Abstract
Mechanisms of protein recognition have been extensively studied for single-domain proteins1, but are less well characterized for dynamic multidomain systems. Ubiquitin chains represent a biologically important multidomain system that requires recognition by structurally diverse ubiquitin-interacting proteins2,3. Ubiquitin chain conformations in isolation are often different from conformations observed in ubiquitin-interacting protein complexes, indicating either great dynamic flexibility or extensive chain remodelling upon binding. Using single-molecule fluorescence resonance energy transfer, we show that Lys 63-, Lys 48- and Met 1-linked diubiquitin exist in several distinct conformational states in solution. Lys 63- and Met 1-linked diubiquitin adopt extended ‘open’ and more compact ‘closed’ conformations, and ubiquitin-binding domains and deubiquitinases (DUBs) select pre-existing conformations. By contrast, Lys 48-linked diubiquitin adopts predominantly compact conformations. DUBs directly recognize existing conformations, but may also remodel ubiquitin chains to hydrolyse the isopeptide bond. Disruption of the Lys 48–diubiquitin interface changes conformational dynamics and affects DUB activity. Hence, conformational equilibria in ubiquitin chains provide an additional layer of regulation in the ubiquitin system, and distinct conformations observed in differently linked polyubiquitin may contribute to the specificity of ubiquitin-interacting proteins.
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Acknowledgements
We would like to thank members of the Komander, Jackson and Klenerman laboratories, R. Williams, S. Freund, C. Johnson, S. McLaughlin and A. Fersht for discussions. Work in the Komander laboratory is supported by the Medical Research Council (U105192732) and the EMBO Young Investigator Program. G.B. and S.I. were supported by the BBSRC, the Newton Trust and an EMBO YIP small grant to D.Ko. Work in the Klenerman laboratory is supported by EPSRC.
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Y.Y., G.B. and M.H.H. designed and performed the experiments, including single-molecule measurements, and analysed the data. Y.Y. and G.B. generated all proteins used in this study. Y.Y. performed kinetic experiments. M.H.H. and S.I. built the PAX instrument and A.A.Z. programmed the control for PAX measurements. S.I. performed single molecule experiments and contributed to data analysis. M.J.R.-R. and A.O. performed lifetime measurements. D.Kl., S.E.J. and D.Ko. directed the research and analysed the results. All authors contributed to the writing of the manuscript.
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Ye, Y., Blaser, G., Horrocks, M. et al. Ubiquitin chain conformation regulates recognition and activity of interacting proteins. Nature 492, 266–270 (2012). https://doi.org/10.1038/nature11722
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DOI: https://doi.org/10.1038/nature11722
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Clive Bagshaw
Ye et al. use single molecule FRET and TCCD measurements to characterize populations of protein states that otherwise could be masked in ensemble measurements. Unfortunately, many of the quantitative aspects of their model are reserved for the supplementary section which makes for difficult reading, particularly as the scheme in Fig 4f is not labeled with equilibrium constants, K1, K2 etc for direct cross reference. As far as I understand, from the supplementary section with regards to USP21i binding to K48NC:
K1 = L/H K2 = L . U/LU K3 = N . U/NU
Let me add for completeness
K4 = N/L K5 = NU/LU
where H, L, and N represent the concentration of high-, low- and non-FRET forms of K48NC respectively, U is the concentration of free USP21i and LU and NU the concentration of the bound complexes.
K1 = 0.1 based on Fig 2a. The values of K2 and K3 are not defined explicitly, but the authors imply they are 16 nM and 4nM respectively based on the data in Fig S9b. In the main text they state ?Estimation of binding constants for the low and non-FRET species indicated a slightly higher affinity of USP21i for the open non-FRET conformation (Supplementary Fig. 9).? The Kd estimates in Fig. S9b clearly have a large error and a more conservative conclusion would be that both apparent Kd?s are < 20 nM. The ensemble data of Fig. S7a appears to have better precision, but here the K48NC concentration (700 nM) exceeds the apparent Kd. Thus, with a 16 nM Kd, the expected profile would be quadratic (with a breakpoint at 700 nM) and not hyperbolic, so there is an inconsistency here . However, there is a more important point. The equilibria of Fig. 4f are coupled and hence the observed Kd ?s for binding are a function of more than one equilibrium constant e.g. if the apparent Kd for U binding to L was 16 nM, the actual value of K2 would be around 1.6 nM since it has to ?pull? the unfavorable H to L transition over (K1=0.1). Furthermore, K5 is defined by the ratio of NU and LU at saturating U and has a value of around 0.6 based on Fig S9b inset. In fact, because of coupling, the apparent Kd?s in Fig S9b should be the same, as LU and NU rise in a constant ratio defined by K5. The value of K5 is more robustly defined than the estimates of K2 and K3 because several data points contribute to its measurement. We can also conclude that K4 < 0.5 based on the statement that populations of FRET states can be defined to within 5% and no N is detected in the absence of U. If we assume K2 = 1.6 nM, then from thermodynamic balance K3=K2.K4/K5 and therefore K3 = or < 1.3 nM. Regardless of the value of K2, the general conclusion is that K2 and K3 could be similar (since K4 and K5 could be similar) or K3 < K2 if K4 <K5. There is no information in the data presented to say more. In any event, even if these equilibria could be defined exactly, they contain no information about whether the predominant flux is through ?conformational selection? or ?remodeling? routes since the overall energetics of these pathways are identical. Kinetic measurements are required to address this.
Clive R. Bagshaw