MUTATIONS LEADING TO ALTERED CHEA BINDING CLUSTER ON A FACE OF CHEY

CheY is the response regulator of Escherichia coli chemotaxis and is o ne of the best studied response regulators of the two-component signal ing system. CheY can receive phosphate from the histidine kinase, CheA . Phospho-CheY interacts with the motor-switch complex to induce clock wise flagellar rotation, thus causing the cell to tumble. We used an e nzyme-linked immunosorbent assay to study the direct interaction betwe en the kinase, CheA, and the regulator, CheY. The products of random, suppressor, and site-specific cheY mutants were assayed for their abil ity to bind CheA. Nine mutants showed altered binding. We sequenced an d mapped these point mutations on the crystal structure of CheY, and a high degree of spatial clustering was revealed, indicating that this region of CheY is involved in CheA binding. Interestingly, five of the se altered binding mutants were previously defined as being involved i n motor-switch binding interactions. This suggested a possible overlap between the motor-switch binding and CheA binding surfaces of CheY. U sing CheY (Trp-58) fluorescence quenching, we determined the equilibri um dissociation constants of CheA (124-257) binding for these CheY mut ants. The results from the fluorescence quenching are in close agreeme nt with our initial enzyme-linked immunosorbent assay results. Therefo re, we propose that the CheA and the motor binding surfaces on cheY pa rtially overlap and that this overlap allows CheY to interact with eit her the CheA or the flagellar motor, depending on its signaling (phosp horylation) state.