Allosteric switch regulates protein–protein binding through collective motion

Abstract

Many biological processes depend on allosteric communication between different parts of a protein, but the role of internal protein motion in propagating signals through the structure remains largely unknown. Through an experimental and computational analysis of the ground state dynamics in ubiquitin, we identify a collective global motion that is specifically linked to a conformational switch distant from the binding interface. This allosteric coupling is also present in crystal structures and is found to facilitate multispecificity, particularly binding to the ubiquitin-specific protease (USP) family of deubiquitinases. The collective motion that enables this allosteric communication does not affect binding through localized changes but, instead, depends on expansion and contraction of the entire protein domain. The characterization of these collective motions represents a promising avenue for finding and manipulating allosteric networks.allostery | protein dynamics | concerted motion | relaxation dispersion | nuclear magnetic resonance I ntermolecular interactions are one of the key mechanisms by which proteins mediate their biological functions. For many proteins, these interactions are enhanced or suppressed by allosteric networks that couple distant regions together (1). The mechanisms by which these networks function are just starting to be understood (2-4), and many of the important details have yet to be uncovered. In particular, the role of intrinsic protein motion and kinetics remains particularly poorly characterized. A number of structural ensembles representing ubiquitin motion have been recently proposed (5-9). Additionally, it has been suggested that through motion at the binding interface, its free state visits the same conformations found in complex with its many binding partners (5, 10). However, it remains an unanswered question if the dynamics that enable this multispecificity are only clustered around the canonical binding interface or whether this motion is allosterically coupled to the rest of the protein, especially given the presence of motion at distal sites (11). ResultsTo answer this question and to provide a detailed structural picture of the underlying mechanism, we applied recently developed high-power relaxation dispersion (RD) experiments (12, 13) to both the backbone amide proton ( 1 H N ) and nitrogen ( 15 N) nuclei of ubiquitin. This survey yielded a nearly twofold increase in the number of nuclei where RD had been previously observed (11)(12)(13)(14) (from 17 to 31; Fig. 1A and Fig. S1). When fit individually, the full set of backbone and side-chain nuclei shows a consistent time scale of motion [exchange lifetime (τ ex ) = 55 μs; Fig. 1B]. Furthermore, the nuclei showing exchange are spread throughout the structure (Fig. 1C). Put together, these data suggest that the motions are not independent but share a common molecular mechanism.To determine whether the RD data could be modeled using a single collective motion, we developed a computational method to take a set of molecu...

A Small Molecule Causes a Population Shift in the Conformational Landscape of an Intrinsically Disordered Protein

“…However, $R_{2,0}^{\mathit{\text{app}}}$ cannot be directly compared to a conventional R 2,0 relaxation rate. Our ability to sample the contributions of R ex for 1 H N nuclei was enhanced through the use of large amplitude ω eff RD methods 45–48 , which overcame limitations associated with measurements of either 13 C or 15 N relaxation (Figure S2). Application of large amplitude ω eff 1 H N RD methods in the future will allow detection of low microsecond motions within other IDPs, IDRs, and unfolded proteins ( e.g.…”

Flexibility and Design: Conformational Heterogeneity along the Evolutionary Trajectory of a Redesigned Ubiquitin

“…Residues Asp52 and Gly53 have been previously identified as a structural switch in ubiquitin that undergoes a discrete peptide flip (Huang et al, 2011) that exchanges on the microsecond timescale (Sidhu et al, 2011; Smith et al, 2016). The original crystal structure of wild-type ubiquitin (1ubq), a standard in computational benchmarking studies, shows the peptide in the “NH-out” state with the Asp52 carbonyl making a hydrogen bond to the backbone of the α1-helix starting residue Glu24.…”

“…Moreover, this flip is thought to be a key structural switch between different states of ubiquitin. The flip state of the peptide can be predicted from the backbone coordinates of other residues clear across the protein (Smith et al, 2016). …”

“…In our final model, the peptide flip is modeled in both states (Figure 6b) for both structures at occupancies of 60–70% for the major NH-in and 30–40% for the NH-out. Interestingly, the NH-in state has been implicated in the binding mode of ubiquitin to the USP class of deubiquitinases which includes the target, USP7 (Smith et al, 2016). While the population of the NH-in state may have been increased relative to WT, it remains surprising that both peptide flip states are observed in our structures given the strong association of this peptide flip with the binding of USP7.…”

Combining experimental and simulation data of molecular processes via augmented Markov models

“…This finding prompted us to analyze the molecular kinetics of the AMMs and MSMs more quantitatively by comparing predictions of NMR spin-relaxation data (14) with recently reported experimental measurements (54). These data are sensitive to the correlation times of conformational transitions, the structures of the metastable configurations involved in the transitions, and their relative populations (55).…”