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

Alternative architecture of the E. coli chemosensory array

Chemotactic responses in motile bacteria are the result of sophisticated signal transduction by large, highly organized arrays of sensory proteins. Despite tremendous progress in the understanding of chemosensory array structure and function, a structural basis for the heightened sensitivity of networked chemoreceptors is not yet complete. Here, we present cryo-electron tomography visualisations of native-state chemosensory arrays in E. coli minicells. Strikingly, these arrays appear to exhibit a p2-symmetric array architecture that differs markedly from the p6-symmetric architecture previously described in E. coli. Based on this data, we propose molecular models of this alternative architecture and the canonical p6-symmetric assembly. We evaluate our observations and each model in the context of previously published data, assessing the functional implications of an alternative architecture and effects for future studies

The unconventional cytoplasmic sensing mechanism for ethanol chemotaxis in Bacillus subtilis

Motile bacteria sense chemical gradients using chemoreceptors, which consist of distinct sensing and signaling domains. The general model is that the sensing domain binds the chemical and the signaling domain induces the tactic response. Here, we investigated the unconventional sensing mechanism for ethanol taxis in Bacillus subtilis. Ethanol and other short-chain alcohols are attractants for B. subtilis. Two chemoreceptors, McpB and HemAT, sense these alcohols. In the case of McpB, the signaling domain directly binds ethanol. We were further able to identify a single amino-acid residue Ala431 on the cytoplasmic signaling domain of McpB, that when mutated to a serine, reduces taxis to ethanol. Molecular dynamics simulations suggest ethanol binds McpB near residue Ala431 and mutation of this residue to serine increases coiled-coil packing within the signaling domain, thereby reducing the ability of ethanol to bind between the helices of the signaling domain. In the case of HemAT, the myoglobin-like sensing domain binds ethanol, likely between the helices encapsulating the heme group. Aside from being sensed by an unconventional mechanism, ethanol also differs from many other chemoattractants because it is not metabolized by B. subtilis and is toxic. We propose that B. subtilis uses ethanol and other short-chain alcohols to locate prey, namely alcohol-producing microorganisms

A bacterial inflammation sensor regulates c-di-GMP signaling, adhesion, and biofilm formation

The reactive oxygen species produced during inflammation through the neutrophilic respiratory burst play profound roles in combating bacterial pathogens and regulating the microbiota. Among these, the neutrophilic oxidant bleach, hypochlorous acid (HOCl), is the most prevalent and strongest oxidizer and kills bacteria through non-specific oxidation of proteins, lipids, and DNA. Thus, HOCl can be viewed as a host-specific cue that conveys important information about what bacterial physiology and lifestyle programs may be required for successful colonization. Nevertheless, bacteria that colonize animals face a molecular challenge in how to achieve highly selective detection of HOCl due to its reactive and transient nature and chemical similarity to more benign and non-host-specific oxidants like hydrogen peroxide (H2O2). Here, we report that in response to increasing HOCl levels E. coli regulates biofilm production via activation of the diguanylate cyclase DgcZ. We show the molecular mechanism of this activation to be specific oxidation of a conserved cysteine that coordinates the zinc of its regulatory chemoreceptor zinc-binding (CZB) domain, forming a zinc-cysteine redox switch 685-fold more sensitive to oxidation by HOCl over H2O2. Dissection of the signal transduction mechanism through quantum mechanics, molecular dynamics, and biochemical analyses reveal how the cysteine redox state alters the delicate equilibrium of competition for Zn++ between the CZB domain and other zinc binders to relay the presence of HOCl through activating the associated GGDEF domain to catalyze c-di-GMP. We find biofilm formation and HOCl-sensing in vivo to be regulated by the conserved cysteine, and point mutants that mimic oxidized CZB states increase production of the biofilm matrix polymer poly-N-acetylglucosamine and total biofilm. We observe CZB-regulated diguanylate cyclases and chemoreceptors in phyla in which host-associated bacteria are prevalent and are possessed by pathogens that manipulate host inflammation as part of their colonization strategy. A phylogenetic survey of all known CZB sequences shows these domains to be conserved and widespread across diverse phyla, suggesting CZB origin predates the bacterial last universal common ancestor. The ability of bacteria to use CZB protein domains to perceive and thwart the host neutrophilic respiratory burst has implications for understanding the mechanisms of diseases of chronic inflammation and gut dysbiosis

Membraneless channels sieve cations in ammonia-oxidizing marine archaea

Nitrosopumilus maritimus is an ammonia-oxidizing archaeon that is crucial to the global nitrogen cycle1, 2. A critical step for nitrogen oxidation is the entrapment of ammonium ions from a dilute marine environment at the cell surface and their subsequent channelling to the cell membrane of N. maritimus. Here we elucidate the structure of the molecular machinery responsible for this process, comprising the surface layer (S-layer), using electron cryotomography and subtomogram averaging from cells. We supplemented our in situ structure of the ammonium-binding S-layer array with a single-particle electron cryomicroscopy structure, revealing detailed features of this immunoglobulin-rich and glycan-decorated S-layer. Biochemical analyses showed strong ammonium binding by the cell surface, which was lost after S-layer disassembly. Sensitive bioinformatic analyses identified similar S-layers in many ammonia-oxidizing archaea, with conserved sequence and structural characteristics. Moreover, molecular simulations and structure determination of ammonium-enriched specimens enabled us to examine the cation-binding properties of the S-layer, revealing how it concentrates ammonium ions on its cell-facing side, effectively acting as a multichannel sieve on the cell membrane. This in situ structural study illuminates the biogeochemically essential process of ammonium binding and channelling, common to many marine microorganisms that are fundamental to the nitrogen cycle

