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Role of HAMP Domains in Chemotaxis Signaling by Bacterial Chemoreceptors

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Role of HAMP Domains in Chemotaxis Signaling by Bacterial Chemoreceptors Role of HAMP domains in chemotaxis signaling by bacterial chemoreceptors Cezar M. Khursigara*†, Xiongwu Wu†‡, Peijun Zhang*†§, Jonathan Lefman*, and Sriram Subramaniam*¶ *Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and ‡Laboratory of Computational Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892 Edited by David J. DeRosier, Brandeis University, Waltham, MA, and approved September 4, 2008 (received for review July 2, 2008) Bacterial chemoreceptors undergo conformational changes in re- sites for the CheA kinase (11, 14, 15), respectively. The confor- sponse to variations in the concentration of extracellular ligands. mational changes resulting from binding of an attractant result These changes in chemoreceptor structure initiate a series of in decreased kinase activity (7, 9). Chemoreceptor methylation signaling events that ultimately result in regulation of rotation of reverses these conformational changes and results in an increase the flagellar motor. Here we have used cryo-electron tomography in kinase activity (7, 16, 17). combined with 3D averaging to determine the in situ structure of Although atomic structures for several chemoreceptor domain chemoreceptor assemblies in Escherichia coli cells that have been fragments have been determined (8, 10, 11), the molecular engineered to overproduce the serine chemoreceptor Tsr. We architectures of intact chemoreceptor homodimers, or of the demonstrate that chemoreceptors are organized as trimers of functionally relevant trimer-of-dimers configuration (3–6), have receptor dimers and display two distinct conformations that differ remained inaccessible to direct structural approaches. More- principally in arrangement of the HAMP domains within each over, the conformational changes involved in signaling are poorly trimer. Ligand binding and methylation alter the distribution of understood. Here we present the architecture of the full-length chemoreceptors between the two conformations, with serine bind- serine chemoreceptor, Tsr from E. coli. We show that Tsr forms ing favoring the ‘‘expanded’’ conformation and chemoreceptor trimers of receptor dimers that display two distinct states in methylation favoring the ‘‘compact’’ conformation. The distinct which the HAMP domains are present in either compact or positions of chemoreceptor HAMP domains within the context of expanded conformations, and we demonstrate how ligand bind- a trimeric unit are thus likely to represent important aspects of ing and methylation modulate this conformational equilibrium. chemoreceptor structural changes relevant to chemotaxis signal- ing. Based on these results, we propose that the compact and Results and Discussion expanded conformations represent the ‘‘kinase-on’’ and ‘‘kinase- Cryo-electron Tomography of Overproduced TsrQEQE in Whole E. coli off’’ states of chemoreceptor trimers, respectively. Cells. To determine the architecture of full-length chemorecep- tors, we first carried out cryo-electron tomography on frozen- cryo-electron tomography ͉ molecular architecture ͉ signal transduction hydrated cells engineered to overproduce wild-type Tsr (TsrQEQE). The high levels of chemoreceptor expressed under otile bacteria such as Escherichia coli respond to changes these conditions result in the formation of small two-dimensional Min their chemical environment by recognizing variations in crystalline chemoreceptor patches (18, 19), which were evident the ligand occupancy of a series of transmembrane chemore- in projection images (Fig. 1). Within these patches, chemore- ceptors (also known as methyl-accepting chemotaxis proteins, or ceptors are organized in ‘‘zippers’’ (Fig. 1 Inset) that are formed MCPs). Ligand binding to chemoreceptors initiates a signaling by invaginations of the cytoplasmic membrane in which the cascade that modulates the activity of the histidine kinase CheA, cytoplasmic ends of chemoreceptors in the upper layer interact with the cytoplasmic ends of the chemoreceptors in the lower which ultimately regulates the rotation of the flagellar motor layer (Fig. 1 Inset) (18). The intrinsic order in chemoreceptor through a diffusible intracellular signal (1, 2). Chemoreceptors assemblies was observed in reconstructed tomograms, as illus- exist as homodimers and have been shown to function as trimers trated in a 4-nm slice from a tomogram of a region of the cell of chemoreceptor dimers both in vitro (3) and in vivo (4–6). containing a chemoreceptor assembly (Fig. 2A). The order (unit Chemoreceptor homodimers can be divided into three func- cell