Specificity and affinity of binding of phosphate-containing compounds to CheY protein

• Published in: Biochemical journal Vol. 287; no. 2; pp. 533 - 543
• Main Authors: KAR, L, DE CROOS, P. Z, ROMAN, S. J, MATSUMURA, P, JOHNSON, M. E
• Format: Journal Article
• Language: English
• Published: Colchester Portland Press 15-10-1992
• Subjects: Adenine - metabolism; Adenosine - metabolism; Adenosine Diphosphate - metabolism; Adenosine Monophosphate - metabolism; Adenosine Triphosphate - metabolism; Bacterial Proteins; Bacteriology; Binding Sites; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Guanosine Triphosphate - metabolism; Kinetics; Magnetic Resonance Spectroscopy - methods; Membrane Proteins - metabolism; Methyl-Accepting Chemotaxis Proteins; Microbiology; Motility, taxis; Nucleotides - metabolism; Phosphates - metabolism; Phosphoric Monoester Hydrolases - metabolism; Phosphorus; Phosphorylation; Protein Binding; Protons; Sensitivity and Specificity; Thymine Nucleotides - metabolism; Uridine Triphosphate - metabolism; Proteins; Phosphates; Specificity; Metal ion; Molecular interaction; NMR spectrometry; ATP; Chemotaxis
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2870533

1H- and 31P-n.m.r. have been used to study the interaction of the bacterial chemotaxis protein, CheY, with ATP and a variety of other phosphates in the presence and absence of bivalent metal ions. In the metal-bound conformation, CheY will bind nucleotide phosphates and phosphates in general, while in the metal-free conformation CheY loses its affinity for phosphates. In the presence of low concentrations of nitroxide-spin-labelled ATP (SL-ATP), specific proton resonances of metal-bound CheY are suppressed, indicating that ATP binds to a specific site on this metal-bound form of the protein. These studies also show that the same resonances are affected by the binding of SL-ATP and Mn2+, indicating that the phosphate- and metal-binding sites are close to each other and to Asp-57 (the site of phosphorylation in CheY). 1H- and 31P-n.m.r. studies using ATP, GTP, TTP, UTP, ADP, AMP and inorganic phosphates show that the binding is not specific for adenine, and does not involve the base directly, but is mediated primarily by the phosphate groups. Experiments with a phosphorylation mutant (Asp-13-->Asn) suggest that the observed phosphate binding and activation of CheY by phosphorylation may be related. Our results indicate that the conformational change and charge interactions brought about by the binding of a metal ion at the active site are required for CheY to interact with a phosphate. These studies also demonstrate the utility of spin-label-induced relaxation in conjunction with two-dimensional-n.m.r. measurements for exploring ligand-binding sites.