Bacterial chemoreceptor signaling complexes control kinase activity by stabilizing the catalytic domain of CheA.

Motile bacteria have a chemotaxis system that enables them to sense their environment and direct their swimming toward favorable conditions. Chemotaxis involves a signaling process in which ligand binding to the extracellular domain of the chemoreceptor alters the activity of the histidine kinase, CheA, bound ~300 Å away to the distal cytoplasmic tip of the receptor, to initiate a phosphorylation cascade that controls flagellar rotation. The cytoplasmic domain of the receptor is thought to propagate this signal via changes in dynamics and/or stability, but it is unclear how these changes modulate the kinase activity of CheA. To address this question, we have used hydrogen deuterium exchange mass spectrometry to probe the structure and dynamics of CheA within functional signaling complexes of the Escherichia coli aspartate receptor cytoplasmic fragment, CheA, and CheW. Our results reveal that stabilization of the P4 catalytic domain of CheA correlates with kinase activation. Furthermore, differences in activation of the kinase that occur during sensory adaptation depend on receptor destabilization of the P3 dimerization domain of CheA. Finally, hydrogen exchange properties of the P1 domain that bears the phosphorylated histidine identify the dimer interface of P1/P1' in the CheA dimer and support an ordered sequential binding mechanism of catalysis, in which dimeric P1/P1' has productive interactions with P4 only upon nucleotide binding. Thus stabilization/destabilization of domains is a key element of the mechanism of modulating CheA kinase activity in chemotaxis, and may play a role in the control of other kinases.

Hydrogen exchange of chemoreceptors in functional complexes suggests protein stabilization mediates long-range allosteric coupling.

Bacterial chemotaxis receptors form extended hexagonal arrays that integrate and amplify signals to control swimming behavior. Transmembrane signaling begins with a 2-Å ligand-induced displacement of an α helix in the periplasmic and transmembrane domains, but it is unknown how the cytoplasmic domain propagates the signal an additional 200 Å to control the kinase CheA bound to the membrane-distal tip of the receptor. The receptor cytoplasmic domain has previously been shown to be highly dynamic as both a cytoplasmic fragment (CF) and within the intact chemoreceptor; modulation of its dynamics is thought to play a key role in signal propagation. This hydrogen deuterium exchange-MS (HDX-MS) study of functional complexes of CF, CheA, and CheW bound to vesicles in native-like arrays reveals that the CF is well-ordered only in its protein interaction region where it binds CheA and CheW. We observe rapid exchange throughout the rest of the CF, with both uncorrelated (EX2) and correlated (EX1) exchange patterns, suggesting the receptor cytoplasmic domain retains disorder even within functional complexes. HDX rates are increased by inputs that favor the kinase-off state. We propose that chemoreceptors achieve long-range allosteric control of the kinase through a coupled equilibrium: CheA binding in a kinase-on conformation stabilizes the cytoplasmic domain, and signaling inputs that destabilize this domain (ligand binding and demethylation) disfavor CheA binding such that it loses key contacts and reverts to a kinase-off state. This study reveals the mechanistic role of an intrinsically disordered region of a transmembrane receptor in long-range allostery.

His-Tag-Mediated Dimerization of Chemoreceptors Leads to Assembly of Functional Nanoarrays.

Transmembrane chemotaxis receptors are found in bacteria in extended hexagonal arrays stabilized by the membrane and by cytosolic binding partners, the kinase CheA and coupling protein CheW. Models of array architecture and assembly propose receptors cluster into trimers of dimers that associate with one CheA dimer and two CheW monomers to form the minimal "core unit" necessary for signal transduction. Reconstructing in vitro chemoreceptor ternary complexes that are homogeneous and functional and exhibit native architecture remains a challenge. Here we report that His-tag-mediated receptor dimerization with divalent metals is sufficient to drive assembly of nativelike functional arrays of a receptor cytoplasmic fragment. Our results indicate receptor dimerization initiates assembly and precedes formation of ternary complexes with partial kinase activity. Restoration of maximal kinase activity coincides with a shift to larger complexes, suggesting that kinase activity depends on interactions beyond the core unit. We hypothesize that achieving maximal activity requires building core units into hexagons and/or coalescing hexagons into the extended lattice. Overall, the minimally perturbing His-tag-mediated dimerization leads to assembly of chemoreceptor arrays with native architecture and thus serves as a powerful tool for studying the assembly and mechanism of this complex and other multiprotein complexes.

