The retinoblastoma gene product (pRB) participates in regulating mammalian cell replication. The mechanism responsible for pRB's growth regulatory activity is uncertain. However, pRB is known to bind
viral transforming proteins including the
papilloma virus E7
protein, cellular
proteins, and
DNA. pRB contains a critical domain termed the "binding pocket" which is required for binding activities. This binding pocket contains 8
cysteine residues. A naturally occurring mutation affecting one of these cysteines is known to eliminate pRB's
protein and
DNA binding activities. To investigate the
cysteine residues in pRB's binding pocket, each residue was mutated to
alanine,
phenylalanine, or
serine. These mutant genes were used to prepare pRBs harboring specific amino acid substitutions. Individual mutations at positions 407, 553, 666, and 706 depressed pRB binding to E7
protein,
DNA, and a conformation-specific anti-pRB antibody, XZ133. Combinations of these inhibitory mutations exhibited additive inhibitory effects on pRB's binding properties. Mutations at positions 438, 489, 590, 712, and 853 did not affect pRB binding to E7
protein,
DNA, or the XZ133 antibody. Combination of these five neutral mutations yielded a pRB species with full E7
protein,
DNA, and XZ133 binding activities. These studies indicate that the
cysteine residues at positions 407, 553, 666, and 706 contribute to the E7
protein and
DNA binding properties of pRB and appear to do so by maintaining pRB's normal conformation.