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• W2140156945 abstract "In bacteria, two signal-sequence-dependent secretion pathways translocate proteins across the cytoplasmic membrane. Although the mechanism of the ubiquitous general secretory pathway is becoming well understood, that of the twin-arginine translocation pathway, responsible for translocation of folded proteins across the bilayer, is more mysterious. TatC, the largest and most conserved of three integral membrane components, provides the initial binding site of the signal sequence prior to pore assembly. Here, we present two crystal structures of TatC from the thermophilic bacteria Aquifex aeolicus at 4.0 Å and 6.8 Å resolution. The membrane architecture of TatC includes a glove-shaped structure with a lipid-exposed pocket predicted by molecular dynamics to distort the membrane. Correlating the biochemical literature to these results suggests that the signal sequence binds in this pocket, leading to structural changes that facilitate higher order assemblies. In bacteria, two signal-sequence-dependent secretion pathways translocate proteins across the cytoplasmic membrane. Although the mechanism of the ubiquitous general secretory pathway is becoming well understood, that of the twin-arginine translocation pathway, responsible for translocation of folded proteins across the bilayer, is more mysterious. TatC, the largest and most conserved of three integral membrane components, provides the initial binding site of the signal sequence prior to pore assembly. Here, we present two crystal structures of TatC from the thermophilic bacteria Aquifex aeolicus at 4.0 Å and 6.8 Å resolution. The membrane architecture of TatC includes a glove-shaped structure with a lipid-exposed pocket predicted by molecular dynamics to distort the membrane. Correlating the biochemical literature to these results suggests that the signal sequence binds in this pocket, leading to structural changes that facilitate higher order assemblies. Crystal structures of TatC from the thermophile Aquifex aeolicus are presented The architecture of TatC generates a glove-shaped pocket buried in the bilayer Molecular dynamics reveal destabilization of the membrane around the pocket Correlation to biochemical results suggest the signal sequence binds in this pocket Prokaryotic organisms secrete protein via two main pathways: the general secretory (SEC) pathway, where the Sec translocon facilitates passage of unfolded protein across the bilayer, and the twin arginine translocation (TAT) pathway, which is involved in targeting and translocation of fully folded proteins across the inner membrane of bacteria (see the following for recent reviews: for the SEC pathway, see Park and Rapoport, 2012Park E. Rapoport T.A. Mechanisms of Sec61/SecY-mediated protein translocation across membranes.Annu. Rev. Biophys. 2012; 41: 21-40Crossref PubMed Scopus (271) Google Scholar, and for the TAT pathway, see Fröbel et al., 2012aFröbel J. Rose P. Müller M. Twin-arginine-dependent translocation of folded proteins.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2012; 367: 1029-1046Crossref PubMed Scopus (68) Google Scholar and Palmer and Berks, 2012Palmer T. Berks B.C. The twin-arginine translocation (Tat) protein export pathway.Nat. Rev. Microbiol. 2012; 10: 483-496PubMed Google Scholar). TAT pathway substrates are characterized by a critical pair of arginines in a consensus sequence (Berks, 1996Berks B.C. A common export pathway for proteins binding complex redox cofactors?.Mol. Microbiol. 