ABP-based profiling and ABP-crosslinking MS.sup.7 (FIG. 2b, FIG. 5b, c,). Data were in support of C4520 being a putative catalytic residue. We next assayed wild type (WT) E3 activity but were unable to detect autoubiquitination or free Ub chain formation. However, we observed rapid E3-dependent discharge of Ub from E2Ub suggestive of the presence of an unknown small molecule nucleophilic acceptor (FIG. 2c). Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that Ub was being quantitatively converted into two species with masses corresponding to condensation products with tris(hydroxymethyl)aminomethane (Tris) and glycerol (8639 and 8668 Da), both of which were present in our assay buffer at routinely employed concentrations of 50 mM and 65 mM, respectively. Due to the common hydroxy functionality within these nucleophiles, MYCBP2 appeared to have esterification activity (FIG. 9a, b). Activity was found to be dependent on C4520, consistent with it forming a thioester-linked E3Ub intermediate.sup.4,5 (FIG. 4c).

[0226] Unexpectedly, a MYCBP2 C4572S mutant retained activity yet formed a discrete mono Ub adduct that was resistant to thiolysis but reversible after base treatment (FIG. 4c, d and FIG. 6c).sup.5. A possibility was the mutated S4572 residue was contributing to catalysis via formation of a less transient (oxy)ester-linked intermediate between Ub and S4572 that retained the ability to modify substrate. We hypothesized that C4520 and C4572 were both catalytic residues that functioned in tandem by relaying Ub from one cysteine to the other through an intramolecular transthiolation reaction. To test this relay mechanism, we carried out gel-based thioester/ester trapping assays.sup.14 (FIG. 4e and FIG. 6d) and observed a thiol-sensitive Ub adduct on WT MYCBP2.sub.cat which was not observed with the C4520S mutant (FIG. 4e). Consistent with earlier experiments (FIG. 4c), C4572S underwent adduct formation that was thiol-resistant but base labile (FIG. 4e). Thus, if a transient thioester intermediate was being formed between Ub and C4520, then an unreactive C4572A mutant should stabilize it. Indeed, thiol-sensitive adduct formation was increased on a C4572A mutant relative to that of the wild type and was presumably linked via a thioester bond (FIG. 4e). Adduct formation was not detected with a C4520A/C4572S double mutant in support of it being linked to the C4520 residue (FIG. 4e). In the absence of direct demonstration of Cys-to-Cys Ub transfer we cannot formally exclude other possibilities. However, the existing data are consistent with the essential cysteines functioning in a relay mechanism. Mutational analysis and size exclusion chromatography-multi angle light scattering (SEC-MALS) of MYCBP2.sub.cat (FIG. 6e, f) suggests the proposed relay mechanism requires both essential cysteines be in the same molecule, consistent with an intramolecular relay mechanism.

[0227] In light of the observed esterification activity we attempted to identify the amino acid substrate of MYCBP2 by screening a panel of amino acids' where discharge activity was strikingly enhanced towards threonine. Product formation was dependent on C4520 (FIG. 7a, b and FIG. 8a-d). MS-based quantification indicated 10-fold selectivity for threonine over serine (FIG. 8e). Although a low level of lysine modification was observed, this was independent of MYCBP2.sub.cat.sup.5. Threonine selectivity (3-fold) was also maintained in a peptide context (FIG. 7c and FIG. 8f-h). Furthermore, basal ubiquitination of a lysine peptide was partially inhibited in the presence of MYCBP2.sub.cat underscoring its lack of lysine activity (FIG. 7c). Taken together, our experiments revealed that MYCBP2 is a novel class of E3 enzyme that operates via two essential cysteines, promotes Ub modification of hydroxyl groups, and esterifies threonine with Ub with selectivity over serine. As MYCBP2 uses a novel mechanism we termed it a RING-Cys-Relay (RCR) E3. We benchmarked the catalytic efficiency of MYCBP2 threonine esterification activity and found it to fall between that of well-characterized HECT and RBR E3 lysine aminolysis activity.sup.5,15 (FIG. 8i-k). E2 mutational analysis.sup.5,16-19, was in further support of MYCBP2.sub.cat being a novel class of E3 (FIG. 9a, b and Methods). To ascertain functional E2 partners, 17 E2s were tested but only UBE2D1, UBE2D3 and UBE2E1 demonstrated robust activity (FIG. 9c).

