Interaction between thrombin mutant W215A/E217A and direct thrombin inhibitor

Tanaka, Kenichi A.; Gruber, András; Szlam, Fania; Bush, Leslie A.; Hanson, Stephen R.; Di, Enrico

doi:10.1097/mbc.0b013e328304e044

Cited by 5 publications

(9 citation statements)

References 13 publications

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“…Cluster 1 contained 22.2%, 24.6%, and 16.2% of the conformations in these systems, respectively, a stark contrast to the 3.1% in wild-type thrombin (Figure 4(a)). This shift potentially elucidates the double mutant’s over 19,000-fold decrease in fibrinogen cleavage capability versus a 7-fold reduction in protein C activation[14, 5, 15, 16]. The catalytic triad exhibits two primary conformations: one in cluster 0 (Figure 4(b)), linked to fibrinogen cleavage, and another in cluster 1 (Figure 4(b)), associated with protein C activation.…”

Section: Resultsmentioning

confidence: 97%

“…In contrast, the TM456-WE system predominantly exhibits conformations in cluster 0, signifying a closed 60s loop (Figure 3(b)), with none in clusters 1 or 2, unlike the 0.3% and 3.64% observed in the WT. This suggests that TM456 binding to the double mutant restricts the 60s loop’s ability to open, potentially explaining the minor 7-fold reduction in protein C activation with TM presence[14, 5, 15, 16]. The consistency of the HDBSCAN results for the 60s loop with our prior study[23] (93.7% and 89.99% conformations of WT and TM456-WT in cluster 0 previously, versus 93.06% and 88.81% currently) underscores the method’s reliability for this specific system.…”

Section: Resultsmentioning

confidence: 99%

“…The WE variant marks a significant shift in thrombin’s function, favoring anticoagulant activities over procoagulant ones[12, 13]. Notably, it demonstrates an over 19,000-fold reduction in fibrinogen cleavage capacity, compared to a 7-fold decrease in protein C activation in conjunction with TM[14, 5, 15, 16]. This paper adheres to sequential numbering for residue identification.…”

Section: Introductionmentioning

confidence: 99%

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Unraveling the Impact of W215A/E217A Mutations on Thrombin’s Dynamics and Thrombomodulin Binding through Molecular Dynamics Simulations

Wu,

Salsbury

2023

Preprint

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Thrombin, a central serine protease in hemostasis, exhibits dual functionality in coagulation processes—favoring fibrinogen cleavage in its native form while shifting towards protein C activation when complexed with thrombomodulin (TM). Thrombin also plays roles in cancer-associated thrombosis and may be involved in metastasis and tumorigenesis. The W215A/E217A (WE) double mutant of thrombin presents a unique case, with its fibrinogen cleavage activity diminished by 19,000-fold, contrasting a modest 7-fold reduction in protein C activation in the presence of TM. The differential substrate specificity of this mutant raises fundamental questions about the underlying molecular mechanisms. In this study, we employed all-atom microsecond-scale molecular dynamics (MD) simulations, complemented by Root Mean Square Fluctuation (RMSF) analysis, clustering algorithms, PCA-based free-energy surfaces, and logistic regression modeling, to dissect the structural and allosteric changes driving thrombin’s substrate specificity. Our results unveil distinct conformational states within the catalytic triad, each optimized for specific substrate interactions. We demonstrate that the WE mutations synergize with TM456 binding, resulting in altered hydrogen bond networks and distinct free energy landscapes. A key finding of our research is the identification of ARG125 as a pivotal element in these interactions, consistently forming critical hydrogen bonds across different thrombin variants. The persistent role of ARG125 not only elucidates aspects of thrombin’s functional plasticity but also positions it as a promising target for novel therapies. This comprehensive analysis enhances our understanding of thrombin’s structural dynamics, paving the way for more effective and targeted therapeutics.

