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Structural basis for RING-Cys-Relay E3 ligase activity and its role in axon integrity

Nature Chemical Biology volume 16, pages 1227–1236 (2020)Cite this article

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Abstract

MYCBP2 is a ubiquitin (Ub) E3 ligase (E3) that is essential for neurodevelopment and regulates axon maintenance. MYCBP2 transfers Ub to nonlysine substrates via a newly discovered RING-Cys-Relay (RCR) mechanism, where Ub is relayed from an upstream cysteine to a downstream substrate esterification site. The molecular bases for E2–E3 Ub transfer and Ub relay are unknown. Whether these activities are linked to the neural phenotypes is also unclear. We describe the crystal structure of a covalently trapped E2~Ub:MYCBP2 transfer intermediate revealing key structural rearrangements upon E2–E3 Ub transfer and Ub relay. Our data suggest that transfer to the dynamic upstream cysteine, whilst mitigating lysine activity, requires a closed-like E2~Ub conjugate with tempered reactivity, and Ub relay is facilitated by a helix–coil transition. Furthermore, neurodevelopmental defects and delayed injury-induced degeneration in RCR-defective knock-in mice suggest its requirement, and that of substrate esterification activity, for normal neural development and programmed axon degeneration.

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Fig. 1: Stabilization strategy and structure determination of the tetrahedral E2~Ub–MYCBP2 transfer intermediate.
Fig. 2: The RCR engages a closed-like E2~Ub conformation to facilitate exclusive transthiolation to cysteine within a transient helix.
Fig. 3: MYCBP2 RING–E2 interface involves a novel RING extension and specificity determinants.
Fig. 4: Alanine and proline scanning of the mediator loop.
Fig. 5: Neurodevelopmental defects and delayed axotomy-induced axon degeneration in Phr1C4629A/C4629A SCG neurons.
Fig. 6: Model for the central role of MYCBP2 RING-Cys-Relay activity in neural development and axon maintenance.

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Data availability

Protein Data Bank coordinates and structure factors for RCR E2–Ub have been deposited, with accession code 6T7F. All DNA constructs were verified by DNA sequencing and are available through the Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, reagents website (https://mrcppureagents.dundee.ac.uk/). The data that supports these findings, including raw mass spectrometry and microscopy data, are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank J.-F. Zhao, G. Gilmour, M. Mohammad, J. Varghese and A. Knebel of the MRC Protein Phosphorylation and Ubiquitylation Unit. We thank the European Synchrotron Radiation Facility. This work was funded by UK Medical Research Council (MC_UU_12016/8) and Biotechnology and Biological Sciences Research Council (BB/P003982/1). A.L. was funded by a Sir Henry Wellcome Postdoctoral Fellowship from the Wellcome Trust (210904/Z/18/Z). We also acknowledge pharmaceutical companies supporting the Division of Signal Transduction Therapy (Boehringer-Ingelheim, GlaxoSmithKline and Merck KGaA).

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Authors and Affiliations

  1. MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK

    Peter D. Mabbitt, Marc-André Déry, Adam J. Fletcher, Kuan-Chuan Pao, Nicola T. Wood & Satpal Virdee

  2. John van Geest Centre for Brain Repair, University of Cambridge, Cambridge, UK

    Andrea Loreto & Michael P. Coleman

  3. Division of Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK

    Mathew Stanley

  4. The Babraham Institute, Babraham Research Campus, Cambridge, UK

    Michael P. Coleman

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Contributions

S.V. conceived the research. P.D.M. designed and carried out experiments. M.P.C. and A.L. designed diaphragm imaging and neuron explant experiments, which were carried out by A.L. M.-A.D. coordinated animal breeding and obtained and characterized MEF samples by immunoblot and ABP gel-shift analysis. A.J.F. prepared MBP-tagged probe and carried out activity-based proteomics and data processing. M.S. and K-C.P. synthesized reagents. N.T.W. designed and made DNA constructs. S.V. and P.D.M. wrote the manuscript with input from other authors.

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Correspondence to Satpal Virdee.