Multidimensional Solid-State Nuclear Magnetic Resonance of a Functional Multiprotein Chemoreceptor Array.

The bacterial chemoreceptor complex governs signal detection and the upstream elements of chemotactic behavior, but the detailed molecular mechanism is still unclear. We have assembled nativelike functional arrays of an aspartate receptor cytoplasmic fragment (CF) with its two cytoplasmic protein partners (CheA and CheW) for solid-state nuclear magnetic resonance (NMR) studies of structural changes involved in signaling. In this initial study of the uniformly (13)C- and (15)N-enriched CF in these >13.8 MDa size arrays, residue-type assignments are made for amino acids that together make up 90% of the protein. We demonstrate that homo- and heteronuclear two-dimensional spectra are consistent with structure-based chemical shift predictions: a number of major assignable correlations are consistent with the predominantly α-helical secondary structure, and minor correlations are consistent with the disordered C-terminal tail. Sub-parts per million line widths and spectral changes upon freezing of samples suggest these arrays are structurally homogeneous and sufficiently immobilized for efficient solid-state NMR.

Lessons in Fundamental Mechanisms and Diverse Adaptations from the 2015 Bacterial Locomotion and Signal Transduction Meeting.

In response to rapid changes in their environment, bacteria control a number of processes, including motility, cell division, biofilm formation, and virulence. Research presented in January 2015 at the biennial Bacterial Locomotion and Signal Transduction (BLAST) meeting in Tucson, AZ, illustrates the elegant complexity of the nanoarrays, nanomachines, and networks of interacting proteins that mediate such processes. Studies employing an array of biophysical, genetic, cell biology, and mathematical methods are providing an increasingly detailed understanding of the mechanisms of these systems within well-studied bacteria. Furthermore, comparisons of these processes in diverse bacterial species are providing insight into novel regulatory and functional mechanisms. This review summarizes research presented at the BLAST meeting on these fundamental mechanisms and diverse adaptations, including findings of importance for applications involving bacteria of medical or agricultural relevance.

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. However, it has been difficult to measure dynamics within the functional system, consisting of extended arrays of receptor complexes with two other proteins, CheA and CheW. We have combined hydrogen exchange mass spectrometry with vesicle template assembly of functional complexes of the receptor cytoplasmic domain to reveal that there are significant signaling-associated changes in exchange, and these changes localize to key regions of the receptor involved in the excitation and adaptation responses. The methylation subdomain exhibits complex changes that include slower hydrogen exchange in complexes in a kinase-activating state, which may be partially consistent with proposals that this subdomain is stabilized in this state. The signaling subdomain exhibits significant protection from hydrogen exchange in complexes in a kinase-activating state, suggesting a tighter and/or larger interaction interface with CheA and CheW in this state. These first measurements of the stability of protein subdomains within functional signaling complexes demonstrate the promise of this approach for measuring functionally important protein dynamics within the various physiologically relevant states of multiprotein complexes.

New insights into bacterial chemoreceptor array structure and assembly from electron cryotomography.

Bacterial chemoreceptors cluster in highly ordered, cooperative, extended arrays with a conserved architecture, but the principles that govern array assembly remain unclear. Here we show images of cellular arrays as well as selected chemoreceptor complexes reconstituted in vitro that reveal new principles of array structure and assembly. First, in every case, receptors clustered in a trimers-of-dimers configuration, suggesting this is a highly favored fundamental building block. Second, these trimers-of-receptor dimers exhibited great versatility in the kinds of contacts they formed with each other and with other components of the signaling pathway, although only one architectural type occurred in native arrays. Third, the membrane, while it likely accelerates the formation of arrays, was neither necessary nor sufficient for lattice formation. Molecular crowding substituted for the stabilizing effect of the membrane and allowed cytoplasmic receptor fragments to form sandwiched lattices that strongly resemble the cytoplasmic chemoreceptor arrays found in some bacterial species. Finally, the effective determinant of array structure seemed to be CheA and CheW, which formed a "superlattice" of alternating CheA-filled and CheA-empty rings that linked receptor trimers-of-dimer units into their native hexagonal lattice. While concomitant overexpression of receptors, CheA, and CheW yielded arrays with native spacing, the CheA occupancy was lower and less ordered, suggesting that temporal and spatial coordination of gene expression driven by a single transcription factor may be vital for full order, or that array overgrowth may trigger a disassembly process. The results described here provide new insights into the assembly intermediates and assembly mechanism of this massive macromolecular complex.

