are cofounders of Expression Therapeutics and own equity in the company. hydrophobic with several aromatic residues and possesses 2429 ?2 of buried surface area. The majority of the binding epitope is within the C1 domain as predicted, and there is a minor binding interface with the A3 domain. The variable domains straddle the Lys2065-Trp2070 surface loop of the fVIII C1 domain (Figure 1B), serving as the centralized region of the epitope for 2A9 and agrees with Palomid 529 (P529) HDX protection patterns Palomid 529 (P529) for other group A anti-C1 inhibitors.7 Here, Phe2068 is completely buried at the fVIII/2A9 interface and makes extensive hydrophobic contacts with Phe48, Phe49, Leu45, and Tyr33 of the variable domain of the light chain, forming a phenylalanine sandwich. This is consistent with mutational analyses showing that a Phe2068His mutant abrogates 2A9 binding.9 The Arg2150-Tyr2156 loop forms additional contacts between the C1 domain and the variable domains of 2A9 (Figure 1C), with Arg2150 and His2152 making direct contacts with the heavy chain and Thr2154-Tyr2156 making direct contacts with the light chain. These contacts are also substantiated by HDX protection patterns in the presence of 2A9 (supplemental Figure 1).7 Additional C1 domain interactions occur between Lys2110 and Trp2112 with the heavy chain of 2A9. Notably, the intermolecular contacts between the C1 domain and 2A9 in our structure are also involved in the fVIII/VWF DD3 complex as shown by HDX.17 Minor interactions also occur between Ile2102 and Thr2122 and the heavy and light variable regions, respectively, which are also consistent with minor HDX protection patterns for the VWF DD3 region. The extent Palomid 529 (P529) of the overlap between the 2A9 epitope and the VWF-binding interface supports the hypothesis that the mechanism of pathology for group A antibody inhibitors is to increase the clearance of fVIII by disrupting the VWF interaction, dramatically decreasing the plasma half-life of fVIII.7,8 With a Keq and koff of 0.1 nM and 2.2 10?4 s?1, respectively, the 2A9 antibody possesses comparable binding characteristics to VWF.17-20 The structure of the 2A9 complex also indicates that the A3 domain contributes to the overall epitope, forming multiple contacts between the Phe1743-Tyr1748 loop of the A3 domain and the 2A9 light chain (Figure 1D). Interestingly, the second N-acetylglucosamine residue at Asn1810 makes electrostatic interactions with Glu1 and Lys95 of the 2A9 light and heavy chains, respectively. The overall structure of the 2A9 antibody Rabbit Polyclonal to Ik3-2 bound to fVIII indicates that glycosylation sites potentially contribute to inhibitor development and illustrates that the epitope definitions are more complex than fVIII domain specificity. Moreover, simultaneous recognition of the C1 and A3 domains further supports the physiological relevance of the relative positioning of the C1 and A3 Palomid 529 (P529) domains in determined fVIII structures.14,21,22 Open in a separate window Figure 1. The structure of the ET3i fVIII/antiCC1 domain 2A9 Fab fragment. (A) Ribbon diagram representation of ET3i in complex with the 2A9 antiCC1 domain Fab antibody fragment (Protein Data Bank identifier [PDBID#] 7K66) (pink, porcine A1 domain; dark magenta, porcine A3 domain; dark teal, human A2, C1, and C2 domains; orange, 2A9 Fab light chain [LC]; green, 2A9 Fab heavy chain [HC]). (B) Direct molecular contacts between the 2A9 antibody and the C1 Palomid 529 (P529) domain 2065-2070 loop. (C) Direct molecular contacts between the 2A9 antibody and the C1 domain 2050-2056 loop. (D) Surface contacts between the 2A9 antibody and the A3 domain 1743-1748 loop and the second N-acetylglucosamine (NAG) residue N-linked to Asn1810. Conformational changes of the fVIII C2 domain.