Abstract
Treatment for hemophilia A consists of repeated intravenous infusions of blood coagulation factor VIII (fVIII), either recombinant or derived from pooled plasma cryoprecipitate. While several recombinant versions are clinically available, all existing fVIII replacement products express at low levels, harbor short half-lives, and are cost intensive to produce for prophylaxis and therapeutic intervention. Here, we report the 3.2 Å structure of a bioengineered construct of fVIII, termed ET3i, which is a human/porcine chimeric B domain-deleted heterodimer with an improved expression profile and slower A2 domain dissociation following proteolytic activation of fVIII (Figure 1A). Previous structural knowledge of fVIII is at 3.7 Å resolution or lower, and these structures display inconsistencies in their atomic details. The structure reported herein shows remarkable conservation amongst the core folds and domain arrangement of the fVIII protein as well as provides a more detailed view of the interface between the A2 domain and the remaining fVIII structure. With two fVIII molecules in the asymmetric unit of the crystal, we independently observe two distinct conformations of the C2 domain relative to the remaining fVIII structure. While one model (chain A) superimposes with previously-determined fVIII structures without significant deviation, the second model (chain B) indicates a 'barrel tilt and roll' conformational change of the C2 domain that may have implications for how proteolytically activated fVIII binds to activated platelet surfaces (Figure 1B). The improved model and stereochemistry of the ET3i A chain was subsequently used as a scaffold to generate an improved, refined model of human fVIII. In the case of both original datasets to 3.7 Å (pdb#: 2R7E) and 3.98 Å (pdb#: 3CDZ), our new model based on the ET3i structure resulted in improved refinement statistics for human fVIII. These improved structures further illustrate the conserved nature of the ET3i protein as well as provide a more confident model of fVIII for next generation engineering efforts to develop fVIII therapeutics with longer half-lives, higher expression levels and lower immunogenicity.
Doering:Expression Therapeutics, LLC: Equity Ownership, Research Funding.
