Identification of contact residues and definition of the CAR-binding site of adenovirus type 5 fiber protein

The binding of adenovirus (Ad) fiber knob to its cellular receptor, the cox sackievirus and Ad receptor (CAR), promotes virus attachment to cells and i s a major determinant of Ad tropism, Analysis of the kinetics of binding of Ad type 5 (Ad5) fiber knob to the soluble extracellular domains of CAR tog ether (sCAR) and each immunoglobulin (Ig) domain (IgV and IgC2) independent ly by surface plasmon resonance demonstrated that the IgV domain is necessa ry and sufficient for binding, and no additional membrane components are re quired to confer high-affinity binding to Ad5 fiber knob. Four Ad5 fiber kn ob mutations, Ser408Glu and Pro409Lys in the AB loop, Tyr477Ala in the DG l oop, and Leu485Lys in beta strand F, effectively abolished high-affinity bi nding to CAR, while Ala406Lys and Arg412Asp in the AB loop and Arg481Glu in beta strand E significantly reduced the level of binding. Circular dichroi sm spectroscopy showed that these mutations do not disorder the secondary s tructure of the protein, implicating Ser408, Pro409, Tyr477, and Leu485 as contact residues, with Ala406, Arg412, and Arg481 being peripherally or ind irectly involved in CAR binding. The critical residues have exposed side ch ains that form a patch on the surface, which thus defines the high-affinity interface for CAR. Additional site-directed mutagenesis of Ad5 fiber knob suggests that the binding site does not extend to the adjacent subunit or t oward the edge of the R sheet. These findings have implications for our und erstanding of the biology of Ad infection, the development of novel Ad vect ors for targeted gene therapy, and the construction of peptide inhibitors o f Ad infection.