Significance of a Posttranslational Modification of the PilA Protein of Geobacter sulfurreducens for Surface Attachment, Biofilm Formation, and Growth on Insoluble Extracellular Electron Acceptors.

, an anaerobic metal-reducing bacterium, possesses type IV pili. These pili are intrinsic structural elements in biofilm formation and, together with a number of -type cytochromes, are thought to serve as conductive nanowires enabling long-range electron transfer (ET) to metal oxides and graphite anodes. Here, we report that a posttranslational modification of a nonconserved amino acid residue within the PilA protein, the structural subunit of the type IV pili, is crucial for growth on insoluble extracellular electron acceptors.

Osmotically-induced tension and the binding of N-BAR protein to lipid vesicles.

The binding affinity of a curvature-sensing protein domain (N-BAR) is measured as a function of applied osmotic stress while the membrane curvature is nearly constant. Varying the osmotic stress allows us to control membrane tension, which provides a probe of the mechanism of binding. We study the N-BAR domain of the Drosophila amphiphysin and monitor its binding on 50 nm-radius vesicles composed of 90 mol% DOPC and 10 mol% PIP.

Hydrogen exchange differences between chemoreceptor signaling complexes localize to functionally important subdomains.

The goal of understanding mechanisms of transmembrane signaling, one of many key life processes mediated by membrane proteins, has motivated numerous studies of bacterial chemotaxis receptors. Ligand binding to the receptor causes a piston motion of an α helix in the periplasmic and transmembrane domains, but it is unclear how the signal is then propagated through the cytoplasmic domain to control the activity of the associated kinase CheA. Recent proposals suggest that signaling in the cytoplasmic domain involves opposing changes in dynamics in different subdomains.

Hydrogen exchange mass spectrometry of functional membrane-bound chemotaxis receptor complexes.

The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (∼2 Å) piston displacement of one helix of the periplasmic and transmembrane domains toward the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away.

Membrane association of a protein increases the rate, extent, and specificity of chemical cross-linking.

Many cellular processes involve interactions between membrane-associated proteins, and those interactions are enhanced by membrane association. We have used cross-linking reactions to compare the extent and specificity of protein interactions in solution versus on a membrane surface. Cysteine mutants of a soluble cytoplasmic fragment (CF) of the aspartate receptor, a transmembrane receptor involved in bacterial chemotaxis, are used in disulfide bond formation with the thiol-specific oxidant diamide and chemical cross-linking reactions with the trifunctional maleimide TMEA.

Ligand affinity and kinase activity are independent of bacterial chemotaxis receptor concentration: insight into signaling mechanisms.

Binding of attractant to bacterial chemotaxis receptors initiates a transmembrane signal that inhibits the kinase CheA bound ~300 Å distant at the other end of the receptor. Chemoreceptors form large clusters in many bacterial species, and the extent of clustering has been reported to vary with signaling state. To test whether ligand binding regulates kinase activity by modulating a clustering equilibrium, we measured the effects of two-dimensional receptor concentration on kinase activity in proteoliposomes containing the purified Escherichia coli serine receptor reconstituted into vesicles over a range of lipid:protein molar ratios.

Two isoforms of Geobacter sulfurreducens PilA have distinct roles in pilus biogenesis, cytochrome localization, extracellular electron transfer, and biofilm formation.

Type IV pili of Geobacter sulfurreducens are composed of PilA monomers and are essential for long-range extracellular electron transfer to insoluble Fe(III) oxides and graphite anodes. A previous analysis of pilA expression indicated that transcription was initiated at two positions, with two predicted ribosome-binding sites and translation start codons, potentially producing two PilA preprotein isoforms. The present study supports the existence of two functional translation start codons for pilA and identifies two isoforms (short and long) of the PilA preprotein.

Change of line tension in phase-separated vesicles upon protein binding.

We measured the effect of a model membrane-binding protein on line tension and morphology of phase-separated lipid-bilayer vesicles. We studied giant unilamellar vesicles composed of a cholesterol/dioleoylphosphatidylcholine/palmitoylsphingomyelin mixture and a controlled mole fraction of a Ni-chelating lipid. These vesicles exhibited two coexisting fluid-phase domains at room temperature.

