On the importance of being aromatic at an antibody-protein antigen interface: Mutagenesis of the extracellular interferon gamma receptor and recognition by the neutralizing antibody A6

A complex formed between the extracellular human interferon gamma receptor alpha-chain (hIFN gamma R) and the Fab fragment of the neutralizing antibod y A6 has been studied by site-directed mutagenesis. Five complementarity de termining regions of the A6 antibody interact primarily with the CC' surfac e loop of the receptor, from Lys47 to Trp56, although contact is also made with residues in the neighbouring F strand, in particular with Trp82. The r elative contribution that individual side-chains make to complex stabilizat ion was assessed with 21 receptors mutants, whose affinity for A6 was monit ored using a surface plasmon resonance biosensor, as well as by solution-ph ase competition ELISA. The results reveal two lysine side-chains (Lys47 and Lys52), an asparagine side-chain (Asn53), and two aromatic side-chains (Ty r49 and Trp82) in the receptor that are important for recognition by A6. Th e role of aromatic side-chains in antibody-antigen recognition is of partic ular interest, not least in this case because 13 aromatic groups (six Tyr, six Trp and one His) are present at the interface (four in V-L, six in V-H and three in the receptor), and several are proximal to the charged and pol ar side-chains of Lys47, Lys52 and Asn53 in the receptor. The results highl ight the possibility for aromatic rings to participate in networks of coope rative interactions with not only hydrophobic, but also charged and hydroge n bond donor and acceptor groups, properties that are well suited for creat ing binding sites for protein epitopes, regardless of the distribution of p olar and non-polar surface residues. These findings may contribute, therefo re, to an understanding of how surface groups on proteins are captured by t he often aromatic-rich hypervariable loops of antibodies, and may be of val ue for the design of molecules with novel recognition properties. (C) 1999 Academic Press.