The Sampling of Conformational Dynamics in Ambient-Temperature Crystal Structures of Arginine Kinase

Godsey, Michael; Davulcu, Omar; Nix, Jay C.; Skalicky, Jack J.; Brüschweiler, Rafael; Chapman, Michael S.

doi:10.1016/j.str.2016.07.013

Cited by 6 publications

(5 citation statements)

References 44 publications

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“…This precludes identification of a single pathway of coupled side chain conformers (via the program CONTACT 19 ). Hence this approach was not useful for our system, as also reported for a different enzyme 22 …”

Section: Resultsmentioning

confidence: 79%

Structural and dynamical description of the enzymatic reaction of a phosphohexomutase

Stiers

Graham

Zhu

et al. 2019

Structural Dynamics

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Enzymes are known to adopt various conformations at different points along their catalytic cycles. Here, we present a comprehensive analysis of 15 isomorphous, high resolution crystal structures of the enzyme phosphoglucomutase from the bacterium Xanthomonas citri . The protein was captured in distinct states critical to function, including enzyme-substrate, enzyme-product, and enzyme-intermediate complexes. Key residues in ligand recognition and regions undergoing conformational change are identified and correlated with the various steps of the catalytic reaction. In addition, we use principal component analysis to examine various subsets of these structures with two goals: (1) identifying sites of conformational heterogeneity through a comparison of room temperature and cryogenic structures of the apo-enzyme and (2) a priori clustering of the enzyme-ligand complexes into functionally related groups, showing sensitivity of this method to structural features difficult to detect by traditional methods. This study captures, in a single system, the structural basis of diverse substrate recognition, the subtle impact of covalent modification, and the role of ligand-induced conformational change in this representative enzyme of the α-D-phosphohexomutase superfamily.

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Section: Resultsmentioning

confidence: 79%

Structural and dynamical description of the enzymatic reaction of a phosphohexomutase

Stiers

Graham

Zhu

et al. 2019

Structural Dynamics

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“…The directionality of anisotropic atomic displacement can be indicative of conformational sampling. − To further investigate the latter, ensemble refinement, that is a combination of X-ray structure refinement and molecular dynamics, was performed to reveal the conformational ensembles of the active site loops. The different analysis results are presented as movies (Supplementary Movies 1–4).…”

Section: Resultsmentioning

confidence: 99%

Changes in Active Site Loop Conformation Relate to the Transition toward a Novel Enzymatic Activity

Jacquet,

Billot,

Shimon

et al. 2024

JACS Au

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Enzymatic promiscuity, the ability of enzymes to catalyze multiple, distinct chemical reactions, has been well documented and is hypothesized to be a major driver of the emergence of new enzymatic functions. Yet, the molecular mechanisms involved in the transition from one activity to another remain debated and elusive. Here, we evaluated the redesign of the active site binding cleft of lactonase SsoPox using structure-based design and combinatorial libraries. We created variants with largely improved catalytic abilities against phosphotriesters, the best ones being >1000-fold better compared to the wild-type enzyme. The observed shifts in activity specificity are large, and some variants completely lost their initial activity. The selected combinations of mutations have considerably reshaped the active site cavity via side chain changes but mostly through large rearrangements of the active site loops and changes to their conformations, as revealed by a suite of crystal structures. This suggests that a specific active site loop configuration is critical to the lactonase activity. Interestingly, analysis of high-resolution structures hints at the potential role of conformational sampling and its directionality in defining the enzyme activity profile.

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“…These two structures, from different crystal forms, are very similar overall, with a backbone RMSD of 0.3 Å. The largest domain level rotations between the two crystal forms involve domains 3 and 5, which differ by 1.2 and 1.0 degrees, respectively (Godsey et al, 2016). These rotations may reflect locations of flexibility within the TSA complex and may be the origin of the R ex observed in these rigid groups.…”

Section: Resultsmentioning

confidence: 99%

Elevated μs-ms timescale backbone dynamics in the transition state analog form of arginine kinase

Davulcu

Peng

Brüschweiler

et al. 2017

Journal of Structural Biology

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Arginine kinase catalyzes reversible phosphoryl transfer between arginine and ATP. Crystal structures of arginine kinase in an open, substrate-free form and closed, transition state analog (TSA) complex indicate that the enzyme undergoes substantial domain and loop rearrangements required for substrate binding, catalysis, and product release. Nuclear magnetic resonance (NMR) has shown that substrate-free arginine kinase is rigid on the ps-ns timescale (average S2=0.84 +/- 0.08) yet quite dynamic on the μs-ms timescale (35 residues with Rex, 12%), and that movements of the N-terminal domain and the loop comprising residues I182-G209 are rate-limiting on catalysis. Here, NMR of the TSA-bound enzyme shows similar rigidity on the ps-ns timescale (average S2=0.91 +/- 0.05) and substantially increased μs-ms timescale dynamics (77 residues; 22%). Many of the residues displaying μs-ms dynamics in NMR Carr-Purcell-Meiboom-Gill (CPMG) 15N backbone relaxation dispersion experiments of the TSA complex are also dynamic in substrate-free enzyme. However, the presence of additional dynamic residues in the TSA-bound form suggests that dynamics extend through much of the C-terminal domain, which indicates that in the closed form, a larger fraction of the protein takes part in conformational transitions to the excited state(s). Conformational exchange rate constants (kex) of the TSA complex are all approximately 2500 s-1, higher than any observed in the substrate-free enzyme (800 - 1900 s-1). Elevated μs-ms timescale protein dynamics in the TSA-bound enzyme is more consistent with recently postulated catalytic networks involving multiple interconnected states at each step of the reaction, rather than a classical single stabilized transition state.

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The Sampling of Conformational Dynamics in Ambient-Temperature Crystal Structures of Arginine Kinase

Cited by 6 publications

References 44 publications

Structural and dynamical description of the enzymatic reaction of a phosphohexomutase

Structural and dynamical description of the enzymatic reaction of a phosphohexomutase

Changes in Active Site Loop Conformation Relate to the Transition toward a Novel Enzymatic Activity

Elevated μs-ms timescale backbone dynamics in the transition state analog form of arginine kinase

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