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. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) measurements of global exchange of the CF demonstrate that the CF exhibits significantly slower exchange in functional complexes than in solution. Because the exchange rates in functional complexes are comparable to those of other proteins with similar structures, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system.

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. CF binding to membranes is mediated by its N-terminal His tag binding to vesicles containing a nickel-chelating lipid, so cross-linking reactions conducted in the presence and absence of vesicles differ only in whether CF is bound to the vesicles or is free in solution. For multiple Cys throughout the CF, membrane association is shown to increase the rate and extent of these reactions. Cross-linking specificity, which is measured as the preference for cross-linking between Cys near each other in the native structure, is also enhanced by membrane association. These results provide an experimental demonstration that membrane binding enhances protein-protein interactions, an important consideration for understanding processes involving membrane-associated proteins. The experiments further demonstrate the importance of cross-linking conditions for these reactions that are often used to probe protein structure and dynamics and the potential of membrane association to restore native interactions of membrane-associated proteins for cross-linking studies.

Diethylpyrocarbonate-Based Covalent Labeling Mass Spectrometry of Protein Interactions in a Membrane Complex System.

Membrane-associated proteins are important because they mediate interactions between a cell's external and internal environment and they are often targets of therapeutics. Characterizing their structures and binding interactions, however, is challenging because they typically must be solubilized using artificial membrane systems that can make measurements difficult. Mass spectrometry (MS) is emerging as a valuable tool for studying membrane-associated proteins, and covalent labeling MS has unique potential to provide higher order structure and binding information for these proteins in complicated membrane systems. Here, we demonstrate that diethylpyrocarbonate (DEPC) can be effectively used as a labeling reagent to characterize the binding interactions between a membrane-associated protein and its binding partners in an artificial membrane system. Using chemotaxis histidine kinase (CheA) as a model system, we demonstrate that DEPC-based covalent labeling MS can provide structural and binding information about the ternary complex of CheA with two other proteins that is consistent with structural models of this membrane-associated chemoreceptor system. Despite the moderate hydrophobicity of DEPC, we find that its reactivity with proteins is not substantially influenced by the presence of the artificial membranes. However, correct structural information for this multiprotein chemoreceptor system requires measurements of DEPC labeling at multiple reagent concentrations to enable an accurate comparison between CheA and its ternary complex in the chemoreceptor system. In addition to providing structural information that is consistent with the model of this complex system, the labeling data supplements structural information that is not sufficiently refined in the chemoreceptor model.

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. The IC(50) of kinase inhibition was unchanged despite a 10-fold change in receptor concentration. Such a change in concentration would have produced a measurable shift in the IC(50) if receptor clustering were involved in kinase regulation, based on a simple model in which the receptor oligomerization and ligand binding equilibria are coupled. These results indicate that the primary signal, ligand control of kinase activity, does not involve a change in receptor oligomerization state. In combination with previous work on cytoplasmic fragments assembled on vesicle surfaces [Besschetnova, T. Y., et al. (2008) Proc. Natl. Acad. Sci. U.S.A.105, 12289-12294], this suggests that binding of ligand to chemotaxis receptors inhibits the kinase by inducing a conformational change that expands the membrane area occupied by the receptor cytoplasmic domain, without changing the number of associated receptors in the signaling complex.

Protein rings are critical to the remarkable signaling properties of bacterial chemotaxis nanoarrays.

