Structural insights into the activation of blood coagulation factor XI zymogen by thrombin: A computational molecular dynamics study.

Activation of human blood coagulation factor XI zymogen to factor XIa plays a significant role in the upstream coagulation pathway, in which factor XIa activates factor IX zymogen. The mechanistic details of the proteolytic activation of factor XI by the activating enzyme thrombin are not well-understood at atomic level. In this study, we employed a combination of molecular docking and microsecond time-scale molecular dynamics simulations to identify the key regions of interaction between fXI and thrombin. The activating complex between the substrate and enzyme was modeled to represent the initial acylation step of the serine-protease hydrolysis mechanism. The proposed solution structural complex, fIX:fIIa, obtained from 3 microseconds of MD refinement, suggests that the activation of factor XI is mediated by thrombin's anion binding exosite-II interactions with A3 and A4 domains. We predict that the two positively charged arginine residues (Arg409 and Arg413) in the exosite-2 region, the ß- and γ-insertion loops of thrombin play an important structural role in the initial activating complex between fXI and thrombin.

The First Intrinsic Tenase Complex Inhibitor with Serine Protease Structure Offers a New Perspective in Anticoagulant Therapy.

Components of the intrinsic blood coagulation pathway, among them factor VIIIa (FVIIIa), have been recognized as suitable therapeutic targets to treat venous thromboembolism, pathological process behind two very serious cardiovascular diseases, deep vein thrombosis and pulmonary embolism. Here, we describe a unique glycoprotein from the nose-horned viper (Vipera ammodytes ammodytes [Vaa]) venom, Vaa serine proteinase homolog 1 (VaaSPH-1), structurally a serine protease but without an enzymatic activity and expressing potent anticoagulant action in human blood. We demonstrated that one of its targets in the blood coagulation system is FVIIIa of the intrinsic tenase complex, where it antagonizes the binding of FIXa. Anticoagulants with such characteristics are intensively sought, as they would be much safer for medical application as the contemporary drugs, which frequently induce excessive bleeding and other complications. VaaSPH-1 is unlikely to be orally available for chronic usage as it has molecular mass of 35 kDa. However, it represents a very promising template to design low molecular mass FVIIIa-directed anticoagulant substances, based on structural features of the interaction surface between VaaSPH-1 and FVIIIa. To this end, we constructed a three-dimensional model of VaaSPH-1 bound to FVIIIa. The model exposes the 157-loop and the preceding α-helix as the most appropriate structural elements of VaaSPH-1 to be considered as a guideline to synthesize small FVIIIa-binding molecules, potential new generation of anticoagulants.

Computational analysis of Asp519 and Glu665 mutations of coagulation factor FVIIIa: Implications for enhanced binding affinity of A2-domain.

The A2-domain of blood coagulation factor VIIIa is non-covalently bound to the A1 and A3 domains via weak intermolecular interactions. Functional instability due to rapid dissociation of A2-domain from the active FVIII in blood presents a major hurdle for the therapeutic applications of FVIIIa to treat Hemophilia-A. To identify the ideal hot-spot residues at the interface of A2 and A1/A3 domains that could enhance the structural stability of FVIIIa, we performed a comprehensive computational mutagenesis study of two A2-domain residues, Asp519 and Glu665, that interface the A1 and A3-domains. Each residue was mutated to 15 uncharged amino-acids and the mutant structures were refined by MD simulations. Based on the estimated relative binding affinities of mutant structures, we predict that the mutation of Asp519 to Leu, Gln, Thr, Val and the mutation of Glu665 to Val, Ile, Met, Asn and Trp enhance the A2-domain binding affinity by more than 20kcal/mol, compared to the WT structure. We anticipate that these predictions will be valuable for enzymatic studies towards the rational design of FVIIIa synthetic constructs with improved A2-domain binding affinity.

Structural insights into the interaction of blood coagulation co-factor VIIIa with factor IXa: a computational protein-protein docking and molecular dynamics refinement study.

Coagulation factor X (FX) zymogen activation by factor IXa (FIXa) enzyme plays a critical role in the middle-phase of coagulation cascade. The activation process is catalytically inert and requires FIXa binding and complex formation with co-factor VIIIa (FVIIIa). In order to understand the structural details of the FVIIIa:FIXa complex, we employed knowledge-driven protein-protein docking and aqueous-phase MD refinement methods to develop a stable structural complex between FVIIIa and FIXa. The model shows that all four domains of FIXa wrap across FVIIIa that spans the co-factor binding surface of A2, A3 and C1 domains. The region surrounding the 558-helix of the A2-domain of FVIIIa is predicted to be the key interaction site with the helical segments of Lys293-Lys301 and Asp332-Arg338 residues of the serine-protease domain of FIXa. The hydrophobic helical stack between the GLA and EGF1 domains of FIXa is predicted to be primary interacting region with the A3-C2 domain interface of FVIIIa.

Role of hydrophobic mutations on the binding affinity and stability of blood coagulation factor VIIIa: a computational molecular dynamics and free-energy analysis.

