Role of HAMP domains in 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 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 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 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.

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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 of the 3D density maps for each class average is Ϸ33 Å as determined by estimates of the resolution at which the Fourier shell correlation coefficient between two halves of the data decreases to a value of 0.5.

Chemoreceptors Adopt Two Distinct Conformations. Inspection of density maps for TsrQEQE confirmed that chemoreceptors are indeed organized as trimers of receptor dimers in the cytoplas- mic membrane, as anticipated from numerous biochemical, C structural, and modeling studies (4, 11, 20). Each repeating unit consists of a longitudinal density Ϸ203 Å in length corresponding to the cytoplasmic domain of the receptor and a 77-Å overlap region where the cytoplasmic ends of apposing chemoreceptor dimers interact. Previous analysis of negatively stained mem- brane assemblies (19) has provided similar estimates of Ϸ189 Å and Ϸ65 Å for lengths of the cytoplasmic domain and the overlap regions, respectively. The locations of the individual chemore- ceptor modules including the signaling region, the HAMP Fig. 2. Cryo-electron tomographic analysis of crystalline Tsr assemblies. (A and domain, and the periplasmic sensing domain were unambigu- B) A 4-nm-thick tomographic slice from a region of a reconstructed tomogram of whole cells containing receptor arrays, and its Fourier transform, respectively. The ously identified based on the shape of intact, membrane- circled diffraction spot in B is at a resolution of Ϸ33 Å. (C) Representative embedded receptors revealed by electron microscopy (19) and by examples of tomographic slices corresponding to local receptor clusters extracted the organization of the chemoreceptor polypeptide (7). We from whole cell tomograms. [Scale bars: 50 nm (A) and 10 nm (C).] therefore placed the corresponding coordinates of these do-

16556 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806401105 Khursigara et al. Downloaded by guest on September 28, 2021 density profile of the membrane along the length of the receptor. AB C Second, components of the receptor in the aqueous phase are detected in cryo-electron microscopic images by virtue of contrast between the protein and the aqueous phase. However, detection of the components of the protein in the lipid bilayer relies on the much poorer contrast between the protein and the rest of the components of the lipid bilayer (mainly lipids). As a result, the map in the region within the lipid bilayer is noisier and therefore less reliable. Thus, chemoreceptor density in the transmembrane regions were approx- imated by using standard ␣-helices following the model suggested by Kim et al. (22) because our map is unreliable in this region. The most striking difference between the two conformations is in the position of the HAMP domains relative to the rest of the chemoreceptors in the trimer unit. In the compact conformation shown in Fig. 4A (cross-sectional views shown in Fig. 4D) the density corresponding to the HAMP domains is continuous with the density arising from the cytoplasmic signaling domain. How- ever, in the expanded conformation shown in Fig. 4B (cross- sectional views shown in Fig. 4E), the HAMP domains from each of the three Tsr dimers within the trimeric unit are displaced outward by Ϸ25 Å. The maps also indicate that there must be significant differences between the two conformations at the junc- D tion between the HAMP and cytoplasmic domains. However, at the present resolution of our maps, the density for the connecting regions is not clearly visible in the expanded conformation, and we have therefore not explicitly interpreted this region in terms of changes in conformation of the cytoplasmic region. The location of the HAMP domains in the expanded conformation shown in Fig. 4B implies that portions of the cytoplasmic region are likely to be splayed outward at the interface joining these two chemoreceptor E modules. The positions of the HAMP domains within the expanded trimer are reminiscent of the splayed-out conformation of the upper portion of the Tsr signaling domain that was determined by using x-ray crystallography (11). The expanded HAMP conforma- tion also demonstrates a small reduction (Ϸ8%) in the overall trimer height when compared to the compact conformation, which may be the result of conformational changes within chemoreceptor Fig. 4. Identification of two trimeric Tsr conformations and interpretation of trimers that occur during signaling. In both conformations, the the density maps in terms of known structures of subdomains. Averaged density density map shows a clockwise rotation when the map is sectioned maps of TsrQEQE in both the compact (A) and expanded (B) conformations, with from the periplasmic region to the HAMP domains (as viewed structural coordinates corresponding to chemoreceptor models fitted to a single from the top, Fig. 4 D and E, sections 1 and 2), which is consistent trimer of receptor dimers. (C) Superposition of the two density maps highlighting with a tilted orientation of the periplasmic domain relative to the overlap in the cytoplasmic domain and differences in the HAMP and periplasmic membrane normal. The extent of the twist is slightly larger in the domains. A schematic view of the lipid bilayer is shown to highlight the locations of the putative transmembrane region in A–C.(D) Sections of the map from compact conformation than in the expanded conformation (com- regions marked 1–3 in A correspond to the periplasmic domain, HAMP domain, pare Fig. 4 A and B, section 1) and is compensated with a further and end of the cytoplasmic domain, respectively. Densities corresponding to each counterclockwise rotation between the HAMP domain and the top of these regions can be unambiguously assigned, although the map is not at a of the cytoplasmic domain, restoring the register of the two resolution where the rotational orientations of the individual domains can be conformations in the cytoplasmic domain [see supporting informa- determined. (E) Sections from the map shown in B displayed as described for D. tion (SI) Movie S1 for greater detail]. The density in the cytoplasmic domain (section 3) was fit with a trimer obtained

