Previously, mutational analyses of the DNA binding domain of the bovine papillomavirus E1 protein (E1DBD) identified several hydrophobic residues that are critical for DNA binding activity (M. West, D. Flanery, K. Woytek, D. Rangasamy, and V. G. Wilson, 2001, J. Virol. 75, 11948-11960). Hydrophobic interactions of nonpolar amino acid side chains can contribute to the function of DNA binding proteins through both conformational effects and direct interaction with nucleotides. To further investigate the role of hydrophobic residues in E1DBD function, a more extensive site-directed mutational analysis of hydrophobic amino acids was conducted. Alanine substitutions were made at residues V196, F197, F217, F, 237, V246, L249, and F276, and the mutants were tested for DNA binding activity in vitro and in vivo. The E1 F237A and F276A mutants were completely defective for site-specific DNA binding, while the other mutants retained partial to full wild-type binding activity. Consistent with their DNA binding defect, the F237A and F276A mutants were severely impaired for the ability to support transient in vivo replication of an origin plasmid. Combined with our previous study, five critical hydrophobic residues have been identified: F175, V193, F237, V246, and F276. These five residues localize to two internal clusters in the E1DBD structure designated hydrophobic clusters A (HCA; includes F175, V193, and F276) and B (HCB; includes F237 and V246). Amino acid side chains from residues in HCA and HCB have little surface accessibility and it is unlikely that they are involved in direct contact with DNA. HCA is distal to the DNA binding surface and presumably contributes to global conformational organization of the E1DBD. HCB is positioned beneath the DNA contact surface and we propose that it serves as an anchor or platform device to stabilize the DNA-binding element. A comparable hydrophobic cluster is present in the corresponding position in the T antigen DBD and likely serves a similar function.
ASJC Scopus subject areas
- Infectious Diseases