Structure and catalytic mechanism of the E . coli chemotaxis phosphatase CheZ

The protein CheZ, which has the last unknown structure in the Escherichia coli chemotaxis pathway, stimulates the dephosphorylation of the response regulator CheY by an unknown mechanism. Here we report the co-crystal structure of CheZ with CheY, Mg2+ and the phosphoryl analog, BeF3. The predominant structural feature of the CheZ dimer is a long four-helix bundle composed of two helices from each monomer. The side chain of Gln 147 of CheZ inserts into the CheY active site and is essential to the dephosphorylation activity of CheZ. Gln 147 may orient a water molecule for nucleophilic attack, similar to the role of the conserved Gln residue in the RAS family of GTPases. Similarities between the CheY–CheZ and Spo0F–Spo0B structures suggest a general mode of interaction for modulation of response regulator phosphorylation chemistry. Over the past 25 years, the molecular events governing chemotaxis in Escherichia coli have emerged in detail1,2. As a consequence, bacterial chemotaxis is one of the most thoroughly characterized biological information processing networks, making it an excellent model for computer simulations in the field of systems biology3,4. Bacterial chemotaxis has also served as a prototype for two-component regulatory systems, a signaling transduction strategy widely used in prokaryotes, plants and fungi5 for response to a broad range of extracellular stimuli. In chemotaxis, chemical attractants or repellants bound to transmembrane receptors regulate the autophosphorylation of the sensor kinase CheA. The phosphoryl group is transferred from a histidyl residue on CheA to an aspartyl residue (Asp 57) on the response regulator CheY. Interaction of phospho-CheY (CheY-P) with the flagellar motor dictates cellular swimming behavior. Efficient removal of the phosphoryl group from CheY-P is essential for the continuous response to environmental changes. Although CheY can catalyze its own dephosphorylation, the protein CheZ in enteric bacteria such as E. coli stimulates this rate and is critical for the rapid response of bacteria to stimuli6, which is essential for chemotaxis. An invaluable component of our current level of understanding of chemotaxis is the atomic structures of six of the seven proteins in the pathway. Genetic and biochemical studies have generated considerable, but fragmented, data about the seventh protein, CheZ, which could be put in context by the determination of its structure. Protease sensitivity suggests that CheZ has at least two structural domains7,8. Disabling single-site substitutions cluster in six regions of the CheZ primary structure8,9. Except for the C-terminus of CheZ, which is involved in binding to the α4β5α5 surface of CheY-P10,11, the functional roles and spatial relationships of the CheZ regions identified by mutagenesis are unknown. Genetic studies suggest that α1 and the β5α5 loop13 of CheY bind to as yet unidentified portions of CheZ. The mechanism by which CheZ catalyzes the dephosphorylation of CheY is also not known. CheY autodephosphorylation is believed to proceed through a direct reversal of the phosphorylation reaction14, which involves an in-line substitution mechanism mediated by an active site Mg2+ (ref. 15). Whether CheZ acts as an allosteric effector of CheY autodephosphorylation activity or as a conventional phosphatase is not known. Possible regulation of CheZ activity by CheY-P16 or a truncated form of CheA17 have been reported but their molecular mechanisms have not been elucidated. The lack of detailed structural or mechanistic information concerning CheZ has made our understanding of chemotaxis and, hence, its use as a model system, incomplete. Our goal was to determine the three dimensional structure of CheZ with the hope that the structure would provide insight into its catalytic mechanism. The recent discovery that BeF3 acts as a stable functional18 and structural analog19 of the phosphoryl group in CheY-P has allowed us to determine the co-crystal structure of CheZ and CheY–BeF3 –Mg2+. The structure completes the set of atomic structures for the seven chemotaxis proteins and reveals the fundamental mechanism of CheZ-stimulated dephosphorylation of CheY.