Effects of common cancer mutations on stability and DNA binding of full-length p53 compared with isolated core domains.
Common cancer mutations of p53 tend either to lower the stability or distort the core domain of the protein or weaken its DNA binding affinity. We have previously analyzed in vitro the effects of mutations on the core domain of p53. Here, we extend those measurements to full-length p53, using either the wild-type protein or a biologically active superstable construct that is more amenable to accurate biophysical measurements to assess the possibilities of rescuing different types of mutations by anticancer drugs. The tetrameric full-length proteins had similar apparent melting temperatures to those of the individual domains, and the structural mutations lowered the melting temperature by similar amounts. The thermodynamic stability of tetrameric p53 is thus dictated by its core domain. We determined that the common contact mutation R273H weakened binding to the gadd45 recognition sequence by approximately 700-1000 times. Many mutants that have lowered melting temperatures should be good drug targets, although the common R273H mutant binds response elements too weakly for simple rescue.
Ang
Hwee Ching
HC
Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom.
Joerger
Andreas C
AC
Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom.
Mayer
Sebastian
S
Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom.
Fersht
Alan R
AR
Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom. Electronic address: arf25@cam.ac.uk.
D018797:Cell Cycle Proteins / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009154:Mutation / Q000502:physiology*; D009369:Neoplasms / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism*; D000097642:GADD45 Proteins
D018797:Cell Cycle Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016159:Tumor Suppressor Protein p53; C439070:GADD45A protein, human; D000097642:GADD45 Proteins