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

ABSTRACT: 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.

SUBMITTER: Venkateswarlu D

PROVIDER: S-EPMC4179997 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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

Venkateswarlu Divi D

Biochemical and biophysical research communications 20140823 3

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 m ...[more]

PMID: 25157807

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Project description:A critical step in injury-induced initiation of blood coagulation is the formation of the complex between the trypsin-like protease coagulation factor VIIa (FVIIa) and its cofactor tissue factor (TF), which converts FVIIa from an intrinsically poor enzyme to an active protease capable of activating zymogens of downstream coagulation proteases. Unlike its constitutively active ancestor trypsin, FVIIa is allosterically activated (by TF). Here, ensemble refinement of crystallographic structures, which uses multiple copies of the entire structure as a means of representing structural flexibility, is applied to explore the impacts of inhibitor binding to trypsin and FVIIa, as well as cofactor binding to FVIIa. To assess the conformational flexibility and its role in allosteric pathways in these proteases, main-chain hydrogen bond networks are analyzed by calculating the hydrogen-bond propensity. Mapping pairwise propensity differences between relevant structures shows that binding of the inhibitor benzamidine to trypsin has a minor influence on the protease flexibility. For FVIIa, in contrast, the protease domain is "locked" into the catalytically competent trypsin-like configuration upon benzamidine binding as indicated by the stabilization of key structural features: the nonprime binding cleft and the oxyanion hole are stabilized, and the effect propagates from the active site region to the calcium-binding site and to the vicinity of the disulphide bridge connecting with the light chain. TF binding to FVIIa furthermore results in stabilization of the 170 loop, which in turn propagates an allosteric signal from the TF-binding region to the active site. Analyses of disulphide bridge energy and flexibility reflect the striking stability difference between the unregulated enzyme and the allosterically activated form after inhibitor or cofactor binding. The ensemble refinement analyses show directly, for the first time to our knowledge, whole-domain structural footprints of TF-induced allosteric networks present in x-ray crystallographic structures of FVIIa, which previously only have been hypothesized or indirectly inferred.

Project description:Essentials Consensus sequence and biochemical data suggest a Na+ -site in the factor (F) IXa protease domain. X-ray structure of the FIXa EGF2/protease domain at 1.37 Å reveals a Na+ -site not observed earlier. Molecular dynamics simulations data support that Na+ ± Ca2+ promote FIXa protease domain stability. Sulfate ions found in the protease domain mimic heparin sulfate binding mode in FIXa. SUMMARY: Background Activated coagulation factor IX (FIXa) consists of a γ-carboxyglutamic acid domain, two epidermal growth factor-like (EGF) domains, and a C-terminal protease domain. Consensus sequence and biochemical data support the existence of a Na+ -site in the FIXa protease domain. However, soaking experiments or crystals grown in high concentration of ammonium sulfate did not reveal a Na+ -site in wild-type or mutant FIXa EGF2/protease domain structure. Objective Determine the structure of the FIXa EGF2/protease domain in the presence of Na+ ; perform molecular dynamics (MD) simulations to explore the role of Na+ in stabilizing FIXa structure. Methods Crystallography, MD simulations, and modeling heparin binding to FIXa. Results Crystal structure at 1.37-Å resolution revealed that Na+ is coordinated to carbonyl groups of residues 184A, 185, 221A, and 224 in the FIXa protease domain. The Na+ -site in FIXa is similar to that of FXa and is linked to the Asp189 S1-site. In MD simulations, Na+ reduced fluctuations in residues 217-225 (Na+ -loop) and 70-80 (Ca2+ -loop), whereas Ca2+ reduced fluctuations only in residues of the Ca2+ -loop. Ca2+ and Na+ together reduced fluctuations in residues of the Ca2+ -loop and Na+ -loop (residues 70-80, 183-194, and 217-225). Moreover, we observed four sulfate ions that make salt bridges with FIXa protease domain Arg/Lys residues, which have been implicated in heparin binding. Based upon locations of the sulfate ions, we modeled heparin binding to FIXa, which is similar to the heparin binding in thrombin. Conclusions The FIXa Na+ -site in association with Ca2+ contributes to stabilization of the FIXa protease domain. The heparin binding mode in FIXa is similar to that in thrombin.

Dataset Information

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

Publications

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

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