title: Quantitative analysis of multisite protein-ligand interactions by NMR: binding of intrinsically disordered p53 transactivation subdomains with the TAZ2 domain of CBP.
abstract: Determination of affinities and binding sites involved in protein-ligand interactions is essential for understanding molecular mechanisms in biological systems. Here we combine singular value decomposition and global analysis of NMR chemical shift perturbations caused by protein-protein interactions to determine the number and location of binding sites on the protein surface and to measure the binding affinities. Using this method we show that the isolated AD1 and AD2 binding motifs, derived from the intrinsically disordered N-terminal transactivation domain of the tumor suppressor p53, both interact with the TAZ2 domain of the transcriptional coactivator CBP at two binding sites. Simulations of titration curves and line shapes show that a primary dissociation constant as small as 1-10 nM can be accurately estimated by NMR titration methods, provided that the primary and secondary binding processes are coupled. Unexpectedly, the site of binding of AD2 on the hydrophobic surface of TAZ2 overlaps with the binding site for AD1, but AD2 binds TAZ2 more tightly. The results highlight the complexity of interactions between intrinsically disordered proteins and their targets. Furthermore, the association rate of AD2 to TAZ2 is estimated to be 1.7 × 10(10) M(-1) s(-1), approaching the diffusion-controlled limit and indicating that intrinsic disorder plus complementary electrostatics can significantly accelerate protein binding interactions.
authors.affiliation: Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Munehito
authors.initials: M
authors.lastname: Arai
authors.forename: Josephine C
authors.initials: JC
authors.lastname: Ferreon
authors.forename: Peter E
authors.initials: PE
authors.lastname: Wright
mesh_terms: D001665:Binding Sites; D050882:CREB-Binding Protein / Q000737:chemistry*; D008024:Ligands; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D054730:Protein Interaction Domains and Motifs; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry*
chemical_list: D008024:Ligands; D016159:Tumor Suppressor Protein p53; D050882:CREB-Binding Protein