Mutations in the DG loop of adenovirus type 5 fiber knob protein abolish high-affinity binding to its cellular receptor CAR

The amino acid residues in adenovirus type 5 (Ad5) fiber that interact with its cellular receptor, the coxsackie PS virus and Ad receptor (CAR), have not been defined, To investigate this, multiple mutations were constructed in the region between residues 479 and 497 in Ad5 fiber (beta-strands E and F and the adjacent region of the DG loop). The effects of these mutations on binding to CAR were determined by use of cell-binding competition experi ments, surface plasmon resonance, and direct binding studies. The mutation effects on the overall folding and secondary structure of the protein were assessed by circular dichroism (CD) spectroscopy. Deletions of two consecut ive amino acids between residues 485 and 493 abolished high-affinity bindin g to CAR; the CD spectra indicated that although there was no disruption of the overall folding and secondary structure of the protein, local conforma tional changes did occur. Moreover, single site mutations in this region of residues with exposed, surface-accessible side chains, such as Thr492, Asn 493, and Val495, had no effect on receptor binding, which demonstrates that these residues are not in contact with CAR themselves. This implies the in volvement of residues in neighboring loop regions. Replacement of the segme nt containing the two very short beta-strands E and F and the turn between them (residues 479 to 486) with the corresponding sequence from Ad3 (beta E FAd3 --> 5 mutation) resulted in the loss of receptor binding. The identica l CD spectra for beta EFAd3 --> 5 and wild-type proteins suggest that these substitutions caused no conformational rearrangement and that the loss of binding may thus be due to the substitution of one or more critical contact residues. These findings have implications for our understanding of the in teraction of Ad5 fiber with CAR and for the construction of targeted recomb inant Ad5 vectors for gene therapy purposes.