1996; 22: 393-404Crossref PubMed Scopus (561) Google Scholar; Chaddock et al., 1995Chaddock A.M. Mant A. Karnauchov I. Brink S. Herrmann R.G. Klösgen R.B. Robinson C. A new type of signal peptide: central role of a twin-arginine motif in transfer signals for the delta pH-dependent thylakoidal protein translocase.EMBO J. 1995; 14: 2715-2722Crossref PubMed Scopus (226) Google Scholar). The components of the pathway were identified in the thylakoid membrane of the chloroplast (Settles et al., 1997Settles A.M. Yonetani A. Baron A. Bush D.R. Cline K. Martienssen R. Sec-independent protein translocation by the maize Hcf106 protein.Science. 1997; 278: 1467-1470Crossref PubMed Scopus (233) Google Scholar) and in E. coli (Sargent et al., 1998Sargent F. Bogsch E.G. Stanley N.R. Wexler M. Robinson C. Berks B.C. Palmer T. Overlapping functions of components of a bacterial Sec-independent protein export pathway.EMBO J. 1998; 17: 3640-3650Crossref PubMed Scopus (443) Google Scholar; Weiner et al., 1998Weiner J.H. Bilous P.T. Shaw G.M. Lubitz S.P. Frost L. Thomas G.H. Cole J.A. Turner R.J. A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins.Cell. 1998; 93: 93-101Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). Although the TAT system is broadly conserved, it is not essential for viability under standard lab conditions in bacteria (Bogsch et al., 1998Bogsch E.G. Sargent F. Stanley N.R. Berks B.C. Robinson C. Palmer T. An essential component of a novel bacterial protein export system with homologues in plastids and mitochondria.J. Biol. Chem. 1998; 273: 18003-18006Crossref PubMed Scopus (329) Google Scholar; Jongbloed et al., 2004Jongbloed J.D. Grieger U. Antelmann H. Hecker M. Nijland R. Bron S. van Dijl J.M. Two minimal Tat translocases in Bacillus.Mol. Microbiol. 2004; 54: 1319-1325Crossref PubMed Scopus (159) Google Scholar). In TAT-pathway-containing organisms, approximately 10% of the total secretome are TAT substrates. The most significant exceptions are halophilic archaea, in which the majority of secreted proteins appear to utilize the TAT pathway (Bolhuis, 2002Bolhuis A. Protein transport in the halophilic archaeon Halobacterium sp. NRC-1: a major role for the twin-arginine translocation pathway?.Microbiology. 2002; 148: 3335-3346PubMed Google Scholar; Rose et al., 2002Rose R.W. Brüser T. Kissinger J.C. Pohlschröder M. Adaptation of protein secretion to extremely high-salt conditions by extensive use of the twin-arginine translocation pathway.Mol. Microbiol. 2002; 45: 943-950Crossref PubMed Scopus (225) Google Scholar; Thomas and Bolhuis, 2006Thomas J.R. Bolhuis A. The tatC gene cluster is essential for viability in halophilic archaea.FEMS Microbiol. Lett. 2006; 256: 44-49Crossref PubMed Scopus (22) Google Scholar) and the pathway is required for viability (Dilks et al., 2005Dilks K. Giménez M.I. Pohlschröder M. Genetic and biochemical analysis of the twin-arginine translocation pathway in halophilic archaea.J. Bacteriol. 2005; 187: 8104-8113Crossref PubMed Scopus (65) Google Scholar). Most TAT substrates are complex, containing cofactors and/or oligomeric assemblies, and must be correctly folded and assembled in the cytoplasm prior to translocation, necessitating a large pore that can translocate a diversity of folded proteins. Example secretion substrates include respiratory redox enzymes, bacterial virulence factors (Kassem et al., 2011Kassem I.I. Zhang Q. Rajashekara G. The twin-arginine translocation system: contributions to the pathobiology of Campylobacter jejuni.Future Microbiol. 2011; 6: 1315-1327Crossref PubMed Scopus (7) Google Scholar; van der Ploeg et al., 2011van der Ploeg R. Mäder U. Homuth G. Schaffer M. Denham E.L. Monteferrante C.G. Miethke M. Marahiel M.A. Harwood C.R. Winter T. et al.Environmental salinity determines the specificity and need for Tat-dependent secretion of the YwbN protein in Bacillus subtilis.PLoS ONE. 2011; 6: e18140Crossref PubMed Scopus (35) Google Scholar), lipoproteins (Shruthi et al., 2010Shruthi H. Anand P. Murugan V. Sankaran K. Twin arginine translocase pathway and fast-folding lipoprotein biosynthesis in E. coli: interesting implications and applications.Mol. Biosyst. 