Abstract
During chemotaxis toward asparagine by Bacillus subtilis, the ligand is thought to bind to the chemoreceptor McpB on the exterior of the cell and induce a conformational change. This change affects the degree of phosphorylation of the CheA kinase bound to the cytoplasmic region of the receptor. Until recently, the sensing domains of the B. subtilis receptors were thought to be structurally similar to the well studied Escherichia coli four-helical bundle. However, sequence analysis has shown the sensing domains of receptors from these two organisms to be vastly different. Homology modeling of the sensing domain of the B. subtilis asparagine receptor McpB revealed two tandem PAS domains. McpB mutants having alanine substitutions in key arginine and tyrosine residues of the upper PAS domain but not in any residues of the lower PAS domain exhibited a chemotactic defect in both swarm plates and capillary assays. Thus, binding does not appear to occur across any dimeric surface but within a monomer. A modified capillary assay designed to determine the concentration of attractant where chemotaxis is most sensitive showed that when Arg-111, Tyr-121, or Tyr-133 is mutated to an alanine, much more asparagine is required to obtain an active chemoreceptor. Isothermal titration calorimetry experiments on the purified sensing domain showed a K(D) to asparagine of 14 mum, with the three mutations leading to less efficient binding. Taken together, these results reveal not only a novel chemoreceptor sensing domain architecture but also, possibly, a different mechanism for chemoreceptor activation.
Highlights
McpB mutantKD seemed to indicate that the presence of the other nine che-OI1085 ⌬10mcp ⌬10mcp ⌬10mcp ⌬10mcp ⌬10mcpNone; wild-type B. subtilisNone; wild-type mcpB mcpB(R111A) mcpB(Y121A) mcpB(Y133A) mcpB(Y133F) M84 460 8200 2100 11,000 460 moreceptors increases the proportion of McpB in the active conformation, confirmed by the fact that the wild type exhibits a random bias, whereas the ⌬10mcp amyE::mcpB strain is tumbly [30].Isothermal Titration Calorimetry of the McpB Sensing Domain—To further characterize the physical binding properplate results, in that mcpB(R111A) and mcpB(Y133A) showed ties of the McpB chemoreceptor and asparagine, the aminovirtually no chemotactic accumulation in the capillary tubes terminal sensing domain was overexpressed and purified
The crystal structures of the Vibrio harveyi LuxQ sensor kinase sensing domain [23], the putative sensory box/ GGDEF family protein from Vibrio parahemeolyticus (Protein Data Bank code 2P7J), the Vibrio cholerae DctB sensing domain [25, 26], and the sensing domain of the V. cholerae McpN chemoreceptor (Protein Data Bank code 3C8C) revealed a novel architecture for the sensing domains of both bacterial twocomponent sensor kinases and chemoreceptors. These crystal structures revealed a dual PAS domain architecture and suggest that this architecture is a more common sensing module than the E. coli four-helical bundle. This current study aims to elucidate the architecture of the sensing domain of the asparagine receptor McpB
It should be noted that His-216 was previously identified as a possible active site residue in the Cache domain, and Phe-205 is highly conserved in all Cache domains [19]. In addition to these residues, Glu-142 was mutated to an alanine because it aligns with the E. coli Tar binding site, and Thr-168 was mutated to an alanine because it is highly conserved in the B. subtilis sensing domains
Summary
Bacterial Strains and Plasmids—All B. subtilis strains are derived from the chemotactic strain (Cheϩ) OI1085 [28]. The B. subtilis strains were created by using QuikChange (Stratagene) mutagenesis on the pAIN750 plasmid, which contained the full-length mcpB receptor gene under control of its native promoter. The GST fusion pGEX-6P-2 plasmids were made by amplifying the desired receptor fragment (spanning residues 35–279) from a pAIN750::mcpB plasmid containing the appropriate mutation by PCR with engineered 5Ј EcoRI and 3Ј NotI sites. The region of sequence between TM1 and TM2, which spans residues 35–279, for each receptor was submitted, and the resulting Protein Data Bank files were examined These servers use homology modeling to produce a structural match, the I-TASSER server used de novo modeling to achieve a final model. The model generated using the LOMETS server showed high confidence (Z-score 26.67), and the model from the I-TASSER server had a C-score of 1.06 and a TM-score of 0.86 Ϯ .07
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