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

PyLipID : A Python package for analysis of protein-lipid interactions from MD simulations

Lipids play important modulatory and structural roles for membrane proteins. Molecular dynamics simulations are frequently used to provide insights into the nature of these protein–lipid interactions. Systematic comparative analysis requires tools that provide algorithms for objective assessment of such interactions. We introduce PyLipID, a Python package for the identification and characterization of specific lipid interactions and binding sites on membrane proteins from molecular dynamics simulations. PyLipID uses a community analysis approach for binding site detection, calculating lipid residence times for both the individual protein residues and the detected binding sites. To assist structural analysis, PyLipID produces representative bound lipid poses from simulation data, using a density-based scoring function. To estimate residue contacts robustly, PyLipID uses a dual-cutoff scheme to differentiate between lipid conformational rearrangements while bound from full dissociation events. In addition to the characterization of protein–lipid interactions, PyLipID is applicable to analysis of the interactions of membrane proteins with other ligands. By combining automated analysis, efficient algorithms, and open-source distribution, PyLipID facilitates the systematic analysis of lipid interactions from large simulation data sets of multiple species of membrane proteins

Zoster-Associated Prothrombotic Plasma Exosomes and Increased Stroke Risk

Herpes zoster (HZ; shingles) caused by varicella zoster virus reactivation increases stroke risk for up to 1 year after HZ. The underlying mechanisms are unclear, however, the development of stroke distant from the site of zoster (eg, thoracic, lumbar, sacral) that can occur months after resolution of rash points to a long-lasting, virus-induced soluble factor (or factors) that can trigger thrombosis and/or vasculitis. Herein, we investigated the content and contributions of circulating plasma exosomes from HZ and non-HZ patient samples. Compared with non-HZ exosomes, HZ exosomes (1) contained proteins conferring a prothrombotic state to recipient cells and (2) activated platelets leading to the formation of platelet-leukocyte aggregates. Exosomes 3 months after HZ yielded similar results and also triggered cerebrovascular cells to secrete the proinflammatory cytokines, interleukin 6 and 8. These results can potentially change clinical practice through addition of antiplatelet agents for HZ and initiatives to increase HZ vaccine uptake to decrease stroke risk

Structure of the native chemotaxis core signaling unit from phage E-protein lysed E. coli cells

Motile bacteria employ conserved chemotaxis networks to detect chemical gradients in their surroundings and effectively regulate their locomotion, enabling the location of essential nutrients and other important biological niches. The sensory apparatus of the chemotaxis pathway is an array of core-signaling units (CSUs) composed of transmembrane chemoreceptors, the histidine kinase CheA and an adaptor protein, CheW. Although chemotaxis pathways represent the best understood signaling systems, a detailed mechanistic understanding of signal transduction has been hindered by the lack of a complete structural picture of the CSU and extended array. In this study, we present the structure of the complete CSU from phage φX174 E protein lysed Escherichia coli cells, determined using cryo-electron tomography and sub-tomogram averaging to 12-Å resolution. Using AlphaFold2, we further predict the atomic structures of the CSU’s constituent proteins as well as key protein-protein interfaces, enabling the assembly an all-atom CSU model, which we conformationally refine using our cryo-electron tomography map. Molecular dynamics simulations of the resulting model provide new insight into the periplasmic organization of the complex, including novel interactions between neighboring receptor ligand-binding domains. Our results further elucidate previously unresolved interactions between individual CheA domains, including an anti-parallel P1 dimer and non-productive binding mode between P1 and P4, enhancing our understanding of the structural mechanisms underlying CheA signaling and regulation. IMPORTANCE Bacterial chemotaxis is a ubiquitous behavior that enables cell movement toward or away from specific chemicals. It serves as an important model for understanding cell sensory signal transduction and motility. Characterization of the molecular mechanisms underlying chemotaxis is of fundamental interest and requires a high-resolution structural picture of the sensing machinery, the chemosensory array. In this study, we combine cryo-electron tomography and molecular simulation to present the complete structure of the core signaling unit, the basic building block of chemosensory arrays, from Escherichia coli . Our results provide new insight into previously poorly-resolved regions of the complex and offer a structural basis for designing new experiments to test mechanistic hypotheses

A bacterial inflammation sensor regulates c-di-GMP signaling, adhesion, and biofilm formation

Bacteria that colonize animals must overcome, or coexist, with the reactive oxygen species products of inflammation, a front-line defense of innate immunity. Among these is the neutrophilic oxidant bleach, hypochlorous acid (HOCl), a potent antimicrobial that plays a primary role in killing bacteria through nonspecific oxidation of proteins, lipids, and DNA. Here, we report that in response to increasing HOCl levels, Escherichia coli regulates biofilm production via activation of the diguanylate cyclase DgcZ. We identify the mechanism of DgcZ sensing of HOCl to be direct oxidation of its regulatory chemoreceptor zinc-binding (CZB) domain. Dissection of CZB signal transduction reveals that oxidation of the conserved zinc-binding cysteine controls CZB Zn2+ occupancy, which in turn regulates the catalysis of c-di-GMP by the associated GGDEF domain. We find DgcZ-dependent biofilm formation and HOCl sensing to be regulated in vivo by the conserved zinc-coordinating cysteine. Additionally, point mutants that mimic oxidized CZB states increase total biofilm. A survey of bacterial genomes reveals that many pathogenic bacteria that manipulate host inflammation as part of their colonization strategy possess CZB-regulated diguanylate cyclases and chemoreceptors. Our findings suggest that CZB domains are zinc-sensitive regulators that allow host-associated bacteria to perceive host inflammation through reactivity with HOCl