dimensions Ϸ 75 Å) in individual patches typically extends tional modules, each of which plays a critical role in receptor- to Ϸ33 Å (Fig. 2B), which represents a lower limit to the in-plane mediated signaling (7). The transmembrane-sensing module is resolution of the tomogram. composed of both the sensing domain, which resides in the To obtain structural information for the chemoreceptors, we periplasm and contains the ligand binding site (8), and the BIOPHYSICS started with tomograms of individual patches from cells over- transmembrane portion of the chemoreceptor. It has been producing Tsr and applied image-averaging methods sim- deduced that ligand binding to the periplasmic domain results in QEQE ilar to those used in electron crystallography of two-dimensional a piston-like sliding motion of one of the four transmembrane protein crystals combined with three-dimensional alignment 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 Author contributions: C.M.K., P.Z., and S.S. designed research; C.M.K., X.W., P.Z., and J.L. kinase, adenylyl cyclases, methyl-binding proteins, and phospha- performed research; C.M.K., X.W., P.Z., and S.S. analyzed data; and C.M.K. and S.S. wrote tases) domain, and a kinase control module, which contains an the paper. adaptation region and a protein interaction region (7, 9). The The authors declare no conflict of interest. HAMP domain is the proposed site of signal conversion between This article is a PNAS Direct Submission. the transmembrane-sensing and kinase control modules (7). The Freely available online through the PNAS open access option. recent solution structure of an archaeal HAMP domain dem- †C.M.K., X.W., and P.Z. contributed equally to this work. onstrates that it adopts a homodimeric four-helical parallel §Present address: Department of Structural Biology, University of Pittsburgh School of coiled-coil conformation (10). The adaptation and protein in- Medicine, Pittsburgh, PA 15260. teraction regions are composed of a continuous four-helix ¶To whom correspondence should be addressed. E-mail: [email protected]. antiparallel coiled coil with a hairpin at its distal end (11) and This article contains supporting information online at www.pnas.org/cgi/content/full/ contain key methyl-accepting residues (12, 13) and interaction 0806401105/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806401105 PNAS ͉ October 28, 2008 ͉ vol. 105 ͉ no. 43 ͉ 16555–16560 Downloaded by guest on September 28, 2021 A B C D Fig. 3. Cryo-electron tomography of Tsr chemoreceptor assemblies in whole Fig. 1. Cryo-electron microscopy of Tsr chemoreceptor assemblies in whole E. E. coli cells. Shown are tomographic slices (Ϸ10 nm thick) generated from E. coli cells. Shown is a projection image recorded from a plunge-frozen E. coli cell coli cells engineered to overproduce TsrQEQE in the absence (A) or presence (B) engineered to overproduce TsrQEQE (in the absence of serine) by using low-dose of serine (Ser), and TsrQQQQ in the in the absence (C) or presence (D) of serine. cryo-electron microscopy. The projection image shows patches of chemoreceptor Chemoreceptor assemblies (CA) are observed as crystalline patches embedded assemblies in the native cytoplasmic membrane (CM) contained within a cell with in cytoplasmic membrane (CM). The crystalline patches are observed through- an intact outer membrane (OM). The cell is close to the edge of a hole in a holey out the cells and are contained by the outer membrane (OM); an expanded carbon grid containing vitreous ice; the black dots are 15-nm gold particles added view of the patches is shown in the Inset to each panel. (Scale bars: 100 nm.) for use as fiducial markers in tomogram reconstruction. (Inset) A schematic perspective view of ‘‘zipper’’-like chemoreceptor assemblies from a region such as that enclosed by the white box. (Scale bar: 100 nm.) corresponding to the repeating units (Fig. 2C). These subvol- umes were then subjected to several rounds of alignment, first within a single patch and subsequently over many patches procedures similar to those used in single-particle analysis (see derived from multiple cells. The aligned subvolumes were then Materials and Methods). Guided by the local lattice vectors of the subjected to 3D classification to determine the major confor- partially ordered patches, we first extracted the subvolumes mations present in the chemoreceptor population. Ordered arrays were observed in cells expressing both TsrQEQE and TsrQQQQ and in the absence or presence of added serine (Fig. 3). A B The classification analysis resulted in the identification of two major clusters, and units within each cluster were then averaged separately to generate the corresponding conformational states (Fig. 4). The resolution
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