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

Biel, J.T.; Thompson, Michael C.; Cunningham, Christian N.; Corn, Jacob E.; Fraser, James S.

doi:10.1016/j.str.2017.03.009

Cited by 27 publications

(32 citation statements)

References 57 publications

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“…To evaluate changes to the HG3 conformational ensemble along its evolutionary trajectory, we solved room-temperature (277 K) X-ray crystal structures of all HG-series Kemp eliminases, both in the presence and absence of bound 6NT. Room-temperature X-ray crystallography can reveal conformational heterogeneity in protein structures that would not be visible at cryogenic temperatures and thereby provide insights into the conformational ensemble that is sampled by a protein in solution 19 . All five enzymes yielded crystals under similar conditions (Supplementary Table 2), and these diffracted at resolutions of 1.35-1.99 Å (Supplementary Table 3).…”

Section: Resultsmentioning

confidence: 99%

Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico

et al. 2020

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The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M−1s−1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M−1s−1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.

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Section: Resultsmentioning

confidence: 99%

Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico

et al. 2020

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“…Previous studies have focused extensively on the role of changes in conformational dynamics during evolution. 45–47 Often, B-factor analysis is used as a proxy for protein dynamics with the assumption that larger B-factors indicate higher conformational motion. 47 We performed B-factor analysis for each structure and found a surprisingly poor correlation with the heterogeneity that is best illustrated with Pf TrpB 2B9 (Figure S12).…”

Section: Resultsmentioning

confidence: 99%

Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble

et al. 2018

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Allosteric enzymes contain a wealth of catalytic diversity that remains distinctly underutilized for biocatalysis. Tryptophan synthase is a model allosteric system and a valuable enzyme for the synthesis of noncanonical amino acids (ncAA). Previously, we evolved the β-subunit from Pyrococcus furiosus, PfTrpB, for ncAA synthase activity in the absence of its native partner protein PfTrpA. However, the precise mechanism by which mutation activated TrpB to afford a stand-alone catalyst remained enigmatic. Here, we show that directed evolution caused a gradual change in the rate-limiting step of the catalytic cycle. Concomitantly, the steady-state distribution of the intermediates shifts to favor covalently bound Trp adducts, which have increased thermodynamic stability. The biochemical properties of these evolved, stand-alone TrpBs converge on those induced in the native system by allosteric activation. High-resolution crystal structures of the wild-type enzyme, an intermediate in the lineage, and the final variant, encompassing five distinct chemical states, show that activating mutations have only minor structural effects on their immediate environment. Instead, mutation stabilizes the large-scale motion of a subdomain to favor an otherwise transiently populated closed conformational state. This increase in stability enabled the first structural description of Trp covalently bound in a catalytically active TrpB, confirming key features of catalysis. These data combine to show that sophisticated models of allostery are not a prerequisite to recapitulating its complex effects via directed evolution, opening the way to engineering stand-alone versions of diverse allosteric enzymes.

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“…1a). In contrast with other proteins previously used to study the evolution of dynamics, 10,15,[27][28][29][30] DANCERs are unique in that the designed Trp43 conformational exchange did not result from alteration of exchange rates and amplitudes between existing low-occupancy conformational states observed in the parent. WT Gβ1 and DANCERs therefore comprise an ideal system to study how conformational exchange can arise in a globular protein, helping us to gain insights on the link between protein sequence and dynamics.…”

Section: Introductionmentioning

confidence: 92%

Origin of Conformational Dynamics in a Globular Protein

Damry

Mayer

Broom

et al. 2019

Preprint

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Protein structures are dynamic, undergoing specific motions that can play a vital role in function.However, the link between primary sequence and conformational dynamics remains poorly understood. Here, we studied how conformational dynamics can arise in a globular protein by evaluating the impact of individual substitutions of core residues in DANCER-3, a streptococcal protein G domain β1 (Gβ1) variant that we previously designed to undergo a specific mode of conformational exchange that has never been observed in the wild-type protein. Using a combination of solution NMR experiments and molecular dynamics simulations, we demonstrate that only two mutations are necessary to create this conformational exchange, and that these mutations work synergistically, with one destabilizing the native Gβ1 structure and the other allowing two new conformational states to be accessed on the energy landscape. Overall, our results show how conformational dynamics can appear in a stable globular fold, a critical step in the molecular evolution of new dynamics-linked functions.

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

Cited by 27 publications

References 57 publications

Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico

Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico

Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble

Origin of Conformational Dynamics in a Globular Protein

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