[0228] MYCBP2 promotes Wallerian axon degeneration through destabilization of Nicotinamide Mononucleotide Adenyltransferase (NMNAT2).sup.20. We next tested whether MYCBP2.sub.cat can ubiquitinate NMNAT2 by esterification in vitro (FIG. 7d). Despite containing 13 lysine residues, NMNAT2 underwent hydroxide labile but thiol resistant ubiquitination, demonstrating that MYCBP2 can target hydroxy residues within one of its putative substrates.sup.20. Cellular substrate recognition is mediated by a Skp1/Fbox45 substrate receptor co-complex that binds to a site 1940 residues N-terminal to the MYCBP2.sub.cat region (FIG. 7a).sup.21. NMNAT2 also undergoes palmitoylation and rapid axonal transport.sup.22 making reconstitution and cellular study of its ubiquitination extremely challenging. However, to establish whether MYCBP2.sub.cat retains non-lysine activity in cells we looked at its autoubiquitination after transient transfection into human embryonic kidney 293 cells. Base-labile (but thiol-resistant) ubiquitination was observed that was dependent on C4520 (FIG. 7e). This demonstrates that MYCBP2 can retain specificity for hydroxy amino acids in cells and that this activity remains dependent on the upstream catalytic residue we implicate with a Ub relay mechanism.

[0229] To further validate the RING-Cys-relay model and the serine/threonine activity we crystallized MYCBP2.sub.cat (residues 4378-4640) and solved a crystal structure to a resolution 1.75 (Table 1 and FIG. 10a-c). Residues 4388-4441 at the N-terminus correspond to the predicted cross-brace C3H2C3 RING domain (FIG. 4a and FIG. 10d). Following the RING domain is a long -helix (4447-4474) that leads into small helix-turn-helix motif (residues 4475-4500) (FIG. 11a and FIG. 10e) and further C-terminal is a structurally unprecedented globular domain that binds four Zn ions (residues 4501-4638) (FIG. 11b and FIG. 10f, g). Since this domain also contains the two essential catalytic residues we term it the Tandem Cysteine (TC) domain. Between the A2 strand and helix 3.sub.10A is an unstructured region (4519-4526) which projects out to the side of the core Zn-binding fold. The upstream C4520 residue resides within this unstructured region, which together with flanking residues, forms a mobile region we term the mediator loop. The Zn coordination configuration (C5HC7HC2) of the TC domain is semi-contiguous and does not adopt cross-brace architecture (FIG. 10c).

[0230] Crystal packing revealed that T4380, within the N-terminus of a symmetry-related MYCBP2.sub.cat molecule (T4380.sub.sym), was placed proximal to the esterification site where it forms a number of substrate-like interactions (FIG. 12a & b). Firstly, the -hydroxy group of T4380.sub.sym complements E4534 and H4583 and forms a potential triad (FIG. 12a). Thus the n-oxygen atom of T4380.sub.sym appears to be primed for deprotonation and nucleophilic attack. A catalytically productive electrophilic center is the C-terminus of Ub when thioester-linked to C4572. Even though this Ub molecule is absent in our structure, the C4572 sulfur atom is 3.8 away from the -oxygen atom of T4380.sub.sym. Thus, the structure appears to accurately reflect a catalytic intermediate poised to undergo threonine ubiquitination by esterification of its -hydroxy group (FIG. 12a). Furthermore, a sub-cluster of Phe residues (F4573, F4578 and F4586), proximal to the -methyl group of T4380.sub.sym, (FIGS. 12a and b) forms a well-defined hydrophobic pocket which the T4380.sub.sym -methyl group docks into and appears to be a positive selectivity determinant for the threonine side chain. The proposed roles of these residues were validated in threonine discharge assays (FIG. 12c). An H4583N mutation abolished activity consistent with a role as a general base. The H4583N mutant also underwent enhanced, thiol-sensitive, Ub adduct formation in accordance with the anticipated defect in rendering substrate nucleophiles reactive towards the C4572 thioester (FIG. 13a). Conservative perturbation of the phenylalanine cluster also markedly reduced threonine discharge activity (FIG. 5c). Perturbation of E4534 did not reduce activity hence its precise role remains unclear.

