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The interaction between the acidic transactivation domain of the human tumor suppressor protein p53 (p53TAD) and the 70 kDa subunit of human replication protein A (hRPA70) was investigated using heteronuclear magnetic resonance spectroscopy. A 1H-15N heteronuclear single quantum coherence (HSQC) titration experiment was performed on a 15N-labeled fragment of hRPA70, containing the N-terminal 168 residues (hRPA701-168) and p53TAD. HRPA701-168 residues important for binding were identified and found to be localized to a prominent basic cleft. This binding site overlapped with a previously identified single-stranded DNA-binding site, suggesting that a competitive binding mechanism may regulate the formation of p53TAD-hRPA70 complex. The amide 1H and 15N chemical shifts of an uniformly 15N-labeled sample of p53TAD were also monitored before and after the addition of unlabeled hRPA701-168. In the presence of unlabeled hRPA701-168, resonance lineshapes increased and corresponding intensity reductions were observed for specific p53TAD residues. The largest intensity reductions were observed for p53TAD residues 42-56. Minimal binding was observed between p53TAD and a mutant form of hRPA701-168, where the basic cleft residue R41 was changed to a glutamic acid (R41E), demonstrating that ionic interactions play an important role in specifying the binding interface. The region of p53TAD most affected by binding hRPA701-168 was found to have some residual alpha helical and beta strand structure; however, this structure was not stabilized by binding hRPA701-168. 15N relaxation experiments were performed to monitor changes in backbone dynamics of p53TAD when bound to hRPA701-168. Large changes in both the transverse (R2) and rotating frame (R1) relaxation rates were observed for a subset of the p53TAD residues that had 1H-15N HSQC resonance intensity reductions during the complex formation. The folding of p53TAD upon complex formation is suggested by the pattern of changes observed for both R2 and R1. A model that couples the formation of a weak encounter complex between p53TAD and hRPA701-168 to the folding of p53TAD is discussed in the context of a functional role for the p53-hRPA70 complex in DNA repair.

NMR chemical shift and relaxation measurements provide evidence for the coupled folding and binding of the p53 transactivation domain