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Unraveling the Impact of W215A/E217A Mutations on Thrombin’s Dynamics and Thrombomodulin Binding through Molecular Dynamics Simulations

Wu,

Salsbury

2023

Preprint

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“…As has been shown for WT thrombin, the W215A/E217A thrombin mutant still requires TM to exhibit anticoagulative activity. 9,14,15 TM binding to ABE1 allosterically decreases the dynamics of the 170s CT , 180s CT , and 220s CT loops, which are 20−30 Å from ABE1. 21 In fact, these loops showed decreased H/D exchange in WT thrombin upon TM binding in the work presented here.…”

Section: ■ Discussionmentioning

confidence: 99%

“…The W215A/E217A double mutant thrombin has been shown to act as a safe and efficacious anticoagulant intervention in vivo . − The W215A/E217A thrombin is called an anticoagulant thrombin because it shifts the balance of these two thrombin activities away from procoagulative and toward anticoagulative substrates. , The loss of ability to cleave fibrinogen is much more significant than the loss of activity toward protein C, which still requires TM. , A crystallographic study suggested that the W215A/E217A mutation causes the primary substrate binding pocket of thrombin to collapse providing an explanation for its strongly decreased activity toward procoagulative substrates. However, it is not clear how TM renders this mutant with a closed substrate pocket capable of cleaving protein C. We hypothesized that the collapsed substrate binding pocket and/or the gain of function upon TM binding may indicate that the W215A/E217A thrombin is dynamic, and perhaps TM binding shifts the dynamic ensemble of thrombin states allowing protein C cleavage.…”

Section: Introductionmentioning

confidence: 99%

How Thrombomodulin Enables W215A/E217A Thrombin to Cleave Protein C but Not Fibrinogen

et al. 2022

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The W215A/E217A mutant thrombin is called "anticoagulant thrombin" because its activity toward its procoagulant substrate, fibrinogen, is reduced more than 500-fold whereas in the presence of thrombomodulin (TM) its activity toward its anticoagulant substrate, protein C, is reduced less than 10-fold. To understand how these mutations so dramatically alter one activity over the other, we compared the backbone dynamics of wild type thrombin to those of the W215A/E217A mutant thrombin by hydrogen−deuterium exchange coupled to mass spectrometry (HDX-MS). Our results show that the mutations cause the 170s, 180s, and 220s C-terminal β-barrel loops near the sites of mutation to exchange more, suggesting that the structure of this region is disrupted. Far from the mutation sites, residues at the N-terminus of the heavy chain, which need to be buried in the Ile pocket for correct structuring of the catalytic triad, also exchange much more than in wild type thrombin. TM binding causes reduced H/D exchange in these regions and also alters the dynamics of the β-strand that links the TM binding site to the catalytic Asp 102 in both wild type thrombin and in the W215A/E217A mutant thrombin. In contrast, whereas TM binding reduces the dynamics the 170, 180 and 220 s C-terminal β-barrel loops in WT thrombin, this region remains disordered in the W215A/E217A mutant thrombin. Thus, TM partially restores the catalytic activity of W215A/E217A mutant thrombin by allosterically altering its dynamics in a manner similar to that of wild type thrombin.

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Residues W215, E217 and E192 control the allosteric E*-E equilibrium of thrombin

Pelc

Koester

Chen

et al. 2019

Sci Rep

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A pre-existing, allosteric equilibrium between closed (E*) and open (E) conformations of the active site influences the level of activity in the trypsin fold and defines ligand binding according to the mechanism of conformational selection. Using the clotting protease thrombin as a model system, we investigate the molecular determinants of the E*-E equilibrium through rapid kinetics and X-ray structural biology. The equilibrium is controlled by three residues positioned around the active site. W215 on the 215–217 segment defining the west wall of the active site controls the rate of transition from E to E* through hydrophobic interaction with F227. E192 on the opposite 190–193 segment defining the east wall of the active site controls the rate of transition from E* to E through electrostatic repulsion of E217. The side chain of E217 acts as a lever that moves the entire 215–217 segment in the E*-E equilibrium. Removal of this side chain converts binding to the active site to a simple lock-and-key mechanism and freezes the conformation in a state intermediate between E* and E. These findings reveal a simple framework to understand the molecular basis of a key allosteric property of the trypsin fold.

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Interaction between thrombin mutant W215A/E217A and direct thrombin inhibitor

Cited by 5 publications

References 13 publications

Unraveling the Impact of W215A/E217A Mutations on Thrombin’s Dynamics and Thrombomodulin Binding through Molecular Dynamics Simulations

Unraveling the Impact of W215A/E217A Mutations on Thrombin’s Dynamics and Thrombomodulin Binding through Molecular Dynamics Simulations

How Thrombomodulin Enables W215A/E217A Thrombin to Cleave Protein C but Not Fibrinogen

Residues W215, E217 and E192 control the allosteric E*-E equilibrium of thrombin

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