2010; 6: 999-1007Crossref PubMed Scopus (22) Google Scholar), and proteins involved in maintaining cell wall integrity and cell motility (Stanley et al., 2001Stanley N.R. Findlay K. Berks B.C. Palmer T. Escherichia coli strains blocked in Tat-dependent protein export exhibit pleiotropic defects in the cell envelope.J. Bacteriol. 2001; 183: 139-144Crossref PubMed Scopus (142) Google Scholar). Additionally, some inner membrane proteins have been found that can be inserted via this pathway (Hatzixanthis et al., 2003Hatzixanthis K. Palmer T. Sargent F. A subset of bacterial inner membrane proteins integrated by the twin-arginine translocase.Mol. Microbiol. 2003; 49: 1377-1390Crossref PubMed Scopus (103) Google Scholar; Heikkilä et al., 2001Heikkilä M.P. Honisch U. Wunsch P. Zumft W.G. Role of the Tat ransport system in nitrous oxide reductase translocation and cytochrome cd1 biosynthesis in Pseudomonas stutzeri.J. Bacteriol. 2001; 183: 1663-1671Crossref PubMed Scopus (47) Google Scholar; Ochsner et al., 2002Ochsner U.A. Snyder A. Vasil A.I. Vasil M.L. Effects of the twin-arginine translocase on secretion of virulence factors, stress response, and pathogenesis.Proc. Natl. Acad. Sci. USA. 2002; 99: 8312-8317Crossref PubMed Scopus (172) Google Scholar; Schaerlaekens et al., 2001Schaerlaekens K. Schierová M. Lammertyn E. Geukens N. Anné J. Van Mellaert L. Twin-arginine translocation pathway in Streptomyces lividans.J. Bacteriol. 2001; 183: 6727-6732Crossref PubMed Scopus (74) Google Scholar). The TAT pathway, as described in E. coli and chloroplasts, minimally requires three membrane proteins: TatA, TatB, and TatC (Bogsch et al., 1998Bogsch E.G. Sargent F. Stanley N.R. Berks B.C. Robinson C. Palmer T. An essential component of a novel bacterial protein export system with homologues in plastids and mitochondria.J. Biol. Chem. 1998; 273: 18003-18006Crossref PubMed Scopus (329) Google Scholar; Sargent et al., 1998Sargent F. Bogsch E.G. Stanley N.R. Wexler M. Robinson C. Berks B.C. Palmer T. Overlapping functions of components of a bacterial Sec-independent protein export pathway.EMBO J. 1998; 17: 3640-3650Crossref PubMed Scopus (443) Google Scholar; Weiner et al., 1998Weiner J.H. Bilous P.T. Shaw G.M. Lubitz S.P. Frost L. Thomas G.H. Cole J.A. Turner R.J. A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins.Cell. 1998; 93: 93-101Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar), which can all be purified at variable ratios in a complex (Bolhuis et al., 2001Bolhuis A. Mathers J.E. Thomas J.D. Barrett C.M. Robinson C. TatB and TatC form a functional and structural unit of the twin-arginine translocase from Escherichia coli.J. Biol. Chem. 2001; 276: 20213-20219Crossref PubMed Scopus (222) Google Scholar). TatA and TatB contain a single N-terminal transmembrane helix (TM), and TatC contains six TMs (Behrendt et al., 2004Behrendt J. Standar K. Lindenstrauss U. Brüser T. Topological studies on the twin-arginine translocase component TatC.FEMS Microbiol. Lett. 2004; 234: 303-308Crossref PubMed Google Scholar; Gouffi et al., 2002Gouffi K. Santini C.L. Wu L.F. Topology determination and functional analysis of the Escherichia coli TatC protein.FEBS Lett. 2002; 525: 65-70Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). TatC has the highest conservation, performing the crucial role of recognition and initial binding of the signal sequence at the N-terminal end of the preprotein substrate (Allen et al., 2002Allen S.C. Barrett C.M. Ray N. Robinson C. Essential cytoplasmic domains in the Escherichia coli TatC