Transcriptional activation and repression via the transcription factors p53 and p65 are mediated by hydrophobic short linear motifs (FXX phi phi) in their activation domains (ADs). To understand the molecular basis for specificity in binding to disparate biological targets, a series of chimeric peptides was synthesized, with sequences derived from the ADs of p53, which binds both the general transcriptional machinery and the repressor protein MDM2, and p65, which is reported to bind the general transcriptional machinery but not MDM2. The FXX phi phi motifs of p53 and p65 differ by only two residues, whereas the flanking sequences have no sequence identity. The affinities of the chimeric peptides to MDM2(25-117) and hTAF(II)31(1-140) were determined. Specificity for binding MDM2 via FXX phi phi motifs derives almost entirely from Trp23 of p53, with a 3.0 kcal mol(-1) loss of binding energy when Trp23 is changed to p65-derived Leu. The identity of the N-terminal flanking sequence did not significantly affect binding to MDM2. In contrast, replacement of the C-terminal sequence of p53 with that of p65 increased the affinity of the chimera for MDM2 by 1.1 kcal mol(-1), contrary to expectations. Replacement of the highly conserved residue Pro27 of p53 with Ser from p65 resulted in a 2.3 kcal mol(-1) improvement in binding to MDM2, generating a ligand (p53-P27S) (Kd = 4.7 nM) that exhibits the highest MDM2 affinity observed for a genetically encodable ligand. The basis for the increased affinity of p53-P27S over p53 was examined by circular dichroism and nuclear magnetic resonance. Pro27 disrupts the recognition alpha-helix of p53, with p53-P27S significantly more alpha-helical than p53.