Over 50% of all human cancers involve p53 mutations, which occur mostly in the sequence−specific DNA−binding central domain (p53c), yielding little/non–detectable affinity to the DNA consensus site. Despite our current understanding of protein−DNA recognition, the mechanism(s) underlying the loss in protein−DNA binding affinity/specificity upon single−point mutation are not well understood. Our goal is to identify the common factors governing the DNA−binding loss of p53c upon substitution of Arg 273 to His or Cys, which are abundant in human tumours. By computing the free energies of wild–type and mutant p53c binding to DNA and decomposing them into contributions from individual residues, the DNA−binding loss upon charge/noncharge–conserving mutation of Arg 273 was attributed not only to the loss of DNA phosphate contacts, but also to longer–range structural changes caused by the loss of the Asp 281 salt–bridge. The results herein and in previous works suggest that Asp 281 plays a critical role in the sequence−specific DNA−binding function of p53c by

1. orienting Arg 273 and Arg 280 in an optimal position to interact with the phosphate and base groups of the consensus DNA, respectively, and

2. helping to maintain the proper DNA–binding protein conformation.