protein.J. Biol. Chem. 2002; 277: 10362-10366Crossref PubMed Scopus (47) Google Scholar; Jongbloed et al., 2000Jongbloed J.D. Martin U. Antelmann H. Hecker M. Tjalsma H. Venema G. Bron S. van Dijl J.M. Müller J. TatC is a specificity determinant for protein secretion via the twin-arginine translocation pathway.J. Biol. Chem. 2000; 275: 41350-41357Crossref PubMed Scopus (135) Google Scholar). TatB and TatC form a stable complex predicted to contain up to eight copies of each protein in a size range of 360–700 kDa (Bolhuis et al., 2001Bolhuis A. Mathers J.E. Thomas J.D. Barrett C.M. Robinson C. TatB and TatC form a functional and structural unit of the twin-arginine translocase from Escherichia coli.J. Biol. Chem. 2001; 276: 20213-20219Crossref PubMed Scopus (222) Google Scholar; de Leeuw et al., 2002de Leeuw E. Granjon T. Porcelli I. Alami M. Carr S.B. Müller M. Sargent F. Palmer T. Berks B.C. Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes.J. Mol. Biol. 2002; 322: 1135-1146Crossref PubMed Scopus (91) Google Scholar; Kneuper et al., 2012Kneuper H. Maldonado B. Jäger F. Krehenbrink M. Buchanan G. Keller R. Müller M. Berks B.C. Palmer T. Molecular dissection of TatC defines critical regions essential for protein transport and a TatB-TatC contact site.Mol. Microbiol. 2012; 85: 945-961Crossref PubMed Scopus (40) Google Scholar; Lee et al., 2006Lee P.A. Orriss G.L. Buchanan G. Greene N.P. Bond P.J. Punginelli C. Jack R.L. Sansom M.S. Berks B.C. Palmer T. Cysteine-scanning mutagenesis and disulfide mapping studies of the conserved domain of the twin-arginine translocase TatB component.J. Biol. Chem. 2006; 281: 34072-34085Crossref PubMed Scopus (53) Google Scholar; McDevitt et al., 2005McDevitt C.A. Hicks M.G. Palmer T. Berks B.C. Characterisation of Tat protein transport complexes carrying inactivating mutations.Biochem. Biophys. Res. Commun. 2005; 329: 693-698Crossref PubMed Scopus (29) Google Scholar; Tarry et al., 2009Tarry M.J. Schäfer E. Chen S. Buchanan G. Greene N.P. Lea S.M. Palmer T. Saibil H.R. Berks B.C. Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system.Proc. Natl. Acad. Sci. USA. 2009; 106: 13284-13289Crossref PubMed Scopus (79) Google Scholar) in a possible 1:1 stoichiometric ratio (Alami et al., 2003Alami M. Lüke I. Deitermann S. Eisner G. Koch H.G. Brunner J. Müller M. Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli.Mol. Cell. 2003; 12: 937-946Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar; Cline and Mori, 2001Cline K. Mori H. Thylakoid DeltapH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport.J. Cell Biol. 2001; 154: 719-729Crossref PubMed Scopus (244) Google Scholar). This complex binds signal sequences that contain the TAT motif (S/T-R-R-x-F-L-K) and transfers the substrate to a TatA complex (Alami et al., 2003Alami M. Lüke I. Deitermann S. Eisner G. Koch H.G. Brunner J. Müller M. Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli.Mol. Cell. 2003; 12: 937-946Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). TatA is predicted to serve as the protein-conducting translocation channel forming a modular homo-oligomeric ringlike pore for secretion of various sized substrates (Gohlke et al., 2005Gohlke U. Pullan L. McDevitt C.A. Porcelli I. de Leeuw E. Palmer T. Saibil H.R. Berks B.C. The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter.Proc. Natl. Acad. Sci. USA. 2005; 102: 10482-10486Crossref PubMed Scopus (219) Google Scholar; Sargent et al., 2001Sargent F. Gohlke U. De Leeuw E. Stanley N.R. Palmer T. Saibil H.R. Berks B.C. Purified components of the Escherichia coli Tat protein transport system form a double-layered ring structure.Eur. J. Biochem. 2001; 268: 3361-3367Crossref PubMed Scopus (127) Google Scholar). Translocation is powered by the proton motive force (PMF) and can be blocked by PMF inhibitors (Bageshwar and Musser, 2007Bageshwar U.K. Musser S.M. Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery.J. Cell Biol. 2007; 179: 87-99Crossref PubMed Scopus (66) Google Scholar; Gérard and Cline, 2007Gérard F. Cline K. The thylakoid proton gradient promotes an advanced stage of signal peptide binding deep within the Tat pathway receptor complex.J. Biol. Chem. 2007; 282: 5263-5272Crossref PubMed Scopus (60) Google Scholar; Kwan et al., 2008Kwan D.C. Thomas J.R. Bolhuis A. Bioenergetic requirements of a Tat-dependent substrate in the halophilic archaeon Haloarcula hispanica.FEBS J. 2008; 275: 6159-6167Crossref PubMed Scopus (9) Google Scholar; Panahandeh et al., 2008Panahandeh S. Maurer C. Moser M. DeLisa M.P. Müller M. Following the path of a twin-arginine precursor along the TatABC translocase of Escherichia coli.J. Biol. Chem. 2008; 283: 33267-33275Crossref PubMed Scopus (59) Google Scholar). TatA and TatB perform distinct functions in E. coli (Sargent et al., 1998Sargent F. Bogsch E.G. Stanley N.R. Wexler M. Robinson C. Berks B.C. Palmer T. Overlapping functions of components of a bacterial Sec-independent protein export pathway.EMBO J. 1998; 17: 3640-3650Crossref PubMed Scopus (443) Google Scholar, Sargent et al., 1999Sargent F. Stanley N.R. Berks B.C. Palmer T. Sec-independent protein translocation in Escherichia coli. A distinct and pivotal role for the TatB protein.J. Biol. Chem. 1999; 274: 36073-36082Crossref PubMed Scopus (248) Google Scholar); yet, sequence conservation suggests that they are derived from a common ancestor, as some TAT-containing bacteria do not appear to contain a TatB, with TatA taking on a dual role (Dilks et al., 2003Dilks K. Rose R.W. Hartmann E. Pohlschröder M. Prokaryotic utilization of the twin-arginine translocation pathway: a genomic survey.J. Bacteriol. 2003; 185: 1478-1483Crossref PubMed Scopus (203) Google Scholar; Wu et al., 2000Wu L.F. Ize B. Chanal A. Quentin Y. Fichant G. Bacterial twin-arginine signal peptide-dependent protein translocation pathway: evolution and mechanism.J. Mol. Microbiol. Biotechnol. 2000; 2: 179-189PubMed Google Scholar; Yen et al., 2002Yen M.R. Tseng Y.H. Nguyen E.H. Wu L.F. Saier Jr., M.H. Sequence and phylogenetic analyses of the twin-arginine targeting (Tat) protein export system.Arch. Microbiol. 2002; 177: 441-450Crossref PubMed Scopus (146) Google Scholar). Extensive genetic and biochemical studies have been performed to understand the interaction of TatC with the signal sequence and TatA and TatB; however, the aggregate of the data has led to a variety of very different models. The central role of TatC and its high conservation suggests that a structure of this protein will provide a wealth of information toward understanding the TAT pathway. Here, we present a structure of TatC from the thermophile Aquifex aeolicus in two different crystal forms at resolutions of 4.0 Å and 6.8 Å. The structure reveals a membrane protein that is shaped like a baseball glove with the concave pocket exposed to the bilayer. We used molecular dynamics to look at this unusual architecture in a bilayer demonstrating the flexible parts of the protein and a water funnel that lines the pocket. We use computational docking to suggest possible dimerization interfaces. Finally, we correlate these results with existing biochemical data to develop a model for signal sequence binding where the signal sequence docks into the groove. This provocative solution to substrate recognition would allow for subsequent conformational changes required for further complex assembly. Genes for TatC from 26 eubacteria, archaea, and a mitochondrion (Malawimonas jakobiformis) were either codon optimized for E. coli and synthesized