from three monomers derived from the structure of this region in the serine Effects of Ligand Binding and Methylation on Chemoreceptor Confor- BIOPHYSICS chemoreceptor Tsr reported by Kim et al. (11). Note that the packing of the trimer mations. To determine the effects of ligand binding and methylation in our map is different from the organization of monomers reported in the crystals used to determine the structure (37) (PDB ID code 1QU7). Both confor- on the conformation of TsrQEQE, tomographic volumes were gen- mations are interpreted by using the same packing arrangement in the cytoplas- erated for TsrQEQE in the presence of the attractant serine and for mic domain. fully methylated TsrQQQQ in both the absence and the presence of serine. Tomographic volumes for each Tsr variant demonstrated the formation of crystalline chemoreceptor patches with similar mains, which were previously derived by x-ray crystallography (8, hexagonal packing arrangements (Fig. 3 Insets). These patches were 11) and NMR (10) studies, into the density maps. subjected to the same averaging and alignment procedures as Overall, the two conformations that we obtained display similar described above. For each Tsr variant analyzed, the classification features in the C-terminal kinase control module. There are some resulted in the identification of two dominant clusters, and each noticeable differences in the density corresponding to the trans- cluster was then averaged separately to generate the corresponding membrane region, but these are not readily interpretable or nec- conformational states. As in the case of TsrQEQE in the absence of essarily significant because they are the most poorly defined regions serine, refinement of the two clusters led to the generation of of the map for two separate reasons. First, because of the missing density maps that displayed two distinct chemoreceptor arrange- wedge in data collection for electron tomography, the resolution in ments for each Tsr variant, with primary differences in the position the direction along the electron beam is poorer than it is in the plane of the HAMP domains in either compact or expanded conforma- perpendicular to the beam (21), resulting in poorer resolution of the tions (Fig. 5 A–D). Quantitative analysis of the relative amounts of

Khursigara et al. PNAS ͉ October 28, 2008 ͉ vol. 105 ͉ no. 43 ͉ 16557 Downloaded by guest on September 28, 2021 A B C D

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Fig. 5. Effects of ligand binding and chemoreceptor methylation on the distribution of Tsr conformations. Tomographic volumes were generated for TsrQEQE and the fully methylated TsrQQQQ variants of Tsr in the absence and presence of the attractant serine (Ser), and the distribution of compact or expanded conformations of the HAMP domains was compared. Shown are sectional views from superimposed density maps of each Tsr variant, TsrQEQE (A), TsrQEQEϩSer (B), TsrQQQQ (C), and TsrQQQQϩSer (D) demonstrating the compact (cyan) and expanded (magenta) conformation of the HAMP domain (as shown in Fig. 4, section 2). (E) Addition of the ligand serine to the TsrQEQE variant causes an increase in the proportion of receptors with an expanded conformation (magenta bars) of the HAMP domain and a concomitant decrease in the proportion of receptors in the compact conformation (cyan bars). Conversely, methylation of Tsr (TsrQQQQ) causes an increase in the proportion of the receptors in the compact state (compare TsrQEQE from E with TsrQQQQ from F). (F) Ligand binding to the TsrQQQQ variant causes a conformational shift similar to that seen in E for TsrQEQE.

chemoreceptors that adopt either the compact or expanded con- of the aspartate receptor in the ligand-free and ligand-bound formations for each of these conditions reveals the structural forms suggests that ligand binding causes a rotation of the consequences of ligand binding and methylation. In the absence of monomers in the periplasmic domain relative to each other in the added serine, the percentage of chemoreceptors with compact chemoreceptor homodimer (8, 27). Independently, extensive versus expanded HAMP domain conformations in cells expressing biochemical and cross-linking analyses have led to models in TsrQEQE is Ϸ67% and Ϸ33%, respectively (Fig. 5E). This distribu- which the signal of ligand binding is communicated across the tion was altered with the addition of serine (TsrQEQEϩSer), which resulted in an increase in the proportion of chemoreceptors in the expanded conformation to Ϸ44% (Fig. 5E). In fully methylated TsrQQQQ and in the absence of serine, the proportion of chemo- receptors in the expanded and compact conformations is Ϸ25% and Ϸ75%, respectively. However, serine binding to TsrQQQQ results in a change in proportion of the two states similar to that seen with TsrQEQE (Fig. 5F). Thus, we demonstrate that binding of serine causes an increase in the proportion of chemoreceptors that have the HAMP domains in the expanded conformation regardless of the initial methylation state of the receptor. In the absence of added serine, an increase in chemoreceptor methylation causes an increase in the proportion of chemoreceptors that have the HAMP domains in the compact conformation.

