Interface dimensions and packing in the r-fVIII molecule. (A) The light chain and packing of the C domains. The C1 domain (cyan ribbon and pink side chains) is engaged in an extensive docked interaction against the A3 domain (light magenta ribbon and yellow side chains) that involves multiple aromatic, aliphatic, and hydrophilic side chains, several of which are sites of missense mutations associated with protein dysfunction and hemophilia A. An N-linked glycosyl modification (red) is also involved in the C1-A3 interface. In contrast, the C2 domain (blue ribbon and side chains) is loosely tethered to the r-fVIII molecule, displaying small, entirely hydrophilic interfaces with the A3 and C1 domains. Interestingly, a number of residues in these interfaces are also associated with hemophilia. (B) Packing of the trimer of A domains and close up of the packing of the A2 domain (green) against the A3 domain, with residues that have been previously mutated to create engineered disulfide cross-links indicated. These residues are located on 2 adjacent loops in the A domains, which appear to display sufficient flexibility to permit covalent disulfide formation while maintaining r-fVIII activity in vivo.