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.
Highlights
Cyclic AMP receptor protein plays a key role in the regulation of more than 150 genes in prokaryotes [1,2,3,4]
The low solubility, the low stability at high temperature (313 K), and the rapid relaxation time of the cyclic nucleotide-bound Cyclic AMP receptor protein (CRP) prevented us from obtaining diverse threedimensional NMR spectra, such as triple resonance spectra, which are essential for its sequence-specific NMR assignments [9]
Due to the insufficiency of the assignments for the cyclic nucleotide-bound CRP, the present results clearly revealed for the first time that a CRP dimer could bind four cAMP molecules in the absence of DNA in solution, forming two distinct cAMP-binding sites in each monomer
Summary
99.9% D2O, cAMP, and cGMP were purchased from Sigma, and all other materials were either analytical or biotechnological grade. The concentrations of the protein and cyclic nucleotides were determined spectrophotometrically using the following extinction coefficients: 41,000 MϪ1 cmϪ1 at 278 nm for the CRP dimer [8, 9]; 14,650 MϪ1 cmϪ1 at 258 nm for cAMP [30]; 12,950 MϪ1 cmϪ1 at 254 nm for cGMP [30]. The conventional two-dimensional 1H-15N HSQC spectra of 50 M [U-15N]CRP dissolved in 50 mM potassium phosphate buffer (pH 6.0) containing 0.5 M potassium chloride, 7% D2O, and diverse concentrations of cAMP or cGMP were obtained at 313 K on a Bruker DRX 600 spectrometer. Peak intensities were calibrated between spectra by making the Gln resonance intensity of each spectrum equal to each other, since the resonance retained the most constant chemical shift and resonance intensity independently of the addition of cyclic nucleotides
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