Sensorgrams of FVIII-C2 mutants that bound BO2C11 with reduced affinity.

A. The crystal structure of FVIII-C2 (hot pink) complexed with the BO2C11 Fab (cyan) [33] is shown in cartoon representation and with relevant side chains shown explicitly. Sensorgrams corresponding to muteins that bound BO2C11 with k_d > 4.0X that of WT-FVIII-C2 are shown; black lines map each sensorgram to the relevant WT-FVIII-C2 residue. The sensorgrams record the mass of the FVIII-C2 protein that becomes attached to the Fab-coated chip. The orange, blue and purple curves show sensorgrams generated following injections of increasing concentrations of the indicated FVIII-C2 protein. The signals are measured in Resonance Units (RUs), which are arbitrary units. Fits to theoretical curves generated using the experimentally derived rate constants are shown in red. Larger images of these sensorgrams are in S1 Fig. Note that residues 2198–2201 comprise a beta turn that adopted slightly different conformations in the crystal structures of FVIII-C2 [50] and of FVIII-C2 bound to BO2C11 [33]. B. Expanded view of the FVIII-C2 (hot pink)—BO2C11 (cyan) interface. The six FVIII-C2 residues comprising the functional epitope are shown in stick representation (labeled in black font), as are the BO2C11 residues that form hydrogen bonds with the side chains of these six residues (labeled in blue font). The corresponding proteins are shown in light, transparent cartoon representation. Hydrogen bonds are indicated by red dotted lines and the interatomic distances are given.