Bacterial chemoreceptors undergo conformational changes in response to variations in the concentration of extracellular ligands. These changes in chemoreceptor structure initiate a series of signaling events that ultimately result in regulation of rotation of the flagellar motor. Here we have used cryo-electron tomography combined with 3D averaging to determine the in situ structure of chemoreceptor assemblies in Escherichia coli cells that have been engineered to overproduce the serine chemoreceptor Tsr. We demonstrate that chemoreceptors are organized as trimers of receptor dimers and display two distinct conformations that differ principally in arrangement of the HAMP domains within each trimer. Ligand binding and methylation alter the distribution of chemoreceptors between the two conformations, with serine binding favoring the ''expanded'' conformation and chemoreceptor methylation favoring the ''compact'' conformation. The distinct positions of chemoreceptor HAMP domains within the context of a trimeric unit are thus likely to represent important aspects of chemoreceptor structural changes relevant to chemotaxis signaling. Based on these results, we propose that the compact and expanded conformations represent the ''kinase-on'' and ''kinaseoff'' states of chemoreceptor trimers, respectively.cryo-electron tomography ͉ molecular architecture ͉ signal transduction M otile bacteria such as Escherichia coli respond to changes in their chemical environment by recognizing variations in the ligand occupancy of a series of transmembrane chemoreceptors (also known as methyl-accepting chemotaxis proteins, or MCPs). Ligand binding to chemoreceptors initiates a signaling cascade that modulates the activity of the histidine kinase CheA, which ultimately regulates the rotation of the flagellar motor through a diffusible intracellular signal (1, 2). Chemoreceptors exist as homodimers and have been shown to function as trimers of chemoreceptor dimers both in vitro (3) and in vivo (4-6).Chemoreceptor homodimers can be divided into three functional modules, each of which plays a critical role in receptormediated signaling (7). The transmembrane-sensing module is composed of both the sensing domain, which resides in the periplasm and contains the ligand binding site (8), and the transmembrane portion of the chemoreceptor. It has been deduced that ligand binding to the periplasmic domain results in a piston-like sliding motion of one of the four transmembrane helices (7, 9). The highly conserved cytoplasmic region of chemoreceptors can be divided into two functional modules: the signal conversion module, which contains a HAMP (histidine kinase, adenylyl cyclases, methyl-binding proteins, and phosphatases) domain, and a kinase control module, which contains an adaptation region and a protein interaction region (7, 9). The HAMP domain is the proposed site of signal conversion between the transmembrane-sensing and kinase control modules (7). The recent solution structure of an archaeal HAMP domain demonstrates that it adopts a ho...
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. We present an interpretation of the tomogram in terms of the packing arrangement of Tsr using constraints derived from previous X-ray and electron-crystallographic studies of receptor clusters. Our results imply that the interaction between the cytoplasmic ends of Tsr is likely to stabilize the presence of the membrane networks in cells overproducing Tsr. We propose that membrane invaginations that are potentially capable of supporting axial interactions between receptor clusters in apposing membranes could also be present in wild-type E. coli and that such receptor aggregates could play an important role in signal transduction during bacterial chemotaxis.Over the last three decades, methods for three-dimensional reconstruction of objects (5) imaged with an electron microscope have been used to determine the structures of a variety of biological assemblies by two types of approaches. One approach, which has been used extensively in analyses of large macromolecular assemblies, involves three-dimensional reconstruction of a structure by averaging images recorded from several identical copies oriented randomly relative to the electron beam (11, 31). The other approach, which has been used for reconstruction of objects that cannot be easily averaged, such as whole cells, involves tomographic reconstruction by combining projection images of an object recorded with an electron microscope over a range of tilt angles (4). Electron tomography is therefore a potentially powerful tool for threedimensional imaging of the spatial arrangement of proteins that make up complex and dynamic assemblies, such as those involved in bacterial chemotaxis.At least 12 proteins act in concert to convert the signal of ligand binding at the periplasmic end of a chemotaxis receptor into rotation of the flagellar motor (6, 27). The principal protein components at the input end include one of the chemotaxis receptors (Tsr, Tar, Trg, Tap, or Aer), and the cytoplasmic signaling proteins CheA and CheW, which are thought to form a noncovalent complex with the chemotaxis receptors. Knowledge of the structure and spatial arrangement of the chemotaxis receptors is therefore fundamental to understanding the structural biology of signaling. X-ray crystallographic studies of the periplasmic fragments of the aspartate receptor fragments have revealed the dimeric organization of the ligand binding domain, in which the ligand binding pocket is located at...
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