The Two-State Chemoreceptor Model and Conformational Signaling. The coexistence of two distinct trimeric chemoreceptor confor- mations, with major differences in the position of their HAMP domains and with an overall twisted trimer-of-dimers architec- ture, provides an attractive solution to a longstanding question about the conformational mechanisms involved in signal trans- Fig. 6. A two-state model describing conformational signaling in chemore- duction by chemoreceptors. Based on our results and the known ceptor trimers. Trimeric chemoreceptors exist in an equilibrium between two effects of attractant binding and methylation on CheA kinase conformations and adopt either expanded or compact arrangements of the activity (7, 9, 16, 17, 23–26), we are able to propose a model for HAMP signaling domain. Binding of the attractant serine initiates movement conformational signaling via bacterial chemoreceptors. Chemo- in the transmembrane helix (9), which, in turn, shifts the conformational receptors exist in an equilibrium, in which their HAMP domains equilibrium of the HAMP domain in favor of the expanded conformation (magenta). Based on the known effects of serine binding to reduce the activity are in flux between compact and expanded conformations (Fig. of the CheA kinase, we propose that this expanded conformation of the HAMP 6). Binding of the attractant serine to Tsr trimers initiates a domain corresponds to the ‘‘kinase-off’’ state. Conversely, an increase in conformational change that is propagated to the HAMP do- chemoreceptor methylation shifts the equilibrium in favor of the compact main, causing its displacement (Fig. 6). Crystallographic studies HAMP conformation (cyan), corresponding to the ‘‘kinase-on’’ state.

