STRUCTURALBIOLOGY Specificity of the CheR2 Methyltransferase in Pseudomonas aeruginosa is Directed by a C-Terminal Pentapeptide in the McpB Chemoreceptor Cristina García-Fontana, 1,2 Andrés Corral Lugo, 1 Tino Krell 1 * Methyltransferases of the CheR family and methylesterases of the CheB family control chemoreceptor methylation, and this dynamic posttranslational modification is necessary for proper chemotaxis of bacteria. Studies with enterobacteria that contain a single CheR or CheB show that, in addition to binding at the methylation site, some chemoreceptors bind CheR or CheB through additional high-affinity sites at distinct pentapeptide sequences in the chemoreceptors. We investigated the recognition of chemoreceptors by CheR proteins in the human pathogen Pseudomonas aeruginosa PAO1. Of the four methyltransferases in PAO1, we detected an interaction only between CheR2 and the chemoreceptor methyl-accepting chemotaxis protein B (McpB), which contains the pentapeptide GWEEF at its carboxyl terminus. Furthermore, CheR2 was also the only paralog that methylated McpB in vitro, and deletion of the pentapeptide sequence abolished both the CheR2-McpB interaction and the methylation of McpB. When clustered according to protein sequence, bacterial CheR proteins form two distinct families—those that bind pentapeptide-containing chemoreceptors and those that do not. These two families are distinguished by an insertion of three amino acids in the b-subdomain of CheR. Deletion of this insertion in CheR2 pre- vented its interaction with and methylation of McpB. Pentapeptide-containing chemoreceptors are com- mon to many bacteria species; thus, these short, distinct motifs may enable the specific assembly of signaling complexes that mediate different responses. INTRODUCTION Bacteria constantly sense and adapt to changing environmental conditions to assure survival. This important function is primarily mediated by one- component systems, two-component systems, and chemosensory pathways (1–3). Chemosensory pathways are involved in mediating flagellum- and type IV pili–mediated taxis and also carry out alternative cellular functions (3). The proteins of chemosensory pathways have been classified as auxil- iary proteins and core proteins based on the frequency of their occurrence (3). Core proteins are the CheA sensor kinase, CheW coupling protein, CheY response regulator, CheR methyltransferase, CheB methylesterase, and chemoreceptors (3). Pathway function involves the concerted action of the excitatory pathway and adaptational mechanism(s). The canonical excitatory pathway is initiated by signal recognition at the chemoreceptor, which in turn modulates CheA autophosphorylation and, subsequently, the transphosphorylation of CheY. When phosphorylated, CheY under- goes a conformational change, triggering an alteration of its activity (4). A number of adaptation mechanisms have evolved to restore the prestimu- lus behavior in the presence of the signal (5, 6). The canonical adaptation mechanism consists of the methylation and demethylation of chemorecep- tors catalyzed by the CheR methyltransferase and CheB methylesterase, respectively (4). Much of what we know in regard to chemosensory pathways is the result of studies of flagellum-mediated taxis in Escherichia coli and Salmonella typhimurium [reviewed in (4)]. E. coli has five chemoreceptors that feed stimuli into a single chemosensory pathway. However, genome analyses have shown that other bacteria have an elevated number of chemoreceptors and multiple copies of chemosensory signaling proteins that form different chemosensory pathways (3, 7). For such bacteria, the human pathogen Pseudomonas aeruginosa has become a model organism (8). This species has five gene clusters that encode chemosensory signaling proteins that assemble into four chemosensory pathways, termed Che, Che2, Wsp, and Chp. Two of the pathways mediate chemotaxis; whereas the Che pathway is essential for chemotaxis (9), the role of the Che2 pathway in chemotaxis is less clear (10). The Wsp and Chp pathways modulate diverse cellular processes by altering the abundance of cyclic diguanosine monophosphate (c-di-GMP) and cyclic adenosine monophosphate (cAMP), respectively (11, 12). P. aeruginosa has 26 chemoreceptor genes, of which only 4 are found in these gene clusters; the remaining chemoreceptor genes are scat- tered throughout the genome (8). Two chemoreceptors, encoded by mcpA and mcpB, are located in the che2 gene cluster (Fig. 1A). McpB (methyl- accepting chemotaxis protein B) has a PAS (Per-Arnt-Sim)–type sensor do- main, lacks transmembrane regions, and is predicted to be of cytosolic location (10). Attempts to identify the function of McpB have been inconclusive. Initially, a study indicated that McpB was involved in aerotaxis (13), but a subsequent study showed that this was not the case (10). It was demon- strated that CheB2 of this pathway was essential for P. aeruginosa infection in a murine lung infection model, from which the authors concluded that CheB2 and the Che2 pathway are involved in a specific chemotactic re- sponse triggered during infection by a yet unknown signal (14). As mentioned, the CheR methyltransferases are among the core pro- teins of chemosensory pathways. CheR methylates glutamyl residues at the chemoreceptor signaling domain. The extent of methylation, in turn, modulates the capacity of the receptor to control CheA autophosphorylation (15). This mechanism adapts CheA autophosphorylation activity to a given signal concentration. CheR uses S-adenosylmethionine (SAM) as a sub- strate, and the methylation reaction gives rise to S-adenosylhomocysteine (SAH) (16). CheR and CheB bind to the methylation site of the chemo- receptor. However, the high-abundance chemoreceptors in E. coli and S. typhimurium have an additional binding site for CheR and CheB 1 Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/ Prof. Albareda, 1, 18008 Granada, Spain. 2 Bio-Iliberis R&D, Polígono Industrial Juncaril, 18 210 Granada, Spain. *Corresponding author. E-mail: tino.krell@eez.csic.es RESEARCHARTICLE www.SCIENCESIGNALING.org 8 April 2014 Vol 7 Issue 320 ra34 1 on April 13, 2015 http://stke.sciencemag.org/ Downloaded from