The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY help to explain their binding affinities to the FliM and CheZ peptides |
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Authors: | McAdams Kenneth Casper Eric S Matthew Haas R Santarsiero Bernard D Eggler Aimee L Mesecar Andrew Halkides Christopher J |
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Affiliation: | a Department of Chemistry and Biochemistry, University of North Carolina Wilmington, 601 S. College Road, Wilmington, NC 28403, USA b Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois College of Pharmacy, 833 S. Wood Street, Chicago, IL 60612-7231, USA |
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Abstract: | CheY is a response regulator in bacterial chemotaxis. Escherichia coli CheY mutants T87I and T87I/Y106W CheY are phosphorylatable on Asp57 but unable to generate clockwise rotation of the flagella. To understand this phenotype in terms of structure, stable analogs of the two CheY-P mutants were synthesized: T87I phosphono-CheY and T87I phosphono-CheY. Dissociation constants for peptides derived from flagellar motor protein FliM and phosphatase CheZ were determined for phosphono-CheY and the two mutants. The peptides bind phosphono-CheY almost as strongly as CheY-P; however, they do not bind T87I phosphono-CheY or T87I/Y106W phosphono-CheY, implying that the mutant proteins cannot bind FliM or CheZ tightly in vivo. The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY were solved to resolutions of 1.8 and 2.4 Å, respectively. The increased bulk of I87 forces the side-chain of Y106 or W106, into a more solvent-accessible conformation, which occludes the peptide-binding site. |
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Keywords: | Chemical modification Structure/function studies Crystallography Fluorescence Calorimetry Chemotaxis Two component systems Protein phosphorylation |
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