Structural insights into blood coagulation factor VIII: Procoagulant complexes, membrane binding, and antibody inhibition.

Advances in structural studies of blood coagulation factor VIII (FVIII) have provided unique insight into FVIII biochemistry. Atomic detail models of the B domain-deleted FVIII structure alone and in complex with its circulatory partner, von Willebrand factor (VWF), provide a structure-based rationale for hemophilia A-associated mutations which impair FVIII stability and increase FVIII clearance rates. In this review, we discuss the findings from these studies and their implications toward the design of a recombinant FVIII with improved circulatory half-life. Additionally, we highlight recent structural studies of FVIII bound to inhibitory antibodies that have refined our understanding of FVIII binding to activated platelet membranes and formation of the intrinsic tenase complex. The combination of bioengineering and structural efforts to understand FVIII biochemistry will improve therapeutics for treating hemophilia A, either through FVIII replacement therapeutics, immune tolerance induction, or gene therapy approaches.

The 3.2 Å structure of a bioengineered variant of blood coagulation factor VIII indicates two conformations of the C2 domain.

BACKGROUND: Coagulation factor VIII represents one of the oldest protein-based therapeutics, serving as an effective hemophilia A treatment for half a century. Optimal treatment consists of repeated intravenous infusions of blood coagulation factor VIII (FVIII) per week for life. Despite overall treatment success, significant limitations remain, including treatment invasiveness, duration, immunogenicity, and cost. These issues have inspired research into the development of bioengineered FVIII products and gene therapies. OBJECTIVES: To structurally characterize a bioengineered construct of FVIII, termed ET3i, which is a human/porcine chimeric B domain-deleted heterodimer with improved expression and slower A2 domain dissociation following proteolytic activation by thrombin. METHODS: The structure of ET3i was characterized with X-ray crystallography and tandem mass spectrometry-based glycoproteomics. RESULTS: Here, we report the 3.2 Å crystal structure of ET3i and characterize the distribution of N-linked glycans with LC-MS/MS glycoproteomics. This structure shows remarkable conservation with the human FVIII protein and provides a detailed view of the interface between the A2 domain and the remaining FVIII structure. With two FVIII molecules in the crystal, we observe two conformations of the C2 domain relative to the remaining FVIII structure. The improved model and stereochemistry of ET3i served as a scaffold to generate an improved, refined structure of human FVIII. With the original datasets at 3.7 Å and 4.0 Å resolution, this new structure resulted in improved refinement statistics. CONCLUSIONS: These improved structures yield a more confident model for next-generation engineering efforts to develop FVIII therapeutics with longer half-lives, higher expression levels, and lower immunogenicity.

Ribosomal protein L7/L12 is required for GTPase translation factors EF-G, RF3, and IF2 to bind in their GTP state to 70S ribosomes.

Ribosomal protein L7/L12 is associated with translation initiation, elongation, and termination by the 70S ribosome. The guanosine 5' triphosphate hydrolase (GTPase) activity of elongation factor G (EF-G) requires the presence of L7/L12, which is critical for ribosomal translocation. Here, we have developed new methods for the complete depletion of L7/L12 from Escherichia coli 70S ribosomes to analyze the effect of L7/L12 on the activities of the GTPase factors EF-G, RF3, IF2, and LepA. Upon removal of L7/L12 from ribosomes, the GTPase activities of EF-G, RF3, and IF2 decreased to basal levels, while the activity of LepA decreased marginally. Upon reconstitution of ribosomes with recombinant L12, the GTPase activities of all GTPases returned to full activity. Moreover, ribosome binding assays indicated that EF-G, RF3, and IF2 require L7/L12 for stable binding in the GTP state, and LepA retained > 50% binding. Lastly, an EF-G∆G' truncation mutant possessed ribosome-dependent GTPase activity, which was insensitive to L7/L12. Our results indicate that L7/L12 is required for stable binding of ribosome-dependent GTPases that harbor direct interactions to the L7/L12 C-terminal domains, either through a G' domain (EF-G, RF3) or a unique N-terminal domain (IF2). Furthermore, we hypothesize this interaction is concomitant with counterclockwise ribosomal intersubunit rotation, which is required for translocation, initiation, and post-termination.

The tertiary structure and domain organization of coagulation factor VIII.

Factor VIII (fVIII) is a serum protein in the coagulation cascade that nucleates the assembly of a membrane-bound protease complex on the surface of activated platelets at the site of a vascular injury. Hemophilia A is caused by a variety of mutations in the factor VIII gene and typically requires replacement therapy with purified protein. We have determined the structure of a fully active, recombinant form of factor VIII (r-fVIII), which consists of a heterodimer of peptides, respectively containing the A1-A2 and A3-C1-C2 domains. The structure permits unambiguous modeling of the relative orientations of the 5 domains of r-fVIII. Comparison of the structures of fVIII, fV, and ceruloplasmin indicates that the location of bound metal ions and of glycosylation, both of which are critical for domain stabilization and association, overlap at some positions but have diverged at others.