title: Stoichiometry and structural effect of the cyclic nucleotide binding to cyclic AMP receptor protein.
abstract: Cyclic AMP receptor protein (CRP) is a homodimeric protein, which is activated by cAMP binding to function as a transcriptional regulator of many genes in prokaryotes. Until now, the actual number of cAMP molecules that can be bound by CRP in solution has been ambiguous. In this work, we performed a nuclear magnetic resonance study on CRP to investigate the stoichiometry of cyclic nucleotide binding to CRP. A series of (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of the protein in the absence and in the presence of cAMP or cGMP were analyzed. The addition of cAMP to CRP induced a biphasic spectral change up to 4 equivalents, whereas the cGMP addition made a monophasic change up to 2 equivalents. Altogether, the results not only established for the first time that CRP possesses two cyclic AMP-binding sites in each monomer, even in a solution without DNA, but also suggest that the syn-cAMP binding sites of the CRP dimer can be formed by an allosteric conformational change of the protein upon the binding of two anti-cAMPs at the N-terminal domain. In addition, a residue-specific inspection of the spectral changes provides some new structural information about the cAMP-induced allosteric activation of CRP.
authors.affiliation: Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea.
authors.forename: Hyung-Sik
authors.initials: HS
authors.lastname: Won
authors.forename: Tae-Woo
authors.initials: TW
authors.lastname: Lee
authors.forename: Sang-Ho
authors.initials: SH
authors.lastname: Park
authors.forename: Bong-Jin
authors.initials: BJ
authors.lastname: Lee
mesh_terms: D000494:Allosteric Regulation; D000242:Cyclic AMP / Q000378:metabolism*; D019281:Dimerization; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D011953:Receptors, Cyclic AMP / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011953:Receptors, Cyclic AMP; D000242:Cyclic AMP