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Studying bacterial chemosensory array with CryoEM.

ABSTRACT: Bacteria direct their movement in respond to gradients of nutrients and other stimuli in the environment through the chemosensory system. The behavior is mediated by chemosensory arrays that are made up of thousands of proteins to form an organized array near the cell pole. In this review, we briefly introduce the architecture and function of the chemosensory array and its core signaling unit. We describe the in vivo and in vitro systems that have been used for structural studies of chemosensory array by cryoEM, including reconstituted lipid nanodiscs, 2D lipid monolayer arrays, lysed bacterial ghosts, bacterial minicells and native bacteria cells. Lastly, we review recent advances in structural analysis of chemosensory arrays using state-of-the-art cryoEM and cryoET methodologies, focusing on the latest developments and insights with a perspective on current challenges and future directions.

SUBMITTER: Qin Z

PROVIDER: S-EPMC8589424 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Studying bacterial chemosensory array with CryoEM.

Qin Zhuan Z Zhang Peijun P

Biochemical Society transactions 20211101 5

Bacteria direct their movement in respond to gradients of nutrients and other stimuli in the environment through the chemosensory system. The behavior is mediated by chemosensory arrays that are made up of thousands of proteins to form an organized array near the cell pole. In this review, we briefly introduce the architecture and function of the chemosensory array and its core signaling unit. We describe the in vivo and in vitro systems that have been used for structural studies of chemosensory ...[more]

PMID: 34495335

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Project description:Bacteria utilize a large multiprotein chemosensory array to sense attractants and repellents in their environment. The array is a hexagonal lattice formed from three core proteins: a transmembrane receptor, the His kinase CheA, and the adaptor protein CheW. The resulting, highly networked array architecture yields several advantages including strong positive cooperativity in the attractant response and rapid signal transduction through the preformed, integrated signaling circuit. Moreover, when isolated from cells or reconstituted in isolated bacterial membranes, the array possesses extreme kinetic stability termed ultrastability (Erbse and Falke (2009) Biochemistry 48:6975-87) and is the most long-lived multiprotein enzyme complex described to date. The isolated array retains kinase activity, attractant regulation, and its bound core proteins for days or more at 22 °C. The present work quantitates this ultrastability and investigates its origin. The results demonstrate that arrays consist of two major components: (i) a quasi-stable component with a lifetime of 1-2 days that decays due to slow proteolysis of CheA kinase in the lattice and (ii) a truly ultrastable component with a lifetime of ~20 days that is substantially more protected from proteolysis. Following proteolysis of the quasi-stable component the apparent positive cooperativity of the array increases, arguing the quasi-stable component is not as cooperative as the ultrastable component. Introduction of structural defects into the array by coupling a bulky probe to a subset of receptors reveals that modification of only 2% of the receptor population is sufficient to abolish ultrastability, supporting the hypothesis that the ultrastable component requires a high level of array spatial order. Overall, the findings are consistent with a model in which the quasi- and ultrastable components arise from distinct regions of the array, such that the ultrastable regions possess more extensive, better-ordered, multivalent interconnectivities between core components, thereby yielding extraordinary stability and cooperativity. Furthermore, the findings indicate that the chemosensory array is a promising platform for the development of ultrastable biosensors.

Project description:Glycogen and starch are the major carbon and energy reserve polysaccharides in nature, providing living organisms with a survival advantage. The evolution of the enzymatic machinery responsible for the biosynthesis and degradation of such polysaccharides, led the development of mechanisms to control the assembly and disassembly rate, to store and recover glucose according to cell energy demands. The tetrameric enzyme ADP-glucose pyrophosphorylase (AGPase) catalyzes and regulates the initial step in the biosynthesis of both α-polyglucans. AGPase displays cooperativity and allosteric regulation by sensing metabolites from the cell energy flux. The understanding of the allosteric signal transduction mechanisms in AGPase arises as a long-standing challenge. In this work, we disclose the cryoEM structures of the paradigmatic homotetrameric AGPase from Escherichia coli (EcAGPase), in complex with either positive or negative physiological allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP respectively, both at 3.0 Å resolution. Strikingly, the structures reveal that FBP binds deeply into the allosteric cleft and overlaps the AMP site. As a consequence, FBP promotes a concerted conformational switch of a regulatory loop, RL2, from a "locked" to a "free" state, modulating ATP binding and activating the enzyme. This notion is strongly supported by our complementary biophysical and bioinformatics evidence, and a careful analysis of vast enzyme kinetics data on single-point mutants of EcAGPase. The cryoEM structures uncover the residue interaction networks (RIN) between the allosteric and the catalytic components of the enzyme, providing unique details on how the signaling information is transmitted across the tetramer, from which cooperativity emerges. Altogether, the conformational states visualized by cryoEM reveal the regulatory mechanism of EcAGPase, laying the foundations to understand the allosteric control of bacterial glycogen biosynthesis at the molecular level of detail.

Project description:The three core components of the ubiquitous bacterial chemosensory array - the transmembrane chemoreceptor, the histidine kinase CheA, and the adaptor protein CheW - assemble to form a membrane-bound, hexagonal lattice in which receptor transmembrane signals regulate kinase activity. Both the regulatory domain of the kinase and the adaptor protein bind to overlapping sites on the cytoplasmic tip of the receptor (termed the protein interaction region). Notably, the kinase regulatory domain and the adaptor protein share the same fold constructed of two SH3-like domains. The present study focuses on the structural interface between the receptor and the kinase regulatory domain. Two models have been proposed for this interface: Model 1 is based on the crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheW adaptor protein. This model has been used in current models of chemosensory array architecture to build the receptor-CheA kinase interface. Model 2 is based on a newly determined crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheA kinase regulatory domain. Both models present unique strengths and weaknesses, and current evidence is unable to resolve which model best describes contacts in the native chemosensory arrays of Escherichia coli, Salmonella typhimurium, and other bacteria. Here we employ disulfide mapping and tryptophan and alanine mutation to identify docking sites (TAM-IDS) to test Models 1 and 2 in well-characterized membrane-bound arrays formed from E. coli and S. typhimurium components. The results reveal that the native array interface between the receptor protein interaction region and the kinase regulatory domain is accurately described by Model 2, but not by Model 1. In addition, the results show that the interface possesses both a structural function that contributes to stable CheA kinase binding in the array and a regulatory function central to transmission of the activation signal from receptor to CheA kinase. On-off switching alters the disulfide formation rates of specific Cys pairs at the interface, but not most Cys pairs, indicating that signaling perturbs localized regions of the interface. The findings suggest a simple model for the rearrangement of the interface triggered by the attractant signal and for longer range transmission of the signal in the chemosensory array.

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Studying bacterial chemosensory array with CryoEM.

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Studying bacterial chemosensory array with CryoEM.

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