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Extended data

Extended Data Fig. 1 Structure determination and representative views of the E2~Ub-MYCBP2 transfer intermediate.

a, A crosslinked transfer complex was prepared by incubating ABP (200 μM) and E3 (MYCBP2 RCR) (50 μM) for 4 h at 30 °C. Complex formation was assessed by SDS-PAGE (left). The stabilized transfer complex was purified by size-exclusion chromatography (right). b, Domain architecture of MYCBP2 including the catalytic RCR machinery. c, The RCR machinery is in stick representation; RING domain (blue), linker helix (purple), helix-turn-helix (green), TC domain (orange), mediator loop (brown), E2 (mauve) and Ub (gray). The mesh represents a simulated annealing composite omit 2|mFo|-|dFc| electron density map contoured at 1.0 σ. d, As above except the mesh represents the experimental 2|Fo|−|Fc| electron density map contoured at 1.0 σ. e, Close-up view of the three-way crosslink between E2 C85, RCR C4520 and the Ub carboxy terminus. The mesh represents a simulated annealing composite omit 2|mFo|-|dFc| electron density map contoured at 1.0 σ carved around the mediator loop G4515 – D4529, E2 residues C85, and the crosslink. f, As e except the mesh represents the experimental 2|Fo|−|Fc| electron density map contoured at 1.0 σ. g, Close-up view of the Ub-esterification site, in the apo-structure the esterification site is occupied by a Thr residue due to crystal packing. The mesh represents a simulated annealing composite omit 2|mFo|-|dFc| electron density map contoured at 1.0 σ carved around E4534, F4570 – F4573, H4583, F4586 and an ordered water molecule. h, As g except the mesh represents the experimental 2|Fo|−|Fc| electron density map contoured at 1.0 σ.

Extended Data Fig. 2 Superposition of apo-MYCBP2 (PDB 5O6C), E2~Ub bound-MYCBP2 and protein crystal stability.

a, Apo-MYCBP2 residues Asn4379-His4638 were aligned with bound-MYCBP2 residues Asp4387-Asn4636. The RCR, E2 and Ub are in cartoon representation: Coloring (apo-MYCBP2/bound-MYCBP2) zinc ions (light gray/gray), RING domain (light blue/blue), linker helix (pink/purple), helix-turn-helix (light green/green) and tandem cysteine domain (yellow/orange). In the apo structure, 8 residues from the mediator loop are disordered and are represented by a black dashed line. The E2 is colored mauve and Ub is gray. MYCBP2 Residues Ala4518, Gly4527, Cys4520 and Cys4572 are in ball and stick representation. In the bound-MYCBP2 structure E2 residue C85 and the engineered crosslinker are in ball and stick representation. b, Closeup of TC domains, in the E2~Ub bound structure the eight mediator loop residues, that were disordered in the apo structure, adopt a helical conformation in the E2~Ub:RCR transfer complex. c, Closeup of RING domains, in the E2~Ub bound structure the RING domain has twisted towards the linker-helix this results in a 3.0 and 4.1 Å shift of Zn2+ 1 and Zn2+ 2, respectively. d, Representative view of the E2~Ub-MYCBP2 transfer intermediate. e, Representative view of the E2~Ub-MYCBP2 transfer intermediate colored by B-factors (blue thin-cartoon lowest B-factors to red thick-cartoon highest B-factors) indicates that ubiquitin and the mediator loop are the most disordered components of the complex. f, SDS-PAGE gel of purified ABP-MYCBP2 complex and ABP-MYCBP2 complex recovered from a crystal drop containing the productive conditions (0.85 M sodium citrate, 100 mM sodium chloride, 100 mM Tris-HCl pH 8.0). The ABP-labelled transfer complex is stable during crystallization. Experiment was repeated twice with similar results.

Extended Data Fig. 3 Data collection and refinement statistics.

Crystallographic data were collected from a single crystal.

Extended Data Fig. 4 Mutational analysis of E2-E3 Ub transfer was determined by single-turnover isopeptide formation in the context of an RCR C4520K mutant.