Kinase-active signaling complexes of bacterial chemoreceptors do not contain proposed receptor-receptor contacts observed in crystal structures.

The receptor dimers that mediate bacterial chemotaxis form high-order signaling complexes with CheW and the kinase CheA. From the packing arrangement in two crystal structures of different receptor cytoplasmic fragments, two different models have been proposed for receptor signaling arrays: the trimers-of-dimers and hedgerow models. Here we identified an interdimer distance that differs substantially in the two models, labeled the atoms defining this distance through isotopic enrichment, and measured it with (19)F-(13)C REDOR.

Mobile loop mutations in an archaeal inositol monophosphatase: modulating three-metal ion assisted catalysis and lithium inhibition.

The inositol monophosphatase (IMPase) enzyme from the hyperthermophilic archaeon Methanocaldococcus jannaschii requires Mg(2+) for activity and binds three to four ions tightly in the absence of ligands: K(D) = 0.8 muM for one ion with a K(D) of 38 muM for the other Mg(2+) ions. However, the enzyme requires 5-10 mM Mg(2+) for optimum catalysis, suggesting substrate alters the metal ion affinity.

Comparative genomics of Geobacter chemotaxis genes reveals diverse signaling function.

Background: Geobacter species are delta-Proteobacteria and are often the predominant species in a variety of sedimentary environments where Fe(III) reduction is important. Their ability to remediate contaminated environments and produce electricity makes them attractive for further study. Cell motility, biofilm formation, and type IV pili all appear important for the growth of Geobacter in changing environments and for electricity production.

Template-directed self-assembly enhances RTK catalytic domain function.

Receptor tyrosine kinases have become important therapeutic targets because of their involvement in diseases, including cancer. Kinase domains, which are soluble and easily purified, have found widespread use in enzyme inhibitor assays, but these domains do not exhibit full function because they are isolated from the membrane. To address this shortcoming, the authors developed a simple method to restore biologically relevant function by assembling kinase domains on a nanometer-scale template, which imitates the membrane surface.

Receptor density balances signal stimulation and attenuation in membrane-assembled complexes of bacterial chemotaxis signaling proteins.

All cells possess transmembrane signaling systems that function in the environment of the lipid bilayer. In the Escherichia coli chemotaxis pathway, the binding of attractants to a two-dimensional array of receptors and signaling proteins simultaneously inhibits an associated kinase and stimulates receptor methylation--a slower process that restores kinase activity. These two opposing effects lead to robust adaptation toward stimuli through a physical mechanism that is not understood.

Template-directed assembly of signaling proteins: a novel drug screening and research tool.

A multitude of proteins reside at or near the cell membrane, which provides a unique environment for organizing and promoting assemblies of proteins that are involved in a variety of cellular signaling functions. Many of these proteins and pathways are implicated in disease. For example, strong links have been established between receptor tyrosine kinases and disease, most notably, cancer.

Liposome-mediated assembly of receptor signaling complexes.

The reconstitution of membrane-associated protein complexes poses significant experimental challenges. The core signaling complex in the bacterial chemotaxis system is an illustrative example: The soluble cytoplasmic signaling proteins CheW and CheA bind to heterogeneous clusters of transmembrane receptor proteins, resulting in an assembly that exhibits cooperative kinase regulation. An understanding of the basis for the cooperativity inherent in the receptor/CheW/CheA interaction, as well as other membrane phenomena, can benefit from functional studies under defined conditions.

Formation and activity of template-assembled receptor signaling complexes.

Problems in membrane biology require methods to recreate the interactions between receptors and cytoplasmic signaling proteins at the membrane surface. Here, unilamellar vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine and a nickel-chelating lipid were used as templates to direct the assembly of proteins from the Escherichia coli chemotaxis signaling pathway. The bacterial chemoreceptors are known to form clusters, which promote the binding of the adaptor protein (CheW) and the kinase (CheA).

Competitive and cooperative interactions in receptor signaling complexes.

