The active site region plays a critical role in Na+ binding to thrombin

Pelc, Leslie A.; Koester, Sarah; Kukla, Cassandra R.; Chen, Zhiwei; Di, Enrico

doi:10.1016/j.jbc.2021.101458

Cited by 4 publications

(4 citation statements)

References 71 publications

(180 reference statements)

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“…The catalytic activity of all coagulation proteases is enhanced by Na + binding to a site that is defined by the 186 and 220 loops and is located >15 Å from the active site residues. 3 , 11 , 12 , 55 , 56 , 57 , 58 , 59 , 60 Whether a protease can effectively bind Na + depends on the residue that occupies position 225. 61 , 62 Serine proteases with F/Y225 bind Na + , whereas those with P225 do not.…”

Section: Resultsmentioning

confidence: 99%

Comparative sequence analysis of vitamin K‐dependent coagulation factors

Stojanovski

2022

Journal of Thrombosis and Haemostasis

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Background: Prothrombin, protein C, and factors VII, IX, and X are vitamin K (VK)dependent coagulation proteins that play an important role in the initiation, amplification, and subsequent attenuation of the coagulation response. Blood coagulation evolved in the common vertebrate ancestor as a specialization of the complement system and immune response, which in turn bear close evolutionary ties with developmental enzyme cascades. There is currently no comprehensive analysis of the evolutionary changes experienced by these coagulation proteins during the radiation of vertebrates and little is known about conservation of residues that are important for zymogen activation and catalysis. Objectives:To characterize the conservation level of functionally important residues among VK-dependent coagulation proteins from different vertebrate lineages. Methods:The conservation level of residues important for zymogen activation and catalysis was analyzed in >1600 primary sequences of VK-dependent proteins.Results: Functionally important residues are most conserved in prothrombin and least conserved in protein C. Some of the most profound functional modifications in protein C occurred in the ancestor of bony fish when the basic residue in the activation site was replaced by an aromatic residue. Furthermore, during the radiation of placental mammals from marsupials, protein C acquired a cysteine-rich insert that introduced an additional disulfide in the EGF1 domain and evolved a proprotein convertase cleavage site in the activation peptide linker that also became significantly elongated.Conclusions: Sequence variabilities at functionally important residues may lead to interspecies differences in the zymogen activation and catalytic properties of orthologous VK-dependent proteins.

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Section: Resultsmentioning

confidence: 99%

Comparative sequence analysis of vitamin K‐dependent coagulation factors

Stojanovski

2022

Journal of Thrombosis and Haemostasis

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“…86 Allosteric activation by a Na + ion turns it into a 10 times more active "fast" form by shifting the populations of conformations of the catalytic nucleophile. 86,87 Therefore, a possible strategy to enhance TEVp and similar enzymes would be to discourage such a process or to even lock Cys in a constantly deprotonated state. While Cys reprotonation is likely a manifestation of a fold being underoptimized for such a nucleophile, the surroundings of active sites of Cys PA clan proteases are clearly different from their Ser counterparts with Asn44 being an obvious example.…”

Section: ■ Discussionmentioning

confidence: 99%

“…The same behavior was proposed to be the cause behind the existence of the “slow” form of thrombin. 86 Allosteric activation by a Na + ion turns it into a 10 times more active “fast” form by shifting the populations of conformations of the catalytic nucleophile. 86 , 87 Therefore, a possible strategy to enhance TEVp and similar enzymes would be to discourage such a process or to even lock Cys in a constantly deprotonated state.…”

Section: Discussionmentioning

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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“…The equilibrium between the pro‐coagulant and anti‐coagulant functions of αT is mainly regulated by effector proteins interaction and, to a minor extent, by sodium ion binding at a specific protease site (Figure 1 ). Na + binding triggers the conformational transition of the enzyme from an anticoagulant “slow” form to a procoagulant “fast” form (Dang et al, 1995 ; Lechtenberg et al, 2010 ; Pelc et al, 2022 ). The Na + ‐bound (fast) form displays procoagulant properties, since it cleaves more specifically fibrinogen and PAR‐1, whereas the Na + ‐free (slow) form is anticoagulant as it retains normal proteolytic activity toward Protein C, but is unable to promote physiologically acceptable hydrolysis of the procoagulant substrates (Dang et al, 1995 ; De Filippis et al, 2005 ).…”

Section: Introductionmentioning

confidence: 99%

From haemadin to haemanorm: Synthesis and characterization of full‐length haemadin from the leech Haemadipsa sylvestris and of a novel bivalent, highly potent thrombin inhibitor (haemanorm)

Acquasaliente,

Pierangelini,

Pagotto

et al. 2023

Protein Science

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Hirudin from Hirudo medicinalis is a bivalent α‐Thrombin (αT) inhibitor, targeting the enzyme active site and exosite‐I, and is currently used in anticoagulant therapy along with its simplified analogue hirulog. Haemadin, a small protein (57 amino acids) isolated from the land‐living leech Haemadipsa sylvestris, selectively inhibits αT with a potency identical to that of recombinant hirudin (KI = 0.2 pM), with which it shares a common disulphide topology and overall fold. At variance with hirudin, haemadin targets exosite‐II and therefore (besides the free protease) it also blocks thrombomodulin‐bound αT without inhibiting the active intermediate meizothrombin, thus offering potential advantages over hirudin. Here, we produced in reasonably high yields and pharmaceutical purity (>98%) wild‐type haemadin and the oxidation resistant Met5 → nor‐Leucine analogue, both inhibiting αT with a KI of 0.2 pM. Thereafter, we used site‐directed mutagenesis, spectroscopic, ligand‐displacement, and Hydrogen/Deuterium Exchange‐Mass Spectrometry techniques to map the αT regions relevant for the interaction with full‐length haemadin and with the synthetic N‐ and C‐terminal peptides Haem(1–10) and Haem(45–57). Haem(1–10) competitively binds to/inhibits αT active site (KI = 1.9 μM) and its potency was enhanced by 10‐fold after Phe3 → β‐Naphthylalanine exchange. Conversely to full‐length haemadin, haem(45–57) displays intrinsic affinity for exosite‐I (KD = 1.6 μM). Hence, we synthesized a peptide in which the sequences 1–9 and 45–57 were joined together through a 3‐Glycine spacer to yield haemanorm, a highly potent (KI = 0.8 nM) inhibitor targeting αT active site and exosite‐I. Haemanorm can be regarded as a novel class of hirulog‐like αT inhibitors with potential pharmacological applications.This article is protected by copyright. All rights reserved.

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The active site region plays a critical role in Na+ binding to thrombin

Cited by 4 publications

References 71 publications

Comparative sequence analysis of vitamin K‐dependent coagulation factors

Comparative sequence analysis of vitamin K‐dependent coagulation factors

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

From haemadin to haemanorm: Synthesis and characterization of full‐length haemadin from the leech Haemadipsa sylvestris and of a novel bivalent, highly potent thrombin inhibitor (haemanorm)

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