[0231] Conservation of RING domain binding to E2.sup.1618,23 permitted the modelling of an E2-RCR E3 ligase complex which was geometrically compatible with transthiolation between E2Ub and MYCBP2.sub.cat C4520 (FIG. 5d and FIG. 13b). To simulate the conformation required for subsequent Ub relay to C4572 we modelled the missing mediator loop residues with a GlyGly dipeptide thioester linked to C4520, representative of the Ub C-terminus that would be transferred via transthiolation with E2Ub if our relay model was valid (FIG. 13c-e). In support of this mechanism the carbonyl C atom of the Ub thioester could be positioned in proximity (3.3 ) of the C4572 sulfhydryl sulfur atom. To adopt this conformation minor twisting of a GlyGly motif (residues 4515-4516) at the tip of the A2 strand was necessary. Clashes were observed between mediator loop residues further C-terminal with R4533, E4534, N4580, H4583 and D4584 but these could be largely relieved by rotations of their sidechains into available space. As ordered loop residues 4527-4531 required a significant displacement to generate the model, we speculate that the mobile mediator loop region would span residues 4515-4531. As C4520, which resides within this mobile structural element, needs to be engaged by the E2 active site.sup.24 this might account for the uncharacterized E2 residue requirements. An explanation for the inability to render the S4520 mutant catalytic in earlier experiments is its dynamic nature and the absence of a general base which could suppress the pK.sub.a of the otherwise fully protonated S4520 side chain. Hence native C4520 catalytic activity is likely to arise from the sulfhydryl groups intrinsic nucleophilicity (FIG. 13a).

[0232] Although non-lysine ubiquitination has been reported.sup.25-27, a human E3 ligase that preferentially carries out this function has remained elusive. Our characterization of the novel RCR E3 ligase found in MYCBP2 suggests that ubiquitination by esterification is intrinsic to higher eukaryotes and may be a regulator of synapse development and axon degradation. Furthermore, non-protein Ub substrates (e.g. lipids, carbohydrates) have not been reported but considering the high esterification activity of MYCBP2 towards small molecule hydroxy compounds, this remains a possibility. It is not immediately clear why the proposed relay (FIG. 14a) mechanism would have evolved. However, transthiolation is a cofactor independent process providing a facile means to shuttle Ub throughout the ubiquitin system.sup.1. We speculate that on steric grounds, direct E2-E3 transthiolation with the structurally rigid and highly conserved E2 ubiquitin conjugating domain (Ubc).sup.28, and serine/threonine activity, are mutually exclusive at the esterification site and evolution of the mediator loop addresses this compatibility issue. Bioinformatic analysis revealed that MYCBP2 orthologues are found in virtually all animals but human homologues are unlikely to exist (Table 2).

[0233] Discussion

[0234] Stabilization of NMNAT2 through MYCBP2 inhibition is a promising therapeutic strategy for mitigating neuron damage after injury and administration of chemotherapeutics.sup.10,29,30 and slowing the progression of a range of neurodegenerative diseases including Alzheimer's and Parkinson's.sup.29. The delineation of this apparent Ub relay mechanism and the structural characterization of the molecular machinery responsible opens up new medical potential for treating a range of neurological conditions.

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