16558 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806401105 Khursigara et al. Downloaded by guest on September 28, 2021 membrane by small vertical displacement of the transmembrane Quantifoil grid (Micro Tools), which had been precoated with gold beads (15 regions of the receptor (9, 28) and/or by rotational displacements nm) to serve as fiducial markers. The grids were manually blotted and plunged initiated at the periplasmic domain (29) and the HAMP domain into cold liquid ethane maintained at approximately Ϫ180°C. The grids were (10). Because the conformational changes that result from attract- then placed in cartridges and loaded into the cryotransfer system of a Polara G2 microscope (FEI). This microscope is equipped with a field emission gun ant binding are known to lower CheA kinase activity (7, 9), the operating at 300 kV, and a 2K ϫ 2K CCD camera mounted at the end of a GIF expanded conformation of the HAMP domain must correspond to 2000 energy filter (Gatan). A series of low-dose projection images of whole the ‘‘kinase-off’’ state. Similarly, an increase in chemoreceptor cells spanning a tilt angle range from Ϫ70° to 70° in 1–3° intervals was methylation results in structural changes (16, 17) that shift the recorded at liquid nitrogen temperatures in the zero-loss mode with a 30-eV equilibrium in favor of the compact HAMP arrangement, corre- slit at effective magnifications of ϫ44,000 and underfocus values ranging sponding to the ‘‘kinase-on’’ state (Fig. 6). This type of movement from5to8␮m. The total dose used for each tilt series was typically Ϸ60–80 in the HAMP domains is also consistent with the changes observed electrons per square angstrom. in fluorescence anisotropy of YFP-labeled chemoreceptors in response to attractant and repellant signals interpreted to arise Tomographic Reconstruction and Three-Dimensional Averaging. For all Tsr vari- ants, a series of two-dimensional tilted projection images (tilt series) were con- from movements of the trimer arms (26). verted into three-dimensional density maps (tomograms) by using the weighted The approach we have used here is a hybrid between back-projection algorithm from the IMOD software package (33). Using the techniques used to determine membrane protein structure by repeating chemoreceptor units from the TsrQEQE variant, an initial rough model electron crystallography and cellular architecture using cryo- was generated by using an approach for 3D averaging based on principles electron tomography, in combination with 3D classification. described by Winkler (34). Specifically, crystalline patches were selected from the This analysis has led to the identification of two distinct tomogram, and the volume corresponding to a small repeating unit was selected conformational states of the chemoreceptor Tsr in its trimeric to search the entire volume to generate a set of subvolumes. This search was organization and has demonstrated the propensity of HAMP guided by determination of the local lattice vectors of the partially ordered patches. The subvolumes were then subjected to several rounds of alignment. domains to be present in two distinct spatial positions relative The aligned subvolumes were then projected along the z axis, and the resulting to the rest of the trimer unit. The results presented here 2D projection images were classified by using EMAN software (35). The dominant provide the first direct structural evidence for the two-state class was used as a new reference for further alignment of a total of 750 chemoreceptor-signaling model and demonstrate that ligand subvolumes collected from six crystalline patches of the TsrQEQE variant, and these binding and methylation can modulate the conformational aligned volumes were averaged together to generate a single density map that equilibrium of chemoreceptors. However, it remains to be seen served as a rough starting model. whether similar conformational states are also observed in the With this starting model, 3,972 TsrQEQE subvolumes, 23,505 TsrQEQE plus serine presence of CheA and CheW and in the context of wild-type (10 mM) subvolumes, 3,797 TsrQQQQ subvolumes, and 12,783 TsrQQQQ plus serine (10 mM) subvolumes were aligned by using the grid-threading Monte Carlo chemoreceptor arrays (30) and whether the homotypic inter- searching algorithm described previously (36). Analysis of the correlation coeffi- actions of the chemoreceptors at the cytoplasmic end lead to cients among the aligned subvolumes identified two major clusters for each Tsr differences in the extent or nature of the structural change as variant. The average of these two clusters led to two conformational states as compared with chemoreceptor–CheA interactions. This is templates with a weighting factor depending on the correlation coefficient especially interesting because our previous analyses have between a subvolume and the template: shown that chemoreceptor ‘‘zippers’’ can coexist with chemo- 1 receptor/CheA/CheW complexes in cells where Tsr is selec- ͑ ͒ ϭ w i Ϫ ͑͑ Ϫ ៮ ͒ ␦ Ϫ ͒ . tively overproduced (30), suggesting that axial receptor– 1 ϩ e a Ci C / C b receptor interactions in the ‘‘zipper’’ region may mimic receptor–CheA/CheW interactions in the wild-type signaling Here w(i) and Ci are the weight of subvolume i and correlation between it and ៮ array. Furthermore, the ‘‘zipper’’-like arrangement may inhibit the template. C and ␦C are the average and the standard deviation of larger-scale arrangements of the receptors that may include correlation coefficients of all subvolumes. a and b are two parameters, typi- intertwined trimers of dimers that have been proposed to be cally with values of 2 and 0, respectively, that define the dependence of the weight on correlation coefficient distribution. involved in kinase down-regulation (22, 31). Application of the Using these averaged volumes as templates, further alignment and methodologies developed here to chemoreceptor arrays in weighted averaging were performed until no significant changes could be wild-type E. coli cells and those of other Gram-negative observed in alignments and averaged maps (final maps for TsrQEQE are shown bacteria (32) should help further elucidate the conformational in Fig. 3). The largest differences between the two averaged maps exist in the changes involved in chemotaxis signaling. region corresponding to the HAMP domains, which are arranged in either compact or expanded conformations. For each Tsr variant, the distribution Materials and Methods between the compact and expanded conformations was normalized to 100% based on the total number of subvolumes in each class to facilitate compar- Bacterial Strains, Plasmids, and Culture Conditions. All expression studies were

isons between experimental conditions. The atomic models of the chemore- BIOPHYSICS carried out with E. coli strain HCB721, which lacks expression of the chemo- ceptor domains [Tsr periplasmic domain (37) (PDB ID code 1VLS), the HAMP taxis-related proteins Tar, Tsr, Trg, Tap, CheA, CheW, CheR, and CheB and is an domain from an archaeal protein (10) (PDB ID code 2ASW) and a modeled isogenic derivative of E. coli strain K12 strain RP437 (32). HCB721 cells were transmembrane domain (18)] were initially placed manually into the density transformed with pHSe5.tsr or pHSe5.tsr plasmids to overproduce Tsr QEQE QQQQ map and subsequently refined by using the local map-fitting feature in the as described previously (19). The cells were grown at 27°C in tryptone broth software package Chimera (38). (1.0% tryptone and 0.5% NaCl) in the presence of ampicillin (100 ␮g/ml) to OD600 of 0.1, and Tsr expression was induced by the addition of isopropyl-␤-D- ACKNOWLEDGMENTS. We thank Drs. Martin Kessel and Mario J. Borgnia for thiogalactopyranoside (IPTG, 1 mM) for 3–4 h. insightful comments and Ethan Tyler and Alan Hoofring for expert assistance with preparation of the figures. This work was supported by funds from the Cryo-electron Tomography. A3-to5-␮l aliquot of cells (OD600 Ϸ 0.5) was intramural research program of the National Cancer Institute, National Insti- withdrawn directly from a culture and immediately placed on a MultiA tutes of Health.

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