Assembly and Functional Architecture of Bacterial Chemoreceptor 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 homogenous, 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 native-like 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. This discovery may also address a previously observed density-dependent transition between signaling states. To further test this, we implemented a paramagnetic relaxation enhancement (PRE) based solid-state NMR approach to obtain long-range (≥ 20 Å) distance constraints across the trimer of dimers interface. Overall, the work presented here shows that minimally perturbing His-tag mediated dimerization promotes assembly of chemoreceptor arrays with native architecture, and thus enabled us to gain insights into the mode of array assembly and the role of the core functional unit

Assembly and Functional Architecture of Bacterial Chemoreceptor 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 homogenous, 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 native-like 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. This discovery may also address a previously observed density-dependent transition between signaling states. To further test this, we implemented a paramagnetic relaxation enhancement (PRE) based solid-state NMR approach to obtain long-range (≥ 20 Å) distance constraints across the trimer of dimers interface. Overall, the work presented here shows that minimally perturbing His-tag mediated dimerization promotes assembly of chemoreceptor arrays with native architecture, and thus enabled us to gain insights into the mode of array assembly and the role of the core functional unit

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.Microbial Biotechnolog

Application of Computational Molecular Biophysics to Problems in Bacterial Chemotaxis

The combination of physics, biology, chemistry, and computer science constitutes the promising field of computational molecular biophysics. This field studies the molecular properties of DNA, protein lipids and biomolecules using computational methods. For this dissertation, I approached four problems involving the chemotaxis pathway, the set of proteins that function as the navigation system of bacteria and lower eukaryotes. In the first chapter, I used a special-purpose machine for molecular dynamics simulations, Anton, to simulate the signaling domain of the chemoreceptor in different signaling states for a total of 6 microseconds. Among other findings, this study provides enough evidence to propose a novel molecular mechanism for the kinase activation by the chemoreceptor and reconcile previously conflicting experimental data. In the second chapter, my molecular dynamics studies of the scaffold protein cheW reveals the existence and role of a conserved salt-bridge that stabilizes the relative position of the two binding sites in the chew surface: the chemoreceptor and the kinase. The results were further confirmed with NMR experiments performed with collaborators at the University of California in Santa Barbara, CA. In the third chapter, my colleagues and I investigate the quality of homology modeled structures with cheW protein as a benchmark. By subjecting the models to molecular dynamics and Monte Carlo simulations, we show that the homology models are snapshots of a larger ensemble of conformations very similar to the one generated by the experimental structures. In the fourth chapter, I use bioinformatics and basic mathematical modeling to predict the specific chemoreceptor(s) expressed in vivo and imaged with electron cryo tomography (ECT) by our collaborators at the California Institute of Technology. The study was essential to validate the argument that the hexagonal arrangement of transmembrane chemoreceptors is universal among bacteria, a major breakthrough in the field of chemotaxis. In summary, this thesis presents a collection of four works in the field of bacterial chemotaxis where either methods of physics or the quantitative approach of physicists were of fundamental importance for the success of the project

A Constrained Peptide That Targets the TLR4/MD2 Interaction \u26 Investigating the Mechanism of Ultra-stability in Bacterial Chemosensory Arrays

