ROLE OF CYSTEINE RESIDUES IN REGULATION OF P53 FUNCTION

Previous studies of p53 have implicated cysteine residues in site-spec ific DNA binding via zinc coordination and redox regulation (P. Hainau t and J. Milner, Cancer Res. 53:4469-4473, 1993; T. R. Hupp, D. W. Mee k, C. A. Midgley, and D. P. Lane, Nucleic Acids Res. 21:3167-3174, 199 3). We show here that zinc binding and redox regulation are, at least in part, distinct determinants of the binding of p53 to DNA. Moreover, by substituting serine for each cysteine in murine p53, we have inves tigated the roles of individual cysteines in the regulation of p53 fun ction. Substitution of serine for cysteine at position 40, 179, 274, 2 93, or 308 had little or no effect on p53 function. In contrast, repla cement of cysteine at position 173, 235, or 239 markedly reduced in vi tro DNA binding, completely blocked transcriptional activation, and le d to a striking enhancement rather than a suppression of transformatio n by p53. These three cysteines have been implicated in zinc binding b y X-ray diffraction studies (Y. Cho, S. Gorina, P. D. Jeffrey, and N. P. Pavletich, Science 265:346-355, 1994); our studies demonstrate the functional consequences of the inability of the central DNA-binding do main of p53 to bind zinc. Lastly, substitutions for cysteines at posit ion 121, 132, 138, or 272 partially blocked both transactivation and t he suppression of transformation by p53. These four cysteines are loca ted in the loop-sheet-helix region of the site-specific DNA-binding do main of p53. Like the cysteines in the zinc-binding region, therefore, these cysteines may cooperate to modulate the structure of the DNA-bi nding domain. Our findings argue that p53 is subject to more than one level of conformational modulation through oxidation-reduction of cyst eines at or near the p53-DNA interface.