Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray-derived conformational ensembles

Yabukarski, Filip; Biel, J.T.; Pinney, Margaux M.; Doukov, Tzanko; Powers, Alex S; Fraser, James S.; Herschlag, Daniel

doi:10.1101/786327

Cited by 9 publications

(34 citation statements)

References 192 publications

(222 reference statements)

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“…Crystallographic structures projected onto a free energy map show an excellent agreement between the distribution of experimental structures and simulations. These results highlight that the conformational change is governed by a few conserved interactions among BDs, reinforcing previous studies that demonstrate the correspondence between ensembles of crystallographic structures and protein plasticity, both in solution and during enzyme catalysis (56,57). Furthermore, we report that two crystallographic structures, one of ZMYND11 and one of PB1 (6), are stable in the open state.…”

Section: Discussionsupporting

confidence: 91%

Discovery of a hidden transient state in all bromodomain families

Raich

Meier

Günther

et al. 2021

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

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Bromodomains (BDs) are small protein modules that interact with acetylated marks in histones. These posttranslational modifications are pivotal to regulate gene expression, making BDs promising targets to treat several diseases. While the general structure of BDs is well known, their dynamical features and their interplay with other macromolecules are poorly understood, hampering the rational design of potent and selective inhibitors. Here, we combine extensive molecular dynamics simulations, Markov state modeling, and available structural data to reveal a transiently formed state that is conserved across all BD families. It involves the breaking of two backbone hydrogen bonds that anchor the ZA-loop with the αA helix, opening a cryptic pocket that partially occludes the one associated to histone binding. By analyzing more than 1,900 experimental structures, we unveil just two adopting the hidden state, explaining why it has been previously unnoticed and providing direct structural evidence for its existence. Our results suggest that this state is an allosteric regulatory switch for BDs, potentially related to a recently unveiled BD-DNA–binding mode.

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Section: Discussionsupporting

confidence: 91%

Discovery of a hidden transient state in all bromodomain families

Raich

Meier

Günther

et al. 2021

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

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“…These results highlight that the conformational change is governed by a few conserved interactions among BDs, and reinforces previous studies that demonstrate the correspondence between ensembles of crystallographic structures and protein plasticity, both in solution and during enzyme catalysis. 51,52 Furthermore, we report that two crystallographic structures, one of ZMYND11 and one of PB1 (6), are stable in the open state. These structures directly support the existence of the novel state and reveal subtle changes of conserved residues that could help in the rationalization of disease related mutations.…”

Section: Discussionmentioning

confidence: 83%

Discovery of a hidden transient state in all bromodomain families

Raich

Meier

Günther

et al. 2020

Preprint

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Bromodomains (BDs) are small protein modules that interact with acetylated marks in histones.These post-translational modifications are pivotal to regulate gene expression, making BDs promising targets to treat several diseases. While the general structure of BDs is well known, their dynamical features and their interplay with other macromolecules are poorly understood, hampering the rational design of potent and selective inhibitors. Here we combine extensive molecular dynamics simulations, Markov state modeling and structural data to reveal a novel and transiently formed state that is conserved across all BD families. It involves the breaking of two backbone hydrogen bonds that anchor the ZA-loop with the α A helix, opening a cryptic pocket that partially occludes the one associated with histone binding. Our results suggest that this novel state is an allosteric regulatory switch for BDs, potentially related to a recently unveiled BD-DNA binding mode.

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“…These findings have direct implications for HSP ensemble analyses. HSP ensembles obtained from multiple cryogenic X-ray structural models provide valuable depictions of overall protein ensemble properties (20,21). However these ensembles are typically collections of structural models obtained for other purposes, often from crystals that have absorbed relatively high X-ray doses to obtain structural data to high resolution, so that, correspondingly, the amount of X-ray damage can be high and the effects on rotameric distributions generally unknown.…”

Section: Discussionmentioning

confidence: 99%

“…Ensemble information can be extracted from "high-sequence similarity PDB" (HSP) ensembles obtained from dozens of traditional X-ray crystallography models of different protein variants and in different crystal forms (20). The overall ensemble information obtained was shown to generally agree with measurements at room temperature and has provided invaluable insights into conformational landscapes, though there are also temperature-induced conformational effects (20,21). Perhaps the largest limitation to this approach is that tens of new cryo X-ray structures are required, rendering it impractical to obtain ensemble information for new variants as is needed to test models and explore new systems.…”

Section: Introductionmentioning

confidence: 99%

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Damaged goods? Evaluating the impact of X-ray damage on conformational heterogeneity in room temperature and cryo-cooled protein crystals

Yabukarski

Doukov

Mokhtari

et al. 2021

Preprint

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X-ray crystallography is a cornerstone of biochemistry. Traditional freezing of protein crystals to cryo-temperatures mitigates X-ray damage and facilitates crystal handling but provides an incomplete window into the ensemble of conformations at the heart of protein function and energetics. Room temperature (RT) X-ray crystallography provides more extensive ensemble information, and recent developments allow conformational heterogeneity, the experimental manifestation of ensembles, to be extracted from single crystal data. However, high sensitivity to X-ray damage at RT raises concerns about data reliability. To systematically address this critical question, we obtained increasingly X-ray-damaged high-resolution datasets (1.02–1.52 Å) from single thaumatin, proteinase K, and lysozyme crystals. Heterogeneity analyses indicated a modest increase in conformational disorder with X-ray damage. Nevertheless, these effects do not alter overall conclusions and can be minimized by limiting the extent of X-ray damage or eliminated by extrapolation to obtain heterogeneity information free from X-ray damage effects. To compare these effects to damage at cryo temperature and to learn more about damage and heterogeneity in cryo-cooled crystals, we carried out an analogous analysis of increasingly damaged proteinase K cryo datasets (0.9–1.16 Å). We found X-ray damage-associated heterogeneity changes that were not observed at RT. This observation and the scarcity of reported X-ray doses and damage extent render it difficult to distinguish real from artifactual conformations, including those occurring as a function of temperature. The ability to aquire reliable heterogeneity information from single crystals at RT provides strong motivation for further development and routine implementation of RT X-ray crystallography to obtain conformational ensemble information.

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Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray-derived conformational ensembles

Cited by 9 publications

References 192 publications

Discovery of a hidden transient state in all bromodomain families

Discovery of a hidden transient state in all bromodomain families

Discovery of a hidden transient state in all bromodomain families

Damaged goods? Evaluating the impact of X-ray damage on conformational heterogeneity in room temperature and cryo-cooled protein crystals

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