Characterization of the Thermotoga maritima chemotaxis methylation system that lacks pentapeptide‐dependent methyltransferase CheR:MCP tethering

Abstract

SummarySensory adaptation in bacterial chemotaxis is mediated by covalent modifications of specific glutamate and glutamine residues within the cytoplasmic domains of methyl-accepting chemotaxis proteins (MCPs). In Escherichia coli and Salmonella enterica, efficient methylation of MCPs depends on the localization of methyltransferase CheR to MCP clusters through an interaction between the CheR b-subdomain and a pentapeptide sequence (NWETF or NWESF) at the C-terminus of the MCP. In vitro methylation analyses utilizing S. enterica and Thermotoga maritima CheR proteins and MCPs indicate that MCP methylation in T. maritima occurs independently of a pentapeptide-binding motif. Kinetic and binding measurements demonstrate that despite efficient methylation, the interaction between T. maritima CheR and T. maritima MCPs is of relatively low affinity. Comparative protein sequence analyses of CheR b-subdomains from organisms having MCPs that contain and/or lack pentapeptide-binding motifs identified key similarities and differences in residue conservation, suggesting the existence of two distinct classes of CheR proteins: pentapeptidedependent and pentapeptide-independent methyltransferases. Analysis of MCP C-terminal ends showed that only~10% of MCPs contain a putative C-terminal binding motif, the majority of which are restricted to the different proteobacteria classes (a, b, g, d). These findings suggest that tethering of CheR to MCPs is a relatively recent event in evolution and that the pentapeptide-independent methylation system is more common than the well-characterized pentapeptide-dependent methylation system.

Comparative genomics of Geobacter chemotaxis genes reveals diverse signaling function

“…MCPs that contain the CheR-binding pentapeptide are restricted primarily to the Proteobacteria ; the genomes of bacteria in other phyla reveal few, if any, MCPs that contain a recognizable CheR-tethering segment, as defined previously [ 7 ]. In such species – for example B. subtilis and T. maritima , methylation operates through a different, pentapeptide-independent mechanism [ 77 ]. Less than 10% of the ~2500 MCPs listed in the SMART database of completed bacterial genomes contain a recognizable CheR tethering segment; this segment always follows the MA domain (SM00283) in the primary sequence of the MCP, which then ends in a pentapeptide that binds CheR [ 7 ].…”

“…To identify possible class-specific MCP-methyltransferase tethering interactions, we compared the aligned β-subdomain sequences of the Geobacter CheR homologs to the Salmonella and E. coli CheR sequences. The Salmonella CheR structure, co-crystallized with the NWETF pentapeptide has enabled the identification of residues in the β-subdomain that are involved in the peptide-CheR interaction (Q182, G188, R187, G190, G194 and R197, numbered according to Salmonella CheR, PDB# 1bc5) [ 75 , 77 ]. Figure 3 shows aligned sequences from the β-subdomain of all the Geobacter CheR homologs, together with the E. coli and Salmonella sequences (residues 166–199).…”

Direct evidence that the carboxyl‐terminal sequence of a bacterial chemoreceptor is an unstructured linker and enzyme tether

“…[3][4][5][6][7][8][9][10][11][12][13][14] This pentapeptide binds the two adaptation enzymes, 5,10,15,16 and in doing so enhances rates of modification for the sequence-bearing receptor as well as for neighboring receptors in the same membrane via adaptational assistance. 6,17,18 Related pentapeptide sequences are found in receptors of other proteobacteria [19][20][21] and are likely to perform a similar role in enhancing adaptational modification.…”

Structural insight into the low affinity between Thermotoga maritima CheA and CheB compared to their Escherichia coli/Salmonella typhimurium counterparts

“…For instance, some chemoreceptors of E. coli and S. typhimurium contain a conserved pentapeptide sequence (NWETF) that binds to CheR and CheB [33, 34]. Although NWETF sequence is not conserved in T. maritima chemoreceptors [35], there is a possibility that T. maritima CheB is tethered to chemoreceptors in close proximity to CheA, thereby increasing the effective local concentrations of the two proteins. Nevertheless, it is still unexplainable that T. maritima CheB would retain the CheY-like CheA interaction domain, CheB N , but not interact with CheA.…”