A Hydrogen Bond Regulates Slow Motions in Ubiquitin by Modulating a β-Turn Flip

Sidhu, Arshdeep; Surolia, Avadhesha; Robertson, Andrew D.; Sundd, Monica

doi:10.1016/j.jmb.2011.06.044

Cited by 31 publications

(59 citation statements)

References 47 publications

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“…conformational switch in this region of ubiquitin has previously been observed in experiments (74,75). In the remaining 10% of the structures, the α helix is partially frayed at its C terminus; similar unfolding of the C-terminal end of the helix has been observed in experiment both at high pressure (76) and in a mutant in which the helix C-cap has been removed (77), suggesting that ubiquitin may undergo such motion, although the force field appears to overestimate its population.…”

Section: Resultssupporting

confidence: 60%

Atomic-level description of ubiquitin folding

Piana

Lindorff‐Larsen

Shaw

2013

Proc. Natl. Acad. Sci. U.S.A.

305

350

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Equilibrium molecular dynamics simulations, in which proteins spontaneously and repeatedly fold and unfold, have recently been used to help elucidate the mechanistic principles that underlie the folding of fast-folding proteins. The extent to which the conclusions drawn from the analysis of such proteins, which fold on the microsecond timescale, apply to the millisecond or slower folding of naturally occurring proteins is, however, unclear. As a first attempt to address this outstanding issue, we examine here the folding of ubiquitin, a 76-residue-long protein found in all eukaryotes that is known experimentally to fold on a millisecond timescale. Ubiquitin folding has been the subject of many experimental studies, but its slow folding rate has made it difficult to observe and characterize the folding process through all-atom molecular dynamics simulations. Here we determine the mechanism, thermodynamics, and kinetics of ubiquitin folding through equilibrium atomistic simulations. The picture emerging from the simulations is in agreement with a view of ubiquitin folding suggested from previous experiments. Our findings related to the folding of ubiquitin are also consistent, for the most part, with the folding principles derived from the simulation of fast-folding proteins, suggesting that these principles may be applicable to a wider range of proteins. CHARMM22* | energy landscape | enthalpy | phi-value analysis | prefactor U nderstanding the principles that govern protein folding, the self-assembly process that leads from an unstructured polypeptide chain to a fully functional protein, has been one of the major challenges in the area of physical biochemistry over the last 50 years. Naturally occurring proteins typically fold on timescales ranging from milliseconds to minutes, but as our understanding of the principles of protein folding has improved, a number of fastfolding proteins that fold on the microsecond timescale have been engineered and characterized (1-12). The design of such fastfolding proteins was in part intended to narrow the gap between the timescales of protein folding and the timescale accessible to physics-based atomistic molecular dynamics (MD) simulations, thus enabling fruitful combinations of experiments and simulations to study the mechanisms of folding (13-18). We recently studied 12 fast-folding proteins using equilibrium MD simulations, for example, with the aim of elucidating general principles underlying the folding of these proteins (19). The folding of these proteins was found to be a relatively sequential process that follows a few paths, in which the order of formation of native structure is correlated with relative structural stability in the unfolded state. The extent to which these observations pertained to fast-folding proteins only or to protein folding more generally, however, was unclear. Here we address this question by applying the same methodology and physics-based force field used in our previous investigation of fast-folding proteins to the study of ubiquitin, a nat...

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Section: Resultssupporting

confidence: 60%

Atomic-level description of ubiquitin folding

Piana

Lindorff‐Larsen

Shaw

2013

Proc. Natl. Acad. Sci. U.S.A.

305

350

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“…2). In addition, a previous study using mutagenesis and extreme pH values suggested that rotation of this peptide bond may explain the microsecond motion observed in two nearby residues (19). Microsecond motions in this region have also been observed with heteronuclear doubleresonance (20) and solid-state RD (21) experiments.…”

Section: Resultsmentioning

confidence: 69%

“…To investigate the necessity of the peptide flip for this collective ubiquitin motion, we used two mutants, E24A and G53A, that have been shown to inhibit the NH-in state (19). In the presence of these mutants, 1 H N RD is either abolished or significantly attenuated (at least by a factor of 10) at all but one residue ( Fig.…”

Section: Significancementioning

confidence: 99%

Allosteric switch regulates protein–protein binding through collective motion

Smith

Ban

Pratihar

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

105

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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...

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“…Severe line broadening due to ms time scale conformational exchange has been observed in residue G53 in solution, while T55 displays a moderate amount of broadening both in solution and in solid. 24,58,59 We have scanned the trajectory for the evidence of transitions between type II and type I conformations (the indicative angles are w in D52 and / in G53 59 ). Although the current simulation is relatively short, 400 ns, it contains 24 ubiquitin molecules, thus offering respectable statistics.…”

Section: Order Parameters (Continued)mentioning

confidence: 99%

Ensemble MD simulations restrained via crystallographic data: Accurate structure leads to accurate dynamics

Xue

Skrynnikov

2014

Protein Science

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Currently, the best existing molecular dynamics (MD) force fields cannot accurately reproduce the global free-energy minimum which realizes the experimental protein structure. As a result, long MD trajectories tend to drift away from the starting coordinates (e.g., crystallographic structures). To address this problem, we have devised a new simulation strategy aimed at protein crystals. An MD simulation of protein crystal is essentially an ensemble simulation involving multiple protein molecules in a crystal unit cell (or a block of unit cells). To ensure that average protein coordinates remain correct during the simulation, we introduced crystallography-based restraints into the MD protocol. Because these restraints are aimed at the ensemble-average structure, they have only minimal impact on conformational dynamics of the individual protein molecules. So long as the average structure remains reasonable, the proteins move in a native-like fashion as dictated by the original force field. To validate this approach, we have used the data from solid-state NMR spectroscopy, which is the orthogonal experimental technique uniquely sensitive to protein local dynamics. The new method has been tested on the well-established model protein, ubiquitin. The ensemble-restrained MD simulations produced lower crystallographic R factors than conventional simulations; they also led to more accurate predictions for crystallographic temperature factors, solid-state chemical shifts, and backbone order parameters. The predictions for 15 N R 1 relaxation rates are at least as accurate as those obtained from conventional simulations. Taken together, these results suggest that the presented trajectories may be among the most realistic protein MD simulations ever reported. In this context, the ensemble restraints based on high-resolution crystallographic data can be viewed as protein-specific empirical corrections to the standard force fields.

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A Hydrogen Bond Regulates Slow Motions in Ubiquitin by Modulating a β-Turn Flip

Cited by 31 publications

References 47 publications

Atomic-level description of ubiquitin folding

Atomic-level description of ubiquitin folding

Allosteric switch regulates protein–protein binding through collective motion

Ensemble MD simulations restrained via crystallographic data: Accurate structure leads to accurate dynamics

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