To decouple E2-E3 Ub transfer (what our structure is reflective of) from subsequent relay and substrate esterification, we devised a robust assay that results in transfer of Ub from E2 to a dead-end product. To achieve this, we mutated the upstream Cys4520 residue to a lysine (Cys4520Lys). We found Ub was transferred to the lysine forming a stable isopeptide adduct. a, Activity was efficient and only E2’s previously shown to support MYCBP2 E3 activity supported isopeptide bond formation25. Experiment was repeated twice with similar results. b, A subset of hallmark interactions involved in closed E2~Ub stabilization are maintained (coloring as Fig. 2a). c, Representative replicate from single-turnover E2~Ub isopeptide assay used for quantification presented in Fig. 2c (also see Methods). d, The majority of mutants were also tested with wild type MYCBP2 in multiple turnover threonine-discharge assays (Extended Data Fig. 5), which yielded similar activity profiles. However, one exception was with E2 mutation D117A. Whereas D117A was fully active with MYCBP2 WT, it was completely inactive in C4520K isopeptide formation. This is reflective of this residue having a lysine-specific role that is redundant with native MYCBP2 E2-E3 transthiolation. e-h, Kinetic analysis for E2 active site residues (n = 3 independent experiments performed with identical purified proteins). Blue squares and black circles correspond to experiments where E3 was added or withheld, respectively. E2 reloading was blocked by addition of E1 inhibitor and depletion of the E2~Ub species was quantified. i, Observed rates of single-turnover Ub discharge (kobs) from experiments e-h. Observed rate constants were obtained from the one-phase exponential association equation using the routine within Graphpad Prism. Assays were carried out in triplicate using identical purified proteins. The 95 % confidence intervals for kobs are presented. ND* indicates that rates of Ub discharge in the presence of E3 were indistinguishable from background E2~Ub hydrolysis. Observed rates for native transthiolation activity determined from experiments presented in Extended Data Fig. 5 are also tabulated.

Source data

Extended Data Fig. 5 Mutational assessment of native MYCBP2 E2-E3 transthiolation activity and demonstration of attenuated E2~Ub reactivity.

a, Proposed intramolecular role for Ub I36 in maintaining a closed-like E2~Ub conformation. The interaction between Ub I36 and L71 is maintained in the “closed-like” E2~Ub conformation. Superposition of the RCR E2~Ub and RNF4 E2~Ub (PDB 4AP4) complexes. The gap between Ub I36 and L71 has decreased by 0.7 Å in the RCR complex. The RCR, E2 and Ub are in cartoon representation with select residues in ball and stick representation: Ub I36, L71, R72 and the engineered linker (gray), E2 C85 (mauve), RCR K4441 (blue) and C4520 (orange). The RNF4 E2~Ub complex is in cartoon representation with select residues in stick representation: Ub I36, L71, R72 (pink), R181 (purple). b, For the selected mutants, MYCBP2 activity was assessed using single turnover E2~Ub discharge assays mediated by the presence of wild type MYCBP2 and threonine (50 mM). c, Single turnover E2~Ub discharge assay mediated by the presence of wild type MYCBP2 and threonine (50 mM) for E2 Asn114Ala and Asn114Gln mutants. Experiment repeated twice with similar results. d, Quantification for selected mutants, (n = 3-4 independent experiments performed with identical purified proteins). e-i native single-turnover WT MYCBP2 and threonine dependent E2~Ub discharge assay. Observed rate constants tabulated in Extended Data Fig. 4 were obtained from the one-phase exponential association equation using the routine within Graphpad Prism. Blue squares and black circles correspond to experiments where E3 was added or withheld, respectively. (n = 3 independent experiments performed with identical purified proteins) j, Quantification of lysine discharge assay in the presence of a transthiolation-defective RCR A4520 mutant or the canonical RING E3 RNF4 (n = 3 independent experiments performed with identical purified proteins). Although efficient lysine discharge was observed with the RCR C4520K mutant, the structural context of this acceptor lysine templates the reaction which can increase the reaction rate by multiple orders of magnitude, thereby reconciling the lack of activity towards free lysine which would be diffusion-limited39,60.