Bacteria build an extensive sensory nanoarray to collect information that guides their swimming. In this issue of Science Signaling, Piñas et al. demonstrate that a key element of these arrays that enhances chemotaxis responses are hexameric rings of CheW, one of two types of rings that couple the responses of core signaling units to achieve remarkable signaling properties such as single-molecule detection.

A practical guide to teaching with Proteopedia.

Proteopedia (proteopedia.org) is an open resource to explore the structure-function relationship of proteins and other biomolecules. This guide provides practical advice on how to incorporate Proteopedia into teaching the structure and function of proteins and other biomolecules. For 11 activities, we discuss desired outcomes, setting expectations, preparing students for the tasks, using resources within Proteopedia, and evaluating student work. We point out features of Proteopedia that make it especially suitable for teaching and give examples of how to avoid common pitfalls.

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. This was done in two types of receptor samples: isolated bacterial membranes containing overexpressed, intact receptor and soluble receptor fragments reconstituted into kinase-active signaling complexes. In both cases, the distance found was not compatible with the receptor dimer-dimer contacts observed in the trimers-of-dimers or in the hedgerow models. Comparisons of simulated and observed REDOR dephasing were used to deduce a closest approach distance at this interface, which provides a constraint for the possible arrangements of receptor assemblies.

Strategies for identifying dynamic regions in protein complexes: Flexibility changes accompany methylation in chemotaxis receptor signaling states.

Bacterial chemoreceptors are organized in arrays composed of helical receptors arranged as trimers of dimers, coupled to a histidine kinase CheA and a coupling protein CheW. Ligand binding to the external domain inhibits the kinase activity, leading to a change in the swimming behavior. Adaptation to an ongoing stimulus involves reversible methylation and demethylation of specific glutamate residues. However, the exact mechanism of signal propagation through the helical receptor to the histidine kinase remains elusive. Dynamics of the receptor cytoplasmic domain is thought to play an important role in the signal transduction, and current models propose inverse dynamic changes in different regions of the receptor. We hypothesize that the adaptational modification (methylation) controls the dynamics by stabilizing a partially ordered domain, which in turn modulates the binding of the kinase, CheA. We investigated the difference in dynamics between the methylated and unmethylated states of the chemoreceptor using solid-state NMR. The unmethylated receptor (CF4E) shows increased flexibility relative to the methylated mimic (CF4Q). Methylation helix 1 (MH1) has been shown to be flexible in the methylated mimic receptor. Our analysis indicates that in addition to MH1, methylation helix 2 also becomes flexible in the unmethylated receptor. In addition, we have demonstrated that both states of the receptor have a rigid region and segments with intermediate timescale dynamics. The strategies used in this study for identifying dynamic regions are applicable to a broad class of proteins and protein complexes with intrinsic disorder and dynamics spanning multiple timescales.

Carbon-nitrogen REDOR to identify ms-timescale mobility in proteins.

Protein dynamics play key mechanistic roles but are difficult to measure in large proteins and protein complexes. INEPT and CP solid-state NMR experiments have often been used to obtain spectra of protein regions that are mobile and rigid, respectively, on the nanosecond timescale. To complement this approach, we have implemented 13C{15N} REDOR to detect protein regions with backbone dynamics on the millisecond time scale that average the ≈1 kHz carbon-nitrogen dipolar coupling. REDOR-filtering of carbon correlation spectra removes signals from rigid backbone carbons and retains signals from backbone carbons with ms-timescale dynamics that would be missing in dipolar-driven NCA/NCO spectra. We use these experiments to investigate functionally important dynamics within the E coli Asp receptor cytoplasmic fragment (U-13C, 15N-CF) in native-like complexes with CheA and CheW. The CF backbone carbons exhibit only 60-75% of the expected REDOR dephasing, suggesting that 40-25% of the backbone experiences significant mobility that averages the 13C15N dipolar couplings to zero. Furthermore, the extent of this mobility changes with signaling state.

Solid-state NMR studies of the structure and mechanisms of proteins.