Pathological pain is a serious health problem that is initiated and perpetuated by Toll-like Receptors (TLRs) on glial cells. Among the TLRs, Toll-like Receptor 4 (TLR4) is one of most studied and most significant members of the TLR family that organizes an innate immune response by recognizing exogenous and endogenous danger signals. Specifically, TLR4 recognizes lipopolysaccharide (LPS) from the cell wall of Gram-negative bacteria, as well as endogenous signals such as HSP70, HSP90, and HMGB-1. Agonism of the receptor is dependent upon the accessory protein MD2 which is responsible for binding LPS and mediating the interaction between TLR4 receptors in an active signaling unit. The recent crystal structures of the TLR4/MD2 complex demonstrate that all of the critical residues for the MD2 interaction with TLR4 are localized to one stretch (C95-E111) of MD2. Moreover, this stretch of amino acids is constrained by a crucial disulfide bond (C95-C105). The proximity of these critical features suggests that an MD2 based synthetic peptide incorporating these critical elements could compete with full-length MD2 for the TLR4 binding interface and subsequently prevent signaling. This study investigates the feasibility of such an approach and demonstrates that a 17 residue peptide based on the TLR4 binding region of MD2 can prevent full-length MD2 from associating with TLR4 and subsequently prevent TLR4 signaling. Bacteria utilize large multi-protein chemosensory arrays to sense attractants and repellants in their environment. The essential components of these arrays are hexagonally arranged core units consisting of receptor trimer-of-dimers, CheA histidine kinase, and CheW coupling protein. Incorporation of these units into arrays has several advantages including strong cooperativity and high sensitivity in ligand sensing, a large dynamic range and rapid signal transduction through the signalling circuit. Another unique advantage of the array architecture is a striking ultra-stability in vitro: arrays retain kinase activity, attractant sensitivity and bound components for weeks. This work examines this remarkable ultra-stability and its origin in more detail. The results of this study demonstrate that arrays are not homogenous, but rather exhibit two major populations. One population is quasi-stable with a lifetime of 1-2 days, and loss of this population is highly correlated with proteolytic degradation of CheA kinase. The second population is truly ultra-stable with a lifetime of 20 days or more, and is less accessible to proteolysis. Following degradation of the less stable population, the cooperativity of the array increases, arguing that the less stable regions of array are not as well ordered and cooperative as the ultra-stable regions. To test the hypothesis that a well-ordered array is required for ultra-stability, we have introduced a small density of defects into the complex through chemical modification. Notably, even a very low degree of packing defects can abolish array ultra-stability, supporting the hypothesis. These findings are consistent with a model in which cooperativity and ultra-stability arise from extensive interconnectivity between multiple components within a well-ordered array

HYDROGEN EXCHANGE IDENTIFIES PROTEIN INTERFACES AND SIGNALING-RELATED CHANGES IN FUNCTIONAL CHEMORECEPTOR ARRAYS

Chemotaxis is an ideal system for studying membrane protein signal transduction. Chemoreceptors are transmembrane proteins that sense chemicals in the environment and use this information to control a phosphorylation cascade that enables the cell to swim towards favorable environments. The receptors form a ternary complex with a histidine kinase, CheA, and an adaptor protein, CheW. These complexes assemble into membrane-bound hexagonal arrays that transmit the signal that controls CheA. It is widely accepted that ligand binding to the receptor causes a 2Å piston motion of a helix that extends through the periplasmic and transmembrane domains. But it is unclear how the signal then propagates through the cytoplasmic domain to inhibit CheA that is bound to the membrane- distal tip of the receptor, ~200Å away. Previous studies have suggested that signal propagation through the cytoplasmic domain involves inverse changes in the dynamics of the receptor. In this study, we employ hydrogen deuterium exchange mass spectrometry (HDX- MS) to measure differences in structure and dynamics between defined states of the receptor. Functional complexes of a His-tagged cytoplasmic fragment (CF) are assembled on vesicles with CheA and CheW in three states for HDX-MS. Widespread correlated exchange is observed, which indicates that the CF in functional complexes populates a long-lived unfolded state. Exchange is rapid throughout the CF except in the protein interaction region where CF binds CheA and CheW. These observations lead us to propose that signaling involves modulation of a folding equilibrium: binding of CheA (and possibly CheW) stabilizes the receptor, and CheA is bound in a kinase-on conformation. Thus, destabilization of the receptor will release this contact with CheA, which then adopts a kinase-off conformation. Both the kinase-off and demethylated samples of CF complexes exhibit faster HDX and less protection from exchange at long times at the binding interfaces with CheA. Thus we proposed that both the ligand-induced piston and demethylation destabilize the receptor, which releases its contact with CheA to turn off the kinase. Preliminary HDX results for CheA also set a stage for future analysis of the domain interactions of CheA in the functional complexes, and the differences that correlate with kinase activity. Ultimately, HDX-MS results will provide important information for deducing the signaling mechanism