Source data

Extended Data Fig. 6 Consideration of crystal packing effects on adoption of the closed-like E2~Ub conformation and assessment of their significance in solution.

a, Interface between Ub (gray), E2 (mauve) RCR (TC domain, orange; linker helix, purple; helix-turn-helix, green; RING, blue) and two symmetry-related RCR molecules (cyan and light-blue). The side chain of Ub E18 and the first 7 residues of the RCR construct are disordered. b, Closeup of the interface between Ub, E2 and symmetry related E3. Ub K48 and R54 are in close proximity to symmetry related MYCBP2 H4599. c, Superposition of the RCR E2~Ub and RNF4 E2~Ub (PDB 4AP4) complexes highlighting the position of Ub Lys48. The altered Ub packing in the RCR complex (E2, mauve: Ub, gray) results in Ub Lys48 shifting away from E2 D42, relative to the RNF4 complex (E2 wheat; Ub, pink). d, Single-turnover E2~Ub discharge and single-turnover isopeptide formation for the indicated Ub variants. The Ub E18R, A46D, R54E, and N60A mutants were discharged similarly to WT Ub, suggesting that the interface with a second copy of MYCBP2 does not exist in solution or is not required for activity. The Ub K48A and K48E mutants reduced activity, which would not be expected if the interaction with H4599 residue within the symmetry related MYCBP2 contributed to prevention of a canonical closed-conformation in solution. As K48 is proximal to the E2 it seems more likely that it intrinsically contributes to closed and closed-like activation. e, Quantification of single-turnover E2~Ub discharge (n=5 independent experiments performed with identical purified proteins) and single-turnover isopeptide formation (n=3 independent experiments performed with identical purified proteins).

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Extended Data Fig. 7 Representative electron density centered on key E2~Ub-RING interfaces and comparison with E2~Ub-RING1 interfaces.

a, The E2-Ub interface is centred on E2 L104, Ub L8, I44, H68, and V70. E2 (mauve) and Ub (gray) are shown as sticks, the RCR RING domain (blue) is shown as a cartoon. For clarity RCR regions C-terminal of the RING are not shown. The mesh represents a simulated annealing composite omit 2|mFo|-|dFc| electron density map contoured at 1.0 σ. b, As a except the mesh represents the experimental 2|Fo|−|Fc| electron density map contoured at 1.0 σ. c, View of the RING-E2 interface, RCR RING (blue), E2 (mauve) and Ub (gray) are shown as sticks. The RCR equivalent to the ‘linchpin’ residue (K4441) and the functionally important RING extension residue (L4426) are labelled. The mesh represents a simulated annealing composite omit 2|mFo|-|dFc| electron density map contoured at 1.0 σ. d, As c except the mesh represents the experimental 2|Fo|−|Fc| electron density map contoured at 1.0 σ. e, View of the RING-E2 interface focused on RCR residues L4392, F4394 and the interaction between E2 S94 and RING P4438. The mesh represents a simulated annealing composite omit 2|mFo|-|dFc| electron density map contoured at 1.0 σ. f, As e except the mesh represents the experimental 2|Fo|−|Fc| electron density map contoured at 1.0 σ. g, Superposition of MYCBP2 E2~Ub and HOIP E2~Ub complexes (PDB 5EDV) highlighting the shift in E2 binding site for HOIP RING1 (E2s were superposed). MYCBP2 complex RING (blue), E2 (mauve), zinc ions (gray); HOIP complex RING1 (cyan), E2 (wheat), zinc (light gray). h, Superposition of MYCBP2 E2~Ub and HHARI E2~Ub complexes (PDB 5UDH) highlighting a loop insertion in HHARI RING1 that prevents the closed E2~Ub conformation. HHARI His234 would sterically clash with Ub in the “closed” E2~Ub, and similarly, His234 is incompatible with the “closed-like” E2~Ub adopted in the RCR complex. MYCBP2 complex RING (blue), E2 (mauve), Ub (gray), zinc (gray); HHARI complex RING1 (light blue), E2 (wheat) and zinc (light gray).

