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Linkage-specific ubiquitin chain formation depends on a lysine hydrocarbon ruler

Abstract

Virtually all aspects of cell biology are regulated by a ubiquitin code where distinct ubiquitin chain architectures guide the binding events and itineraries of modified substrates. Various combinations of E2 and E3 enzymes accomplish chain formation by forging isopeptide bonds between the C terminus of their transiently linked donor ubiquitin and a specific nucleophilic amino acid on the acceptor ubiquitin, yet it is unknown whether the fundamental feature of most acceptors—the lysine side chain—affects catalysis. Here, use of synthetic ubiquitins with non-natural acceptor site replacements reveals that the aliphatic side chain specifying reactive amine geometry is a determinant of the ubiquitin code, through unanticipated and complex reliance of many distinct ubiquitin-carrying enzymes on a canonical acceptor lysine.

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Fig. 1: UBE2N~UB/UBE2V1/RNF4 RING E3 complex reacts preferentially with free amino acids harboring amine acceptors and various side-chain hydrocarbon linkers.
Fig. 2: K48 and K63 chain-forming E2s show strong preferences for a native lysine acceptor on UB.
Fig. 3: K63 chain-forming HECT E3 ligases show strong preferences for a native lysine acceptor on UB.
Fig. 4: The location of lysine analogs on acceptor UB impacts the distribution of di-UB chain linkage types generated by the E2 enzyme UBE2D3.
Fig. 5: Molecular dynamics (MD) simulations reveal pleiotropic structural effects on UBs harboring lysine analogs.

Data availability

All raw gels are included in source data files. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD021286. Source data are provided with this paper.

Code availability

ROSETTA software can be downloaded from www.rosettacommons.org and is available free to academic users.

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Acknowledgements

This study is dedicated the memory of our inspiring mentor, colleague and beloved friend, Huib Ovaa, whom we miss dearly. We thank J.R. Prabu, J. Kellermann, S. von Gronau, D. Scott, S. Uebel, S. Pettera, V. Sanchez, K. Baek, D. Horn-Ghetko and S. Kostrhon for assistance, reagents and helpful discussions. We also thank C. Talavera-Ormeño and P. Hekking for assistance with peptide synthesis. B.A.S. has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant no. 789016-NEDD8Activate), and from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation—SCHU 3196/1-1). B.A.S. and M.M. are supported by the Max Planck Society. Also, N.P., D.H., N.B. and G.K. were supported by a grant from the National Institutes of Health (R15GM117555-02). G.J.v.d.H.v.N was supported by grants from NWO (VIDI and Off-Road). H.O. was supported by a VICI grant from the Netherlands Foundation for Scientific Research (NWO). Work by M.S. and M.J.B. was performed within the framework of SFB 1035 (German Research Foundation DFG, Sonderforschungsbereich 1035, no. 201302640, project Z01).

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Contributions

Syntheses of UB analogs were designed and executed by G.J.v.d.H.v.N. J.L. performed all biochemical assays. Kinetics experiments were carried out by G.K., N.P., D.H. and N.B. MS experiments were designed and conducted by M.M., F.M.H. and O.K. MD simulations were performed by V.H.T. and J.S.H. NMR was carried out by M.J.B. and M.S. The manuscript was prepared by J.L., D.T.K., G.J.v.d.H.v.N., G.K., H.O. and B.A.S., with input from all authors. The project was supervised by B.A.S., G.K., H.O. and D.T.K.

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Correspondence to Gary Kleiger or Huib Ovaa or Brenda A. Schulman.

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H.O. was a shareholder of UbiqBio. All other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 UBE2N~UB/UBE2V1/RNF4 RING E3 complex reacts preferentially with free amino acids harboring amine acceptors and various side-chain.

a, Fluorescence scan of SDS-PAGE gels demonstrating the discharge of labeled UB (UB*) to L-lysine compared with the absence of amino acid acceptor using wild-type UBE2N. Electrophoresis was performed under both reducing and non-reducing conditions to differentiate thioester bonded complexes from isopeptide bonded E2-donor UB ones. b, Time-courses of fluorescent UB discharge from UBE2N K92R~UB/UBE2V1/RNF4 RING E3 to the indicated amino acids, normalized to starting signal of fluorescent UB thioester-bonded to UBE2N. For all, N=2 independent experiments. For samples derived from the same experiment, gels were processed in parallel. Source data

Extended Data Fig. 2 K48 and K63 chain-forming E2s equally discharge to K63UBC1-C5 and K48UBC1-C5 acceptors respectively.

a, Di-UB formed by K48 UB chain-forming E2 UBE2G1 with K63UBC1-C5 acceptors in the absence (left) or presence (right) of neddylated CRL4 (N8CRL4). b, Di-UB formed by the K63 UB chain-forming E2 UBE2N/UBE2V1 complex with the K48UBC1-C5 acceptors in the absence (left) or presence (right) of the E3 RNF4 RING domain. For all plots graphs, di-UB levels (μMol) represent the final time-points from the reactions (Source Data Extended Data Fig. 2), N=2 independent experiments. For samples derived from the same experiment, gels were processed in parallel. Source data

Extended Data Fig. 3 1D and 2D proton NMR spectra for synthetic UBC4, recombinant UB, and K48UBC5 are highly superimposabe.