Molecular modeling and simulation of bacterial chemosensory arrays

The movement of an organism in response to environmental chemical cues is known as chemotaxis. Motile bacteria use chemotaxis to navigate through their environments, enabling cells to efficiently locate favorable growing conditions while avoiding harmful ones. Central to this ability, bacteria posses a universally conserved sensory apparatus, known as the chemosensory array, which involves the clustering of thousands of proteins into a highly cooperative signaling network. The present dissertation will present my work using techniques in computational modeling and simulation to investigate the molecular structure and function of the bacterial chemosensory array. A brief overview of each chapter follows. Chapter 1 provides an introduction to the systems-level features of chemotaxis in the model organism Escherichia coli as well as an overview of the molecular organization and function of the chemosensory array. Chapter 2 gives an outline of the core methodologies used in my work, specifically all-atom molecular dynamics (MD) simulation and Molecular Dynamics Flexible Fitting (MDFF). In addition, two of the primary techniques used to analyze the MD simulations presented in this dissertation are sketched out, namely structural clustering based on root- mean-square displacement (RMSD) and Principal Component Analysis (PCA). Chapter 3 reports my work, in collaboration with Peijun Zhang’s Lab, to investigate the structural and dynamical features of the extended chemosensory array. Using computational techniques to synthesize multi-scale structural data from X-ray crystallography and cryo-electron tomography (cryo-ET), an atomic model of the cytoplasmic portion of the chemosensory array from Thermotoga maritima is constructed and refined. Through the use of large-scale MD simulations, a novel conformational change in a key signaling protein is identified and subsequently shown to be critical for chemotaxis signaling in live E. coli cells. Chapter 4 details the construction of an atomic model of a complete, transmembrane (TM) chemoreceptor. In particular, I use homology modeling and MD simulations, in- formed by biochemical and X-ray crystallographic data, to derive a model of the E. coli serine receptor (Tsr), including the previously uncharacterized TM four-helix bundle and HAMP domains. In addition, I report a series of MD simulations of a fragment of the resulting Tsr model, investigating the structural and dynamical effects of mutations on a key control cable residue. Preliminary MD simulations of the intact Tsr model are also presented. Chapter 5 reports work in collaboration with Michael Eisenbach’s Lab at the Weizmann Institute, exploring the role of acetylation on CheY activation and the generation of clockwise (CW) flagellar motor rotation. Specifically, I present a series of MD simulations that investigate the effect of a hyperactivating mutation at a key acetylation site and offer a molecular explanation of acetylation-dependent generation of CW flagellar motor rotation. I conclude with a brief description of recent work, expanding upon the results of the previous chapters, which has resulted in the first atomically resolved model of the E. coli transmembrane chemosensory array

CHEMORECEPTOR DYNAMICS AND SIGNALING: NMR MEASUREMENTS WITHIN FUNCTIONAL COMPLEXES

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 widely accepted that ligand binding to the receptor causes a 2 Å piston motion of a helix that extends through the periplasmic and transmembrane domains. However, it is not clear how the signal propagates 200 Å further to control activity of the kinase bound at the tip of the receptor. Dynamic changes within the cytoplasmic domain of the receptor have been proposed to play a key role in signal transmission. To test these proposals, we applied solid-state NMR to study the structure and dynamics of the E coli Asp receptor cytoplasmic fragment (U-13C,15N-CF) in native-like arrays of functional complexes with CheA and CheW. To detect segments that experience motions on the millisecond time scale, we use a 15N{13C} REDOR filter to remove signals from rigid backbone carbons and retain signals from backbone carbons with ms-timescale dynamics. This experiment exhibits only 60-70% of the expected REDOR dephasing, suggesting that 40-30% of the backbone has ms-timescale mobility that averages the ~ 1000 Hz 13C15N dipolar couplings. A novel REDOR-filtered DARR experiment led us to identify MH2 (methylation helix 2) as the region with ms-timescale motion. INEPT spectra reveal dynamics on the ns or shorter timescale. The INEPT-detectable regions are identified through a combination of biochemical and NMR approaches, to establish that MH1 (methylation helix 1) is mobile on the ns timescale. Interestingly, the INEPT and REDOR studies indicate the methylation region has asymmetric mobility: MH1 has ns-timescale dynamics that increase in the kinase-off state, and MH2 has ms-timescale dynamics that decrease in the kinase-off state. Thus these NMR measurements of the dynamics of CF within its functional complexes provide insight into the structural organization and signaling-related changes of the receptor methylation region, which is responsible both for transmitting the ligand-binding signal and for adaptation, both of which are critical to the chemotaxis response

Complete structure of the chemosensory array core signalling unit in an E. coli 1 minicell strain

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays. Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit. Here we introduce an Escherichia coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We use cryo-electron tomography and subtomogram averaging to provide a three-dimensional map of a complete core signalling unit, with visible densities corresponding to the HAMP and periplasmic domains. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields a molecular model of the transmembrane core signalling unit and enables spatial localisation of its individual domains. Our work thus offers a solid structural basis for the interpretation of a wide range of existing data and the design of further experiments to elucidate signalling mechanisms within the core signalling unit and larger array