Functional mapping of factor VIII C2 domain

Abstract

The factor VIII (FVIII) is a cofactor of the coagulation cascade. The FVIII C2 domain is a critical domain that participates in the interactions with the von Willebrand factor and the phospholipidic surfaces. To assess the importance of each residue of this domain in the maintenance of the structure and the function of FVIII, a number (n=139) of mutants were generated by substituting the original residues, from Ser2173 to Gly2325, by an alanine. Mutants were built within a complete B domain-deleted FVIII and expressed in COS-1 cells. Mutant antigen levels and procoagulant activities were measured. Two in silico analyses, a sliding average procedure and an analysis of the mutation energy cost were conducted in parallel on the FVIII structure. Both results were in agreement with the functional data, and illustrated the benefit of using such strategies prior to targeting specific residues in the aim of generating active recombinant molecules. The functional assays identify the residues that are important to maintaining the structure of the C2 domain, mainly those forming β-sheet, and those that can afford substitution, establishing a detailed functional relation with the available crystallographic data. This study provided a comprehensive functional mapping of the FVIII C2 domain and discussed the implication of specific residues in respect to the maintenance in the activity and structure stability, the efficiency in secretion, the binding to phospholipids and the formation of epitope.

Prediction of hemophilia A severity using a small-input machine-learning framework

“…We used an in vitro mutagenesis screening where almost all of the residues of the A2 and the C2 domains were mutated to alanine 30 , 31 . The A2 domain is important for the FVIII activity because in addition to its role in stabilizing the protein structure 32 , it contains a binding site for FIXa (ref.…”

“…The ELISA assay was used to assess the efficiency of FVIII mutant constructs expression and secretion (often referred as antigen assay because they measure both functional and nonfunctional FVIII proteins by the amount of FVIII antigen (protein) immobilized on the ELISA plate. Two commercial kits were used to apply the “sandwich” method, where the antigen (FVIII construct) is captured between two layers of antibodies 30 , 31 .…”

Membrane Interaction of the Factor VIIIa Discoidin Domains in Atomistic Detail

“…This could suggest that the major components necessary for sufficient membrane interaction for FVIIIa in vivo are non-specific contributions, such as hydrophobic partitioning and electrostatics, consistent with the results of the simulations described here. Furthermore, alanine-scanning the FVIII C2 domain within a complete B-domainless FVIII molecule has suggested that primarily resides in structural regions of the domain (β-sheet forming residues) are functionally sensitive to mutation 63 . In addition to these general observations, our results further implicate R2163 in FVIII C1, and R2220 and R2320 in FVIII C2 as potentially critical residues in that they are the only basic residues capable of forming direct interactions with PS lipids.…”

Eukaryotic GPN-loop GTPases paralogs use a dimeric assembly reminiscent of archeal GPN

“…1). Preliminary in silico mutagenesis was performed at this position with every other 19 amino acids (AA) using in-house software 21 . It was found that most AA replacements were deleterious.…”

“…The mutational energy cost for replacing Glu112 residue was performed by computing ΔΔE E→K = ΔE K - ΔE E , 46 where ΔE K and ΔE E are the relative potential energies of the mutant Lys and WT Glu in an optimized protein environment of the crystal structure, respectively 21 . The potential energy is the sum of vdw and Coulombic interactions.…”