Factor VIIIa is a non-covalently bound hetero-trimer among A1, A2 and A3-C1-C2 domains and an essential co-factor for factor IXa enzyme during proteolytic activation of factor X zymogen. The relatively weak interactions between A2 and the interface A1/A3 domains dampen the functional stability of FVIIIa in plasma and results in rapid degradation. We studied the mutational effect of three charged residues (Asp519, Glu665 and Asp666) to several hydrophobic residues by molecular dynamics simulations. Analysis of the binding free energy by MM-PBSA and MM-GBSA methods shows that the mutation of Asp519 and Glu665 residues to either Val or Ala enhance the A2 domain binding affinity in agreement with the experimental site-specific mutagenesis data. Mutation of Asp666 to Val, Tyr, Met and Phe showed largest improvement in the A2-domain binding among the eight hydrophobic mutants studied. Our studies suggest that the enrichment of hydrophobic interactions in the buried surface regions of A2 domain plays crucial role in improving the overall stability of FVIIIa.

Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study.

BACKGROUND: Human blood coagulation factor VIII (fVIII) is a large plasma glycoprotein with sequential domain arrangement in the order A1-a1-A2-a2-B-a3-A3-C1-C2. The A1, A2 and A3 domains are interconnected by long linker peptides (a1, a2 and a3) that possess the activation sites. Proteolysis of fVIII zymogen by thrombin or factor Xa results in the generation of the activated form (fVIIIa) which serves as a critical co-factor for factor IXa (fIXa) enzyme in the intrinsic coagulation pathway. RESULTS: In our efforts to elucidate the structural differences between fVIII and fVIIIa, we developed the solution structural models of both forms, starting from an incomplete 3.7 A X-ray crystal structure of fVIII zymogen, using explicit solvent MD simulations. The full assembly of B-domainless single-chain fVIII was built between the A1-A2 (Ala1-Arg740) and A3-C1-C2 (Ser1669-Tyr2332) domains. The structural dynamics of fVIII and fVIIIa, simulated for over 70 ns of time scale, enabled us to evaluate the integral motions of the multi-domain assembly of the co-factor and the possible coordination pattern of the functionally important calcium and copper ion binding in the protein. CONCLUSIONS: MD simulations predicted that the acidic linker peptide (a1) between the A1 and A2 domains is largely flexible and appears to mask the exposure of putative fIXa enzyme binding loop (Tyr555-Asp569) region in the A2 domain. The simulation of fVIIIa, generated from the zymogen structure, predicted that the linker peptide (a1) undergoes significant conformational reorganization upon activation by relocating completely to the A1-domain. The conformational transition led to the exposure of the Tyr555-Asp569 loop and the surrounding region in the A2 domain. While the proposed linker peptide conformation is predictive in nature and warrants further experimental validation, the observed conformational differences between the zymogen and activated forms may explain and support the large body of experimental data that implicated the critical importance of the cleavage of the peptide bond between the Arg372 and Ser373 residues for the full co-factor activity of fVIII.

Three-dimensional solution structure of Tropidechis carinatus venom extract trocarin: a structural homologue of Xa and prothrombin activator.

Trocarin belongs to group D of prothrombin activators derived from snake venom of Tropidechis carinatus and is a rich non-hepatic source of Xa, the only known hepatic prothrombin activator. The structural and functional similarity with Xa makes trocarin an interesting target for exploring the structure-functional relationship with Xa. Herein we report a predicted complete three-dimensional all-atom structural model of trocarin equilibrated in explicit water using 4 ns of molecular dynamics simulation. The tertiary structure was modeled using the structure of human blood coagulation factor Xa. The conformational and structural features of trocarin are then compared with the X-ray crystal and solution simulation structures of human factor Xa. The modeled structure of trocarin has four individual domains (Gla, EGF1, EGF2 and SP) connected along the long axis with similar secondary structural elements to Xa. The simulations suggest that sodium ion binding in the serine protease domain is impaired in trocarin as compared to Xa. In contrast to Xa, for which the sodium ion forms an octahedral coordination network that brings two loop regions connecting four anti-parallel beta-sheets together, we do not find a similar pattern of network in trocarin. We observe that the difference in the binding pattern of sodium ion leads to a approximately 2-A "shrinkage" of the beta2 strand (B2), in comparison to human Xa, as marked by a shorter distance between 189Asp373 (S1-site residue) and 195Ser379 (active-site residue) in the B2 strand. We propose that these differences may be linked to the experimentally observed lower amidolytic activity of trocarin as compared to Xa.

Structure and dynamics of zymogen human blood coagulation factor X.

The solution structure and dynamics of the human coagulation factor X (FX) have been investigated to understand the key structural elements in the zymogenic form that participates in the activation process. The model was constructed based on the 2.3-A-resolution x-ray crystallographic structure of active-site inhibited human FXa (PDB:1XKA). The missing gamma-carboxyglutamic acid (GLA) and part of epidermal growth factor 1 (EGF1) domains of the light chain were modeled based on the template of GLA-EGF1 domains of the tissue factor (TF)-bound FVIIa structure (PDB:1DAN). The activation peptide and other missing segments of FX were introduced using homology modeling. The full calcium-bound model of FX was subjected to 6.2 ns of molecular dynamics simulation in aqueous medium using the AMBER6.0 package. We observed significant reorientation of the serine-protease (SP) domain upon activation leading to a compact multi-domain structure. The solution structure of zymogen appears to be in a well-extended conformation with the distance between the calcium ions in the GLA domain and the catalytic residues estimated to be approximately 95 A in contrast to approximately 83 A in the activated form. The latter is in close agreement with fluorescence studies on FXa. The S1-specificity residues near the catalytic triad show significant differences between the zymogen and activated structures.