title: NMR chemical shift and relaxation measurements provide evidence for the coupled folding and binding of the p53 transactivation domain.
abstract: 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.
authors.affiliation: Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho PO Box 443052, Life Science South Room 142, Moscow 83844-3052, Idaho, USA.
authors.forename: Pamela D
authors.initials: PD
authors.lastname: Vise
authors.forename: Bharat
authors.initials: B
authors.lastname: Baral
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Latos
authors.forename: Gary W
authors.initials: GW
authors.lastname: Daughdrill
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051716:Replication Protein A; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000596:Amino Acids; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D015534:Trans-Activators; D016159:Tumor Suppressor Protein p53