CheV: CheW-like coupling proteins at the core of the chemotaxis signaling network

Abstract

Microbes have chemotactic signaling systems that enable them to detect and follow chemical gradients in their environments. The core of these sensory systems consists of chemoreceptor proteins coupled to the CheA kinase via the scaffold or coupler protein CheW. Some bacterial chemotaxis systems replace or augment CheW with a related protein, CheV, which is less well understood. CheV consists of a CheW domain fused to a receiver domain that is capable of being phosphorylated. Our review of the literature, as well as comparisons of the CheV and CheW sequence and structure, suggest that CheV proteins conserve CheW residues that are crucial for coupling. Phosphorylation of the CheV receiver domain might adjust the efficiency of its coupling and thus allow the system to modulate the response to chemical stimuli in an adaptation process.

Figure 1. Chemotaxis network architectures in different bacteria

Lines denote types of protein-protein interactions, with arrows indicating activation, blunt-ended lines indicating inactivation, and simple lines indicating interactions that are either not well characterized or that can be either activating or inactivating. Proteins are shown with single connecting lines, although in some cases there may be additional interactions. Abbreviations: L, attractant ligand; R, CheR; B, CheB; Bp, CheB~P; CR, chemoreceptor; W, CheW; V, CheV; A, CheA; Yp, CheY~P; Z, CheZ; C, CheC; and D, CheD. By way of example, the E. coli network can be described as follows. Attractant ligand (L) interacts with chemoreceptor (CR) to deactivate the CheA kinase (A). A is associated with the CR via CheW (W), and phosphorylates CheY to generate CheY~P (Yp). CheZ (Z) dephosphorylates Yp. CheR (R) methylates CR, causing CR to be more able to activate CheA; CheB is phosphorylated by CheA to generate CheB~P (Bp) that in turn removes methyl groups from the CR and causes it to deactivate CheA. S. typhimurium varies from E. coli by having an additional coupling protein CheV (V) [39, 40]. C. jejuni is not as well characterized and so many of the depicted protein interactions are based only on homology [66]; additionally, in ε-proteobacteria, CheA possesses an additional REC domain that may be phosphorylated by CheA [36]. H. pylori contains three CheV proteins (V1, V2, V3); some of these appear to activate CheA while others deactivate it [19]. Additionally, H. pylori encodes no CheR or CheB proteins. B. subtilis contains several additional proteins and activities, including CheD (D) and CheC (C), as well as no predicted CheZ. The three ε-proteobacteria, C. jejuni, H. hepaticus and H. pylori, represent an interesting evolutionary time series of related signaling networks. Their genomic complement of chemotaxis proteins suggests an intriguing hypothesis that remains to be tested experimentally. The hypothesis is that C. jejuni represents the original ε-proteobacterial chemotaxis network, with adaptation mediated mainly through CheB and CheR. Then in H. hepaticus, not shown in the figure, the number of CheV proteins tripled, providing a broader dynamic range over which a hypothesized CheV adaptation mechanism could act. Finally, in H. pylori, the existence of the strengthened CheV-based adaptation mechanism allowed the loss of the main methylation-based adaptation mechanism via deletion of CheR and CheB, with a concomitant loss of conserved sites of methylation on H. pylori chemoreceptors (R. Alexander, unpublished data).

Figure 2. Distribution of genes encoding CheA, CheW, CheV, CheR and CheB chemotaxis proteins

