Three-Dimensional Electron Microscopic Imaging of Membrane Invaginations in
            <i>Escherichia coli</i>
            Overproducing the Chemotaxis Receptor Tsr

Escherichia coli chemotaxis is mediated by membrane receptor͞ histidine kinase signaling complexes. Fusing the cytoplasmic domain of the aspartate receptor, Tar, to a leucine zipper dimerization domain produces a hybrid, lzTar C, that forms soluble complexes with CheA and CheW. The three-dimensional reconstruction of these complexes was different from that anticipated based solely on structures of the isolated components. We found that analogous complexes self-assembled with a monomeric cytoplasmic domain fragment of the serine receptor without the leucine zipper dimerization domain. These complexes have essentially the same size, composition, and architecture as those formed from lzTar C. Thus, the organization of these receptor͞signaling complexes is determined by conserved interactions between the constituent chemotaxis proteins and may represent the active form in vivo. To understand this structure in its cellular context, we propose a model involving parallel membrane segments in receptor-mediated CheA activation in vivo.CheA ͉ histidine kinase ͉ serine receptor ͉ signal transduction

Section: Discussionsupporting

confidence: 65%

“…In other regions, CheA and CheW may be bound to the receptors, but CheA is not activated. This schematic is similar in appearance to some of the membrane invaginations that have been observed in sections of wild-type cells by TEM (32).…”

Section: Discussionsupporting

confidence: 49%

Self-assembly of receptor/signaling complexes in bacterial chemotaxis

Wolanin¹,

Baker

Francis³

et al. 2006

“…To determine the architecture of full-length chemoreceptors, we first carried out cryo-electron tomography on frozenhydrated cells engineered to overproduce wild-type Tsr (Tsr QEQE ). The high levels of chemoreceptor expressed under these conditions result in the formation of small two-dimensional crystalline chemoreceptor patches (18,19), which were evident in projection images (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

Role of HAMP domains in chemotaxis signaling by bacterial chemoreceptors

Khursigara

Zhang

et al. 2008

Self Cite

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...

“…32 for a review). Extended arrays of receptors have been visualized by EM in cells overexpressing Tsr in the absence of CheW and CheA (34,35). In these cells, Tsr tended to form bilaminar structures where the signaling domains are apparently interdigitated in an antiparallel manner.…”

Section: Discussionmentioning

confidence: 99%

Three-dimensional structure and organization of a receptor/signaling complex

Francis

Wolanin

Stock

et al. 2004

Transmembrane signaling in bacterial chemotaxis has become an important model system for experimental and theoretical studies. These studies have provided a wealth of detailed molecular structures, including the structures of CheA, CheW, and the cytoplasmic domain of the serine receptor Tsr. How these three proteins interact to form the receptor͞signaling complex remains unknown. By using EM and single-particle image analysis, we present a three-dimensional reconstruction of the receptor͞signaling complex. The complex contains CheA, CheW, and the cytoplasmic portion of the aspartate receptor Tar. We observe density consistent with a structure containing 24 aspartate-receptor monomers and additional density sufficient to house the expected four CheA monomers and six CheW monomers. Within this bipolar structure are four groups of three receptor dimers that are not threefold symmetric and are therefore unlike the symmetric trimers observed in the x-ray crystal structure of the cytoplasmic domain of the serine receptor. In the latter, the interdimer contacts occur in the signaling domains near the hairpin loop. In our structure, the signaling domains within trimers appear spaced apart by the presence of CheA and CheW. This structure argues against models where one CheA and one CheW bind to the outer face of each of the dimers in the trimer. This structure of the receptor͞signaling complex provides an additional basis for understanding the architecture of the large arrays of chemotaxis receptors, CheA, and CheW found at the cell poles in motile bacteria.chemotaxis ͉ electron microscopy ͉ macromolecular assembly ͉ signal transduction M otile bacteria are capable of detecting and moving in response to gradients of nutrients, oxygen, light, and other stimuli. They do so in response to temporal changes in the stimuli by altering the behavior of the motors that generate the force for cell movement. The signal transduction system controlling bacterial chemotaxis has become an important model for the study of transmembrane signaling, and the most studied system is that in Escherichia coli.The core components of the E. coli chemotaxis signaltransduction system are found in nearly all motile bacteria and consist of transmembrane chemoreceptors; an associated histidine kinase, CheA; and an associated CheA-activator, CheW. Signaling occurs within the context of the assembled receptor͞ CheW͞CheA complexes within a large, densely packed patch of chemoreceptors, which is typically located at one (or both) poles of the cell (1). No gross structural changes in the receptor͞ signaling complex are observed in response to stimulatory ligands (2).Recent studies and modeling of the chemotaxis system have emphasized the importance of interactions among large numbers of receptors in terms of the system's sensitivity and adaptive response (3, 4), and chemoreceptors for different ligands appear to communicate with one another (3). The lack of detailed information about how CheA and CheW interact with the chemoreceptors and each other in the ...