a, 2D Nuclear Overhauser effect spectroscopy (NOESY) recorded at 298 K and 1D spectra (b) for UBC4 (blue), recombinant UB (C4 Bio; pink) and K48UBC5 (purple). The 2D NOESY spectra show NOE interactions between amide protons (x-axis) and amino acid side-chain protons (y-axis), whereas the 1D spectra show signals from methyl protons (-0.5–1.0 ppm), Cα-protons (3.5 – 6 ppm) and amide protons (6 – 10 ppm). The signal from water is at 4.7 ppm1. The observed dispersion of signals demonstrates that all three UBs are well folded, while the comparable overlays indicate that the UBs share a highly similar fold. c, same as (a), except data were recorded at 310 K. d, same as (b), except at 310 K.

Extended Data Fig. 4 Lack of preference for a native lysine on acceptor UBs for the K11 chain-forming E2 UBE2S.

a, Cartoon of experimental scheme monitoring the reactivity of E2s with K11UBC1-C5 acceptors (UBA). Plot of the discharge of labeled UB (UB*) from UBE2S_IsoT to K11RUB, UBC4 Bio or K11UBC1-5 acceptors (left) and representative fluorescence scans of SDS-PAGE gels representing the primary data (right). b, same as (a), except in the presence of the E3 APC/C. c, same as (a), except in the presence of K11UBC2 or UBC4 Bio acceptors or the same harboring an E34D mutation. d, same as (b), except in the presence of K48RUB, UBC4 Bio or K48UBC1-5 acceptors. e, same as (a), except with UBE2N/UBE2V1. f, same as (b), except with UBE2N/UBE2V1 and the RING domain from the E3 RNF4. For all plots graphs, di-UB levels (μMol) represent the final time-points from the reactions (N=2 independent experiments). For samples derived from the same experiment, gels were processed in parallel. Source data

Extended Data Fig. 5 The location of lysine analogues on acceptor UB impacts the distribution of di-UB chain linkage types generated by the E2 enzyme UBE2D3.

a, Plots showing the relative changes in UBE2D3 generated di-UB chain linkages in the presence of the RING domain from the E3 RNF4, comparing products containing K11UBC5 or UBC4 acceptors (N=3 technical replicates). ND, not determined. b, Same as (a), except with K63UBC5 acceptor.

Extended Data Fig. 6 Molecular Dynamics simulations reveal pleiotropic structural effects on UBs harboring lysine analogs.

a, Plot showing the degree of various side-chain rotamer interconversions for K11UBC5, K48UBC5, or K63UBC5 versus UBC4 acceptor UBs. Bins are divided by 120° intervals. b, Distribution of the distances between lysine acceptor amine and Cα atoms for UBC4 versus UBC5 during 25 ns MD simulations (N=3 independent experiments) for the UBE2N~UB/UBE2V1/acceptor UB multi-subunit complex. Bins are divided by 10° intervals. c, Plot showing the dynamics of φ and ψ main-chain torsion angles for UBC4 or UBC5 acceptors in the UBE2N~UB/UBE2V1/acceptor UB multi-subunit complex. Bins are divided by 10° intervals. d, Plot showing the dynamics of the side-chain rotamers for UBC4 or UBC5 acceptors in the UBE2N~UB/UBE2V1/acceptor UB multi-subunit complex. Bins are divided by 120° intervals. e, Rose plot showing the distance and angle of the amine acceptor of UBC4 relative to the active-site during 25 ns MD simulations of the UBE2N~UB/UBE2V1/acceptor UB multi-subunit complex (N=3 independent experiments). Golden star indicated starting position. f, same as (e), but with K63UBC5 (g) RMSD of gate loop during the trajectory for UBC4. h, same as (g), except with K63UBC5.

Extended Data Fig. 7 Di-UB chain formation reactions with UBC4 or UBC5 acceptors produce distinct results depending on the identity of the ubiquitin carrying enzyme.

a, Graph of the reactions velocities as a function of pH. performed in the presence of wild-type UBE2N/UBE2V1, radiolabeled K63R donor UB and either K63UBC4 or K63UBC5 acceptor UBs (b) same as (a), except with K92R UBE2N/UBE2V1. c, Graph of the reaction velocities as a function of the acceptor UB concentration for UBE2N/UBE2V1. The inset shows the fit of the data to the model for reactions containing K63UBC5 acceptor UB since the magnitude of the velocities is far less than reactions containing UBC4. d, same as (c) except in the presence of the RING domain of RNF4. e, Graph of the reaction velocities performed as a function of pH, in the presence of UBE2R2 and radiolabeled K48R donor UB and either UBC4 or K48UBC5 acceptors. f, Graph of the reaction velocities as a function of the acceptor UB concentration and their fit to the Michaelis-Menten model for UBE2R2. g, same as (f), except in the presence of the yeast HECT E3 Rsp5p. N=2 independent experiments. Samples derive from the same experiment, gels were processed parallelly. Source data

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Liwocha, J., Krist, D.T., van der Heden van Noort, G.J. et al. Linkage-specific ubiquitin chain formation depends on a lysine hydrocarbon ruler. Nat Chem Biol 17, 272–279 (2021). https://doi.org/10.1038/s41589-020-00696-0

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