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 (hIFNgammaR) and the Fab fragment of the neutralizing antibody A6 has been studied by site-directed mutagenesis. Five complementarity determining regions of the A6 antibody interact primarily with the CC' surface 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 relative contribution that individual side-chains make to complex stabilization was assessed with 21 receptors mutants, whose affinity for A6 was monitored using a surface plasmon resonance biosensor, as well as by solution-phase competition ELISA. The results reveal two lysine side-chains (Lys47 and Lys52), an asparagine side-chain (Asn53), and two aromatic side-chains (Tyr49 and Trp82) in the receptor that are important for recognition by A6. The role of aromatic side-chains in antibody-antigen recognition is of particular interest, not least in this case because 13 aromatic groups (six Tyr, six Trp and one His) are present at the interface (four in VL, six in VH and three in the receptor), and several are proximal to the charged and polar side-chains of Lys47, Lys52 and Asn53 in the receptor. The results highlight the possibility for aromatic rings to participate in networks of co-operative interactions with not only hydrophobic, but also charged and hydrogen bond donor and acceptor groups, properties that are well suited for creating binding sites for protein epitopes, regardless of the distribution of polar and non-polar surface residues. These findings may contribute, therefore, to an understanding of how surface groups on proteins are captured by the often aromatic-rich hypervariable loops of antibodies, and may be of value for the design of molecules with novel recognition properties.
Hofstädter
K
K
Department of Chemistry, University of Zurich, Winterthurerstrasse 190, Zurich, 8057, Switzerland.
Stuart
F
F
Jiang
L
L
Vrijbloed
J W
JW
Robinson
J A
JA
D000911:Antibodies, Monoclonal / Q000276:immunology; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D005110:Extracellular Space; D006801:Humans; D016296:Mutagenesis; D009500:Neutralization Tests; D017471:Receptors, Interferon / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D020349:Surface Plasmon Resonance; D000096743:Interferon gamma Receptor
D000911:Antibodies, Monoclonal; D017471:Receptors, Interferon; D000096743:Interferon gamma Receptor