Exploiting Structural Constraints of Proteolytic Catalytic Triads for Fast Supercomputer Scaffold Probing in Enzyme Design Studies

Zlobin, Alexander; Ermidis, Alexander-Pavel; Maslova, Valentina; Belyaeva, Julia; Golovin, Andrey V.

doi:10.1007/978-3-030-92864-3_5

Cited by 3 publications

(5 citation statements)

References 26 publications

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“…There exist two major groups of such enzymes, chymotrypsinlike and subtilisin-like serine proteases. 81 Despite this grouping, they share most features relevant to our story: Ser-His-Asp catalytic triad, Ser participating also in the oxyanion hole via its backbone nitrogen. Suppose we want to study the catalytic mechanism of subtilisin, we start with the bare minimal QM system of Ser221-His64-Asp32 sidechains and the substrate peptide.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Challenges in Protein QM/MM Simulations with Intra-Backbone Link Atoms

Zlobin

Belyaeva

Golovin

2023

J. Chem. Inf. Model.

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Hybrid quantum mechanical/molecular mechanical (QM/MM) simulations fuel discoveries in many fields of science including computational biochemistry and enzymology. Development of more convenient tools leads to an increase in the number of works in which mechanical insights into enzymes’ mode of operation are obtained. Most commonly, these tools feature hydrogen-capping (link atom) approach to provide coupling between QM and MM subsystems across a covalent bond. Extensive studies were conducted to provide a solid foundation for the correctness of such an approach when a bond to a nonpolar MM atom is considered. However, not every task may be accomplished this way. Certain scenarios of using QM/MM in computational enzymology encourage or even necessitate the incorporation of backbone atoms into the QM region. Two out of three backbone atoms are polar, and in QM/MM with electrostatic embedding, a neighboring link atom will be hyperpolarized. Several schemes to mitigate this effect were previously proposed alongside a rigorous assessment of quantitative effects on model systems. However, it was not clear whether they may translate into qualitatively different results and how link atom hyperpolarization may manifest itself in a real-life enzymological scenario. Here, we show that the consequences of such an artifact may be severe and may completely overturn the conclusions drawn from the simulations. Our case advocates for the use of charge redistribution schemes whenever intra-backbone QM/MM boundaries are considered. Moreover, we addressed how different boundary types and charge redistribution schemes influence backbone dynamics. We showed that the results are heavily dependent on which boundary MM terms are retained, with charge alteration being of secondary importance. In the worst case, only three intra-backbone boundaries may be used with relative confidence in the adequacy of resulting simulations, irrespective of the hyperpolarization mitigation scheme. Thus, advances in the field are certainly needed to fuel new discoveries. As of now, we believe that issues raised in this work might encourage authors in the field to report what boundaries, boundary MM terms, and charge redistribution schemes they are using, so their results may be correctly interpreted.

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Section: ■ Results and Discussionmentioning

confidence: 99%

“…We decided to do it by studying serine triad proteases. There exist two major groups of such enzymes, chymotrypsin-like and subtilisin-like serine proteases . Despite this grouping, they share most features relevant to our story: Ser-His-Asp catalytic triad, Ser participating also in the oxyanion hole via its backbone nitrogen.…”

Section: Resultsmentioning

confidence: 99%

Challenges in Protein QM/MM Simulations with Intra-Backbone Link Atoms

Zlobin

Belyaeva

Golovin

2023

J. Chem. Inf. Model.

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“…TEVp belongs to the PA clan of proteases and shares a structural similarity with chymotrypsin-like proteases that harbor the canonic Ser–His–Asp triad. This similarity reaches the very orientation of catalytic triad residues in space, despite TEVp having Cys instead of Ser as a nucleophile . As a result, TEVp is sometimes assumed to follow the same catalytic mechanism as Ser-utilizing PA clan proteases.…”

Section: Introductionmentioning

confidence: 98%

“…This similarity reaches the very orientation of catalytic triad residues in space, despite TEVp having Cys instead of Ser as a nucleophile. 24 As a result, TEVp is sometimes assumed to follow the same catalytic mechanism as Ser-utilizing PA clan proteases. These enzymes catalyze acylation, which is a rate-limiting stage, through a tetrahedral intermediate stabilized by interactions with oxyanion hole H-bond donors ( Figure 1 A).…”

Section: Introductionmentioning

confidence: 99%

Between Protein Fold and Nucleophile Identity: Multiscale Modeling of the TEV Protease Enzyme–Substrate Complex

Zlobin

Golovin

2022

ACS Omega

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The cysteine protease from the tobacco etch virus (TEVp) is a well-known and widely utilized enzyme. TEVp's chymotrypsin-like fold is generally associated with serine catalytic triads that differ in terms of a reaction mechanism from the most well-studied papain-like cysteine proteases. The question of what dominates the TEVp mechanism, nucleophile identity, or structural composition has never been previously addressed. Here, we use enhanced sampling multiscale modeling to uncover that TEVp combines the features of two worlds in such a way that potentially hampers its activity. We show that TEVp cysteine is strictly in the anionic form in a free enzyme similar to papain. Peptide binding shifts the equilibrium toward the nucleophile′s protonated form, characteristic of chymotrypsin-like proteases, although the cysteinyl anion form is still present and interconversion is rapid. This way cysteine protonation generates enzyme states that are a diversion from the most effective course of action, with only 13.2% of Michaelis complex sub-states able to initiate the reaction. As a result, we propose an updated view on the reaction mechanism catalyzed by TEVp. We also demonstrate that AlphaFold is able to construct protease−substrate complexes with high accuracy. We propose that our findings open a way for its industrious use in enzymological tasks. Unique features of TEVp discovered in this work open a discussion on the evolutionary history and trade-offs of optimizing serine triad-associated folds to cysteine as a nucleophile.

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“…Proteolytic enzymes are among them, and within this broad group, triad proteases are the most widely used and studied. 20 They act in the widest ranges of pH and are well known for their pronounced substrate specificity. There exist approaches to obtain a specificity profile for a particular protease, both experimental 21 and computational.…”

Section: Introductionmentioning

confidence: 99%