Magic-angle spinning solid-state NMR experiments are well suited to investigating the structures and mechanisms of important proteins that are inaccessible to X-ray crystallography and solution NMR spectroscopy, including membrane proteins and disease-related protein aggregates. Good progress has been made in the development of methods for the complete structure determination of small (<20 kDa) solid proteins using uniformly 13C, 15N-labeled samples. Studies of selectively labeled proteins focusing on labeled active sites have yielded insights into the mechanisms of enzymes and of membrane proteins involved in energy and signal transduction. Studies of selectively labeled synthetic peptides have yielded structural models for biomedically important systems, including amyloid fibrils and surface-associated peptides involved in biomineralization and cell adhesion. Novel NMR and biochemical methods are being developed to target solid-state NMR experiments within large proteins and whole cells. These approaches are being used to investigate mechanisms of transmembrane signaling by membrane receptors and to characterize binding interactions between antibiotics and bacterial cell walls. Thus, solid-state NMR is proving to be a valuable biophysical tool for probing structure and dynamics in a wide range of biomolecules.

Signaling-Related Mobility Changes in Bacterial Chemotaxis Receptors Revealed by Solid-State NMR.

Bacteria employ remarkable membrane-bound nanoarrays to sense their environment and direct their swimming. Arrays consist of chemotaxis receptor trimers of dimers that are bridged at their membrane-distal tips by rings of two cytoplasmic proteins, a kinase CheA and a coupling protein CheW. It is not clear how ligand binding to the periplasmic domain of the receptor deactivates the CheA kinase bound to the cytoplasmic tip ∼300 Šaway, but the mechanism is thought to involve changes in dynamics within the cytoplasmic domain. To test these proposals, we applied solid-state NMR mobility-filtered experiments to functional complexes of the receptor cytoplasmic fragment (U-13C,15N-CF), CheA, and CheW. Assembly of these proteins into native-like, homogeneous arrays is mediated by either vesicle binding or molecular crowding agents, and paramagnetic relaxation enhancement is used to overcome sensitivity challenges in these large complexes. INEPT spectra reveal that a significant fraction of the receptor is dynamic on the nanosecond or shorter time scale, and these dynamics change with signaling state. The mobile regions are identified through a combination of biochemical and NMR approaches (protein truncations and unique chemical shifts). The INEPT spectra are consistent with an asymmetric mobility in the methylation region (N-helix mobility ≫ C-helix mobility) and reveal an increase in the mobility of the N-helix in the kinase-off state. This finding identifies functionally relevant dynamics in the receptor, and suggests that this N-helix segment plays a key role in propagating the signal.

Population level analysis of evolved mutations underlying improvements in plant hemicellulose and cellulose fermentation by Clostridium phytofermentans.

BACKGROUND: The complexity of plant cell walls creates many challenges for microbial decomposition. Clostridium phytofermentans, an anaerobic bacterium isolated from forest soil, directly breaks down and utilizes many plant cell wall carbohydrates. The objective of this research is to understand constraints on rates of plant decomposition by Clostridium phytofermentans and identify molecular mechanisms that may overcome these limitations. RESULTS: Experimental evolution via repeated serial transfers during exponential growth was used to select for C. phytofermentans genotypes that grow more rapidly on cellobiose, cellulose and xylan. To identify the underlying mutations an average of 13,600,000 paired-end reads were generated per population resulting in ∼300 fold coverage of each site in the genome. Mutations with allele frequencies of 5% or greater could be identified with statistical confidence. Many mutations are in carbohydrate-related genes including the promoter regions of glycoside hydrolases and amino acid substitutions in ABC transport proteins involved in carbohydrate uptake, signal transduction sensors that detect specific carbohydrates, proteins that affect the export of extracellular enzymes, and regulators of unknown specificity. Structural modeling of the ABC transporter complex proteins suggests that mutations in these genes may alter the recognition of carbohydrates by substrate-binding proteins and communication between the intercellular face of the transmembrane and the ATPase binding proteins. CONCLUSIONS: Experimental evolution was effective in identifying molecular constraints on the rate of hemicellulose and cellulose fermentation and selected for putative gain of function mutations that do not typically appear in traditional molecular genetic screens. The results reveal new strategies for evolving and engineering microorganisms for faster growth on plant carbohydrates.