In bacterial chemotaxis, clustered transmembrane receptors and the adaptor protein CheW regulate the kinase CheA. Receptors outnumber CheA, yet it is poorly understood how interactions among receptors contribute to regulation. To address this problem, receptor clusters were simulated using liposomes decorated with the cytoplasmic domains of receptors, which supported CheA binding and stimulation.

Inch by inch, row by row.

Bacterial chemotaxis systems have cooperatively interacting clusters of transmembrane receptors and signaling proteins to detect, amplify, integrate and adapt to environmental signals. A recent study provides experimental data to construct a new model of the signaling complex.

12 Reversible methylation of glutamate residues in the receptor proteins of bacterial sensory systems.

The methyltransferase CheR catalyzes methyl group transfer from S-adenosyl-l-methionine to specific glutamic acid side chains of bacterial chemoreceptors, referred to as the methyl-accepting chemotaxis proteins (MCPs). A second enzyme, the methylesterase CheB, catalyzes ester hydrolysis. Together, CheR and CheB facilitate a reversible receptor methylation process that is essential for sensory adaptation.

Site-specific and synergistic stimulation of methylation on the bacterial chemotaxis receptor Tsr by serine and CheW.

Background: Specific glutamates in the methyl-accepting chemotaxis proteins (MCPs) of Escherichia coli are modified during sensory adaptation. Attractants that bind to MCPs are known to increase the rate of receptor modification, as with serine and the serine receptor (Tsr), which contributes to an increase in the steady-state (adapted) methylation level. However, MCPs form ternary complexes with two cytoplasmic signaling proteins, the kinase (CheA) and an adaptor protein (CheW), but their influences on receptor methylation are unknown.

Ligand-specific activation of Escherichia coli chemoreceptor transmethylation.

Adaptation in the chemosensory pathways of bacteria like Escherichia coli is mediated by the enzyme-catalyzed methylation (and demethylation) of glutamate residues in the signaling domains of methyl-accepting chemotaxis proteins (MCPs). MCPs can be methylated in trans, where the methyltransferase (CheR) molecule catalyzing methyl group transfer is tethered to the C terminus of a neighboring receptor. Here, it was shown that E.

Three-dimensional electron microscopic imaging of membrane invaginations in Escherichia coli overproducing the chemotaxis receptor Tsr.

Electron tomography is a powerful method for determining the three-dimensional structures of large macromolecular assemblies, such as cells, organelles, and multiprotein complexes, when crystallographic averaging methods are not applicable. Here we used electron tomographic imaging to determine the molecular architecture of Escherichia coli cells engineered to overproduce the bacterial chemotaxis receptor Tsr. Tomograms constructed from fixed, cryosectioned cells revealed that overproduction of Tsr led to formation of an extended internal membrane network composed of stacks and extended tubular structures.

Distributed subunit interactions in CheA contribute to dimer stability: a sedimentation equilibrium study.

The structural domains of the Escherichia coli CheA protein resemble 'beads on a string', since the N-terminal phosphate-accepting (P) domain is joined to the CheY/CheB-binding (B) domain through a flexible linker, and the B domain is in turn joined to the C-terminal dimerization/catalytic/regulatory domains by a second intervening linker. Dimerization occurs primarily via interactions between two dimerization domains, which is required for CheA trans-autophosphorylation. In this study, sedimentation equilibrium was used to demonstrate significant subunit interactions at secondary sites in the two naturally occurring (full-length and short) forms of CheA (CheA(1-654) or CheA(L), and CheA(98-654) or CheA(S)) by contrasting the dimerization of CheA(L) and CheA(S) to CheA(T), an engineered form that lacked the P domain entirely.

Template-directed assembly of receptor signaling complexes.

Transmembrane receptors in the signaling pathways of bacterial chemotaxis systems influence cell motility by forming noncovalent complexes with the cytoplasmic signaling proteins to regulate their activity. The requirements for receptor-mediated activation of CheA, the principal kinase of the Escherichia coli chemotaxis signaling pathway, were investigated using self-assembled clusters of a receptor fragment (CF) derived from the cytoplasmic domain of the aspartate receptor, Tar. Histidine-tagged Tar CF was assembled on the surface of sonicated unilamellar vesicles via a lipid containing the nickel-nitrilotriacetic acid moiety as a headgroup.