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

Abstract

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.

Bivalent-metal binding to CheY protein. Effect on protein conformation

“…Estimation of the concentration of bound metal in CheYl by AA shows that the binding stoichiometry is 1: 1, as in CheYl'. These two metal-bound forms of the protein also differ in their interaction with phosphates, as described in the following paper (Kar et al, 1992). We have no explanation as yet for this difference.…”

“…However, we have eliminated protein degradation as a cause. CheYl', obtained from freshly prepared CheY2 by the addition of bivalent metals, shows phosphate-binding properties that are distinctly different from those of freshly prepared CheY I (Kar et al, 1992). Also, CheY ', CheY2 and CheYl are all stable at low temperatures (0-10 C) for over 1 month, and their respective metal-and phosphate-binding properties remain unchanged over this time period.…”