Structural Basis for the Localization of the Chemotaxis Phosphatase CheZ by CheA _S

Abstract

CheA-short interacts with CheZ to localize CheZ to cell poles. The fifth helical region (residues 112 to 133) from the phosphotransfer domain of CheA interacts with CheZ and becomes ordered and helical, although it lacks a stable fold in the CheA fragment comprising residues 98 to 150 alone. One CheA molecule binds to one CheZ dimer.During bacterial chemotaxis, transmembrane receptors regulate the activity of the chemotaxis-specific histidine autokinase CheA with the aid of a coupling protein, CheW. CheA acts to phosphorylate the response regulator CheY and the response regulator domain of the methylesterase CheB. Phosphorylated CheY (CheY-P) binds to the "switch complex" in the flagellar motor to regulate the sense of rotation of the motor. CheZ acts as a CheY phosphate phosphatase.Maddock and Shapiro (4) showed that the chemotaxis receptors tend to be clustered and often located at polar ends of bacterial cells. This localization of receptors is in large part dependent on the presence of CheA and CheW, and the clusters that form in wild-type cells contain receptors, CheA, CheW, CheY, and CheZ (8). These clusters are essential for proper communication among receptors and other members of the signal transduction complex.In Escherichia coli and many related bacteria, a naturally occurring short form of CheA (CheA S ) (7) interacts with CheZ, enhances the rate of dephosphorylation of CheY-P (5, 10), and is responsible for the localization of CheZ to the polar assemblies of receptors, CheA, and CheW (1). Having the kinase and the phosphatase colocalized generates more uniform CheY levels within the bacterial cell (9).In order to understand the structural basis of the CheA SCheZ interaction, we examined a CheA fragment containing residues 98 to 150 (CheA ). This fragment begins at the alternative site of translation initiation for CheA S and extends into the linker region joining the histidine phosphotransfer domain to the CheY-binding domain. This fragment includes residues that correspond to the C terminus of the fourth helix and the complete fifth helix of the intact histidine phosphotransfer domain, also known as the P1 domain. H dimension, suggesting a high degree of backbone mobility (2). Complete backbone assignments for the nonproline residues in CheA 98-150 were made using standard HNCACB and CBCA(CO)NH methods (6). We have assigned Glu100 through His154 (a residue of the His 6 tag). Although the nuclear magnetic resonance (NMR) spectra and 15 N relaxation properties (3) suggest that this fragment has no stable structure under these conditions, the central region (Asp112 to Glu133) exhibits positive ( 1 H-) 15 N nuclear Overhauser effects and large, positive (up to 2.6 ppm) C␣ secondary shifts (11), consistent with a partially rigid, helical structure.To establish which residues of CheA 98-150 are involved in CheZ binding, we titrated 15 N-labeled CheA 98-150 with unlabeled wild-type CheZ. The spectra were collected using a mixture of 50 mM sodium phosphate buffer, pH 6.8, 1 mM EDTA, and 2 mM dithiothreit...

Protein Residues and a Novel Motif Involved in the Cellular Localization of CheZ in Azorhizobium caulinodans ORS571

“…In E. coli , the polar localization of CheZ is achieved by binding with a short form of CheA (CheA EC ), which begins at Met-98 of full length CheA EC ( Cantwell et al, 2003 ; Hao et al, 2009 ). There is only one CheA protein encoded in A. caulinodans genome, termed as CheA AC .…”

“…CheZ EC interacts with CheA using a small region of amino acids with most interactions coming from the apical hairpin loop consisting of two aromatic residues, Phe-97 and Trp-98 ( Cantwell and Manson, 2009 ). For CheAs, two hydrophobic residues Leu-123 and Leu-126 in the N-terminus of CheA are responsible for CheZ EC interactions ( Cantwell et al, 2003 ; Hao et al, 2009 ).…”

Auxiliary phosphatases in two-component signal transduction

“…In E. coli , a subpopulation of CheZ is associated with the transmembrane complex composed of chemoreceptors, the CheA sensor kinase, and the CheW scaffolding protein [23,24]. CheZ localization is mediated by interaction between the hairpin end of the four-helix bundle (Figure 2a) and an exposed hydrophobic helix of CheA-short (CheA s )- a truncated version of CheA [25–27]. Association of CheZ with CheA s increases CheZ phosphatase activity by two to three-fold [28].…”

“…Association of CheZ with CheA s increases CheZ phosphatase activity by two to three-fold [28]. Binding between relevant fragments of CheA s and CheZ results in NMR chemical shift perturbations that propagate down the CheZ four-helix bundle to the catalytic site, providing a possible structural explanation for CheZ activation by CheA s [25]. Clustering of CheZ at the cell poles results in a homogenous concentration of CheYp across the length of the cell [29,30] which may be important in synchronization of the rotational direction of flagellar motors.…”

Action at a Distance: Amino Acid Substitutions That Affect Binding of the Phosphorylated CheY Response Regulator and Catalysis of Dephosphorylation Can Be Far from the CheZ Phosphatase Active Site

“…Several observations support this proposed mechanism for the GOF mutants and their suppressors. First, there is direct nuclear magnetic resonance evidence for the propagation of structural changes along the CheZ four-helix bundle upon the binding of the CheA S protein to the hairpin end (7,13). Furthermore, CheA S has been reported to enhance CheZ activity about 2-fold (21,40), an enhancement similar in magnitude to that exhibited by GOF mutants, thus raising the possibility that structural changes in the bundle affect CheZ activity.…”