Discipline: Biological Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Shenna Shearin - North Carolina Agricultural and Technical State University
Blood coagulation factor VIIIa (fVIIIa) is an essential cofactor for factor IXa (fIXa) enzyme. The fVIIIa-fIXa complex, known as intrinsic tenase complex, activates inactive fX zymogen to activated form fXa. This process is essential for generation of a stable clot which stops bleeding and maintains hemostasis. FVIIIa is a 1383 residue length hetero-trimer comprised of A1, A2 and A3/C1/C2 domains that circulates in blood as a non-covalently bound complex. More than 2000 point mutations of fVIIIa are known that reduce the functional stability of fVIIIa and often lead to a mild to severe bleeding disorder called Hemophilia A. Currently, there is no cure for Hemophilia A and the only mode of treatment is to treat the patients with recombinant fVIIIa infusion. Futhermore, fVIIIa is temperature sensitive and unstable product with a half-life of merely 8 to 12 hours. Several fVIII point mutations have been shown to facilitate the rate of dissociation of A2 domain relative to the wild type, and these residues localize to the interface of the A2 domain with either the A1 or A3 domains. Since hydrophobic interactions are the driving force for protein folding, we hypothesized that mutating key interacting residues within the domain-domain binding interface of fVIIIa will lead to improvement in the binding affinity. As a result, fVIIIa will be more stable. This can lead to disovery of key molecular recognition sites between fVIIIa and fIXa and identification of mutational “hotspots” for protein engineering design of novel therapeutic drugs. In our approach, we employed Molecular Dynamics (MD) simulations and MM-PBSA binding free-energy analysis to investigate the structural and energetic impact of hydrophobic resides located within the interface of domains A1 and A2, A2 and A3, and A1 and A3 domains.
In this study, we have identified Glu287 at the A1–A2 interface that differentially contributes to the stabilization of fVIIIa. Our data indicates that the mutation of Glu287 to Ala, Gly, Met, and Try make favorable contributions within the fVIIIa complex. The PBSA method predicts the E287A mutant stabilizes the FVIIIa complex by ~10 Kcal/mol than the Tyr287A mutant. The E287M mutant is as effective as E287A in improving the binding affinity compared to Try287 and Gly287. The M287 side chain buries within a hydrophobic patch surrounded by Tyr1979, Phe671, and Val266. Although E287M and E287A do not differ in binding affinity, the non-polar and gas-phase energies of E287A compared to E287M is significantly improved. Although E287G mutant stabilizes the A1 domain by ~15 Kcal/mol more than E287W mutant suggests that the ideal size of the side-chain of the mutant residues should also be considered as a crucial factor in designing the new variants.
In conclusion, MM/PBSA method is reasonably accurate to reproduce the experimental data of A2 domain binding affinity of Asp519 and Glu665 mutants. We predict that the site of Glu287 residue that interfaces A2 domain is ideal hotspot for mutations. We rank the overall A1 domain binding affinity of hydrophobic mutants at Glu287 in the order of: Ala > Met > Gly > Tyr. Our results also suggest that Isoleucine, Tryptophan, and Phenylalanine mutations at Glu287 may not be good candidates for structural modification of FVIIIa.Abstract_2016.docx
Funder Acknowledgement(s): This work was partly supported by the National Institutes of Health Grant (R15-HL082632). The computing resources were acquired by MRI Grant from National Science Foundation (NSF-ACI-1126543). We also thank Professor Philip Fay, University of Rochester Medical center for providing the experimental Kd values.
Faculty Advisor: Divi Venkateswarlu, firstname.lastname@example.org