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Modeling the transmembrane domain of bacterial chemoreceptors.

ABSTRACT: Bacterial chemoreceptors signal across the membrane by conformational changes that traverse a four-helix transmembrane domain. High-resolution structures are available for the chemoreceptor periplasmic domain and part of the cytoplasmic domain but not for the transmembrane domain. Thus, we constructed molecular models of the transmembrane domains of chemoreceptors Trg and Tar, using coordinates of an unrelated four-helix coiled coil as a template and the X-ray structure of a chemoreceptor periplasmic domain to establish register and positioning. We tested the models using the extensive data for cross-linking propensities between cysteines introduced into adjacent transmembrane helices, and we found that many aspects of the models corresponded with experimental observations. The one striking disparity, the register of transmembrane helix 2 (TM2) relative to its partner transmembrane helix 1, could be corrected by sliding TM2 along its long axis toward the periplasm. The correction implied that axial sliding of TM2, the signaling movement indicated by a large body of data, was of greater magnitude than previously thought. The refined models were used to assess effects of inter-helical disulfides on the two ligand-induced conformational changes observed in alternative crystal structures of periplasmic domains: axial sliding within a subunit and subunit rotation. Analyses using a measure of disulfide potential energy provided strong support for the helical sliding model of transmembrane signaling but indicated that subunit rotation could be involved in other ligand-induced effects. Those analyses plus modeled distances between diagnostic cysteine pairs indicated a magnitude for TM2 sliding in transmembrane signaling of several angstroms.

SUBMITTER: Peach ML

PROVIDER: S-EPMC2373522 | biostudies-literature | 2002 Apr

REPOSITORIES: biostudies-literature

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Modeling the transmembrane domain of bacterial chemoreceptors.

Peach Megan L ML Hazelbauer Gerald L GL Lybrand Terry P TP

Protein science : a publication of the Protein Society 20020401 4

Bacterial chemoreceptors signal across the membrane by conformational changes that traverse a four-helix transmembrane domain. High-resolution structures are available for the chemoreceptor periplasmic domain and part of the cytoplasmic domain but not for the transmembrane domain. Thus, we constructed molecular models of the transmembrane domains of chemoreceptors Trg and Tar, using coordinates of an unrelated four-helix coiled coil as a template and the X-ray structure of a chemoreceptor peripl ...[more]

PMID: 11910034

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Project description:Transmembrane bacterial chemoreceptors are extended, rod-shaped homodimers with ligand-binding sites at one end and interaction sites for signaling complex formation and histidine kinase control at the other. There are atomic-resolution structures of chemoreceptor fragments but not of intact, membrane-inserted receptors. Electron tomography of in vivo signaling complex arrays lack distinct densities for chemoreceptor rods away from the well-ordered base plate region, implying structural heterogeneity. We used negative staining, transmission electron microscopy, and image analysis to characterize the molecular shapes of intact homodimers of the Escherichia coli aspartate receptor Tar rendered functional by insertion into nanodisc-provided E. coli lipid bilayers. Single-particle analysis plus tomography of particles in a three-dimensional matrix revealed two bend loci in the chemoreceptor cytoplasmic domain, (i) a short, two-strand gap between the membrane-proximal, four-helix-bundle HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemoreceptors, and phosphatases) domain and the membrane-distal, four-helix coiled coil and (ii) aligned glycines in the extended, four-helix coiled coil, the position of a bend noted in the previous X-ray structure of a receptor fragment. Our images showed HAMP bends from 0° to ?13° and glycine bends from 0° to ?20°, suggesting that the loci are flexible hinges. Variable hinge bending explains indistinct densities for receptor rods outside the base plate region in subvolume averages of chemotaxis arrays. Bending at flexible hinges was not correlated with the chemoreceptor signaling state. However, our analyses showed that chemoreceptor bending avoided what would otherwise be steric clashes between neighboring receptors that would block the formation of core signaling complexes and chemoreceptor arrays.IMPORTANCE This work provides new information about the shape of transmembrane bacterial chemoreceptors, crucial components in the molecular machinery of bacterial chemotaxis. We found that intact, lipid-bilayer-inserted, and thus functional homodimers of the Escherichia coli chemoreceptor Tar exhibited bends at two flexible hinges along their ?200-Å, rod-like, cytoplasmic domains. One hinge was at the short, two-strand gap between the membrane-proximal, four-helix-bundle HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemoreceptors, and phosphatases) domain and the membrane-distal, four-helix coiled coil. The other hinge was at aligned glycines in the extended, four-helix coiled coil, where a bend had been identified in the X-ray structure of a chemoreceptor fragment. Our analyses showed that flexible hinge bending avoided structural clashes in chemotaxis core complexes and their arrays.

Project description:Chemoreceptor-based signaling pathways are among the major modes of bacterial signal transduction, and Pseudomonas aeruginosa PAO1 is an important model to study their function. Of the 26 chemoreceptors of this strain, PctA has a broad ligand range and responds to most of the proteinogenic amino acids, whereas PctB and PctC have a much narrower range and show strong ligand preference for l-glutamine and γ-aminobutyrate, respectively. Using several comparative genomics approaches, we show that these receptors are paralogs: pctA gene duplication in the common ancestor of the genus Pseudomonas led to pctC, whereas pctB originated through another, independent pctA duplication in the common ancestor of P. aeruginosa Thus, the broad-range amino acid chemoreceptor was evolutionarily older, and chemoreceptors that complemented "missing" amino acid sensing abilities arose later in specific Pseudomonas lineages. Using comparative sequence analysis, newly solved crystal structures of PctA, PctB, and PctC ligand-binding domains, and their molecular dynamics simulations, we identified a conserved amino acid recognition motif and changes in the ligand-binding pocket that led to novel ligand specificities. In addition, we determined major forces driving the evolution of this group of chemoreceptors.IMPORTANCE Many bacteria possess a large number of chemoreceptors that recognize a variety of different compounds. More than 60% of the genomes analyzed in this study contain paralogous chemoreceptors, suggesting that they emerge with high frequency. We provide first insight on how paralogous receptors have evolved and show that two chemoreceptors with a narrow ligand range have evolved from an ancestral protein with a broad chemoeffector spectrum. Protein structures show that multiple changes in the ligand-binding site account for the differences in the ligand spectrum. This work lays the ground for further studies aimed at establishing whether the principles of ligand-binding evolution reported here can be generalized for a wider spectrum of sensory proteins in bacteria.

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Modeling the transmembrane domain of bacterial chemoreceptors.

Publications

Modeling the transmembrane domain of bacterial chemoreceptors.

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