Extended Data Fig. 8 The RCR-helix-turn-helix motif prevents binding of an open E2~Ub conjugate, UBE2L3 activity cannot be imparted by Lys96Ser mutation, and further mediator loop analysis.

a, Superposition of ‘open’ conformation E2~Ub from a HECT E2~Ub complex (PDB 3JVZ) with the RCR E2~Ub complex. The open conformation in the HECT E2~Ub complex is incompatible with the observed RCR conformation as Ub is sterically blocked by the helix-turn-helix motif. HECT E2 (pink) and Ub (light gray) are displayed in cartoon representation. b, Despite introduction of the corresponding lysine residue into UBE2D3 abolishing its activity, we could not impart UBE2L3 activity by substitution of Lys96 to Ser, as found in UBE2D3. Blue squares and black circles correspond to experiments where E3 was added or withheld, respectively (n=3 independent experiments performed with identical purified proteins). c, The mediator loop has high sequence conservation across orthologues. The deletions in Drosophila and C. elegans relative to human MYCBP2 imply the Ub relay process is highly plastic. d, Deletion of mediator loop residue A4518 substantially impairs E2-E3 transthiolation and Ub relay. Experiment was repeated twice with similar results.

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Extended Data Fig. 9 Relaxation of the E2 Ser94-RING Pro4438 interaction is crystallographically observed for RING-linked E3s (RBRs and RCR) relative to canonical RINGs.

Interestingly, the Ser94-Pro4438 H-bond is highly conserved in solved E2-RING structures but for canonical RING E3s an idealized geometry is observed (2.3-3.0 Å). For RBR E3s that undergo transthiolation, this H-bond distance is comparable to that observed for the RCR being ~0.4 Å longer. Thus it would appear that relaxation of this H-bond may be a hallmark of RING-linked E3s but the mechanistic basis for this is not clear. a, Distances between E2 Ser94 (gamma oxygen) and E3 Pro (carbonyl oxygen) for MYCBP2 relative to canonical RING E3s. b, Alignment of the C-terminal portion of the RING including the 7th and 8th zinc coordinating residues. The conserved proline that interacts with E2 serine 94 is shown in blue. Zinc coordinating residues are shown in red. The linchpin residue location is indicated with an asterisk. c, Distances between E2 Ser94 (gamma oxygen), or E2 Lys96 (epsilon amino nitrogen), and E3 Pro (carbonyl oxygen) for MYCBP2 relative to RBR E3s. d, Alignment of the c-terminal portion of the RING including the 7th and 8th zinc coordinating residues. The conserved proline that interacts with E2 serine 94 in MYCBP2 shown in blue. Zinc coordinating residues are shown in red.

Extended Data Fig. 10 Further characterization of Phr1C4629A/C4629A mouse line.

a, Expression levels of Phr1/MYCBP2 in Phr1+/+ (WT), Phr1C4629A/+ (HET) and Phr1C4629A/C4629A (HOM) mouse embryonic fibroblasts (MEFs) and neuroblastoma SH-SY5Y cells (CRISPR KO and WT) (left). An alternative in-house antibody25 was also used to assess Phr1 expression levels across genotypes (right). b, Experimental work-flow used to generate activity-based proteomic data presented in Fig. 5b. c, Extracted proteomes from MEF with indicated genotypes were treated with a maltose-binding protein (MBP)-tagged activity-based probe. MBP tagging was necessary to discern a gel shift upon MYCBP2/Phr1 (0.5 MDa) labelling. ABP labelling was selectively abolished in Phr1C4629A/C4629A MEFs consistent with the E3 ligase activity being disrupted. This experiment was carried out twice with similar results. d, NMNAT2 with a C-terminal HA-tag was transiently transfected into MEFs representing all three genotypes. This experiment was carried out once. e, The number of secondary branches (gray) between the primary branches (black) crossing the red dotted line were counted. Quantification of secondary axonal branches of the right phrenic nerve; n number per genotype are indicated in the figure (mean ±SEM; Kruskal-Wallis test followed by Dunn’s multiple comparison test). Asterisks indicate: * P ≤ 0.05.

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Unprocessed SDS–PAGE gel.

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Unprocessed SDS–PAGE gels.

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Unprocessed SDS–PAGE gels.

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Unprocessed SDS–PAGE gels.

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Unprocessed immunoblots.

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Mabbitt, P.D., Loreto, A., Déry, MA. et al. Structural basis for RING-Cys-Relay E3 ligase activity and its role in axon integrity. Nat Chem Biol 16, 1227–1236 (2020). https://doi.org/10.1038/s41589-020-0598-6

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