Presence of chemotaxis proteins are mapped onto a phylogenetic tree of organisms with completely sequenced genomes. Subtrees are colored by phylogenic group as follows: black, spirochetes; blue, Actinobacteria (high %GC Gram-positive); cyan, Firmicutes (low %GC Gram-positive); green, Cyanobacteria; magenta, Epsilonproteobacteria; brown, Deltaproteobacteria; maroon, Alphaproteobacteria; red, Betaproteobacteria; orange, Gammaproteobacteria; grey, other. The first five rings surrounding the tree show the presence of at least one copy of cheA (red), cheW (blue), cheV (cyan), cheB (orange) and cheR (green), respectively, using the same protein color scheme as in Figure 1. The outer two rings show genomes in which the number of cheW or cheV genes is greater than the number of cheA genes, denoted by additional columns for cheW (blue) or cheV (cyan). The locations of the five species from Figure 1 on the tree are marked by a two-letter species code: Bs, B. subtilis; Ec, E. coli; Cj, C. jejuni; Hp, H. pylori; St, S. typhimurium. Chemotaxis proteins were identified by querying the MicrobesOnline database [67] (http://www.microbesonline.org) in March 2009 using the advanced search function and the following terms, based on Pfam or SMART domain architecture as suggested by Wuichet and colleagues [13]: CheA, domainName:(HATPase_c AND CheW); CheW, domainName:(CheW AND NOT Response_reg AND NOT HATPase_c AND NOT MeTrc AND NOT Hpt); CheV, domainName:(CheW AND Response_reg AND NOT HATPase_c AND NOT MeTrc AND NOT Hpt); CheB, domainName:(CheB_methylest AND NOT HWE_HK AND NOT HisKA); CheR, domainName:((CheR OR MeTrc) AND NOT CheW AND NOT TPR). The phylogenetic tree of all bacteria and archaea was downloaded from the Microbes Online website and visualized in MEGA4 [68]. After pruning leaves for incomplete genomes and multiple strains of the same species, ~490 species remain. Presence/absence of predicted Che proteins was mapped onto the tree using custom Perl scripts.

Figure 3. The CheW domains of various CheW and CheV proteins

The figure shows, from left to right: CheW(Tm), T. maritima CheW; CheW(Ec), E. coli CheW; CheV_W(Bs), B. subtilis CheV_W; CheV_W(St), S. typhimurium CheV_W; CheW(Hp), H. pylori CheW; CheV1_W(Hp), H. pylori CheV1_W; CheV2_W(Hp), H. pylori CheV2_W; CheV3_W(Hp), H. pylori CheV3_W. A conserved arginine necessary for modulating CheA kinase activity is displayed in blue, the receptor binding patch is highlighted in red, and the position of insertion sequences mentioned in Box 2 are marked in green. Alignments were generated with ClustalW [69], and then used to generate structural models of the various CheW domains based on Chain Y from the PDB file 2CH4 [29], using the Swiss-Modeler Deep View Software package with default settings [70].

Figure 4. Amino acid conservation in the coupling and receiver domains of CheV proteins

(a) Coupling domain. A multiple sequence alignment of CheW proteins and the CheW domain of CheV (CheV_W) proteins is illustrated with separate sequence logos for CheV_W (top) and CheW proteins (bottom). Each letter indicates an amino acid using standard single letter code. Height of each amino acid indicates its degree of conservation in that position of the alignment. Amino acid coloring indicates the chemical nature of the residue: small (ASTG) in green, aliphatic (ILMV) in black, aromatic (FHWY) in yellow, negative (DE) in red, negative-related (NQ) in magenta, positive (KR) in blue, and special (CP) in cyan. Sites of amino acid mutations that affect CheA interactions (arrowheads) or chemoreceptor interactions (circles) or both are from [, –29]. The crucial CheA-interacting arginine residue is marked with a blue star. These mutations are summarized with lines above the indicated regions with notations ‘CheA’ or ‘CR’ for chemoreceptor[A1]. The CheA-interacting regions correspond to beta sheets 3–4-5 and intervening loops in domain 2 of CheW. (b) REC domains. A multiple sequence alignment of the REC domain of CheV (CheV_REC) proteins and a selection of single-domain REC proteins is illustrated as in (a) with separate sequence logos for CheV_REC (top) and REC proteins (bottom). Asterisks indicate REC active site residues, with a red star indicating the phosphorylated Asp. Methods: CheW and CheV proteins were identified by their domain content (CheW: CheW domain only; CheV: CheW and Response_reg domains only) [13] and their sequences retrieved from the MicrobesOnline database [67] (http://www.microbesonline.org) in March 2009. A subset of single-domain REC (CheY) proteins was collected from NCBI by searching for proteins from Refseq of length 150 amino acids or less that contain the Pfam Response_reg domain (PF00072). Alignments were done using MUSCLE 3.6 with default parameters [71]. Gap columns absent in 95% or more of the proteins were removed.