based on their protein sequence or amplified from genomic DNA, and they were then cloned into an inducible expression vector. To prevent copurification of native E. coli TAT components, the operon for TatABCD followed by the gene for TatE, a TatA paralog, were deleted from the strain BL21(DE3)GOLD to generate the strain CJMS2. Using this strain, we tested each clone for expression in a variety of conditions. Ultimately, TatC from the hyperthermophile Aquifex aeolicus (AaTatC) proved to give the best expression and was used for structural studies. This protein was well behaved by gel filtration in a variety of detergents including dodecyl-maltoside (DDM) and di-heptanoyl phosphatidylcholine (DHPC). The wild-type AaTatC crystallized in DHPC resulted in diffraction with reflections visible at ∼10 Å resolution after some optimization. To improve the diffraction, mutants were generated to reduce surface entropy, with one combination (K40A, E41A with a C-terminal truncation) resulting in well-formed crystals that diffracted to 7.5 Å. By visual inspection, it became clear that two different kinds of crystal morphology were growing in the same drop. The less frequent of the two crystal forms diffracted better and was used for seeding into clear drops, resulting in only this form appearing. A single crystal of this form that diffracted to high resolution was used to collect a native data set at 4.0 Å resolution in the space group P4122, with cell dimension a = b = 110.43 Å c = 107.42 Å, referred to here as AaDHPC. An alternative approach to obtaining crystals was to generate a lysozyme-AaTatC fusion similar to that used for other membrane protein crystallization (Rosenbaum et al., 2007Rosenbaum D.M. Cherezov V. Hanson M.A. Rasmussen S.G. Thian F.S. Kobilka T.S. Choi H.J. Yao X.J. Weis W.I. Stevens R.C. Kobilka B.K. GPCR engineering yields high-resolution structural insights into β2-adrenergic receptor function.Science. 2007; 318: 1266-1273Crossref PubMed Scopus (1189) Google Scholar). We added a codon-optimized T4 lysozyme at the C terminus of AaTatC. This fusion was well behaved by gel filtration and crystallized in DDM. Refinement of these conditions resulted in a 6.8 Å data set in the space group I4122, with cell dimension a = b = 142.015 Å c = 251.748 Å, referred to here as AaDDM. Although many approaches were attempted, we were unable to obtain phases for either crystal form by isomorphous replacement or related methods; therefore, the recently deposited TatC structure from Berks, Lea, and coworkers from Aquifex aeolicus was used as a molecular replacement search model (Protein Data Bank [PDB] ID 4B4A, referred to here as AaMNG as the protein was crystallized in a maltose-neopentyl glycol detergent). For the AaDHPC crystals, a single molecule was found in the asymmetric unit, while two were identified in the AaDDM asymmetric unit (Figures S1C and S1D available online). For the latter, we were unable to identify a lysozyme in the resulting maps. For the AaDHPC crystals, refinement proceeded through normal processes. In the final model, residues 5 to 232 had continuous density except for residues 133 to 140 in the second periplasmic loop, which were disordered and given occupancies of zero. The final AaDHPC model gave an R/R-free of 28.8/32.3%, while the lower resolution AaDDM gave a final R/R-free of 34.4/42.9%. Complete crystallographic statistics are found in Table 1. Unless noted, the figures and description of AaTatC will use the higher resolution AaDHPC crystal structure and not the lysozyme fusion.Table 1Crystallographic StatisticsAaDHPCAaDDMWavelength (Å)1.081.08Resolution range (Å)29.9–4.0 (4.5–4.0)aValues in parentheses are for the highest resolution shell.30.0–6.80 (7.0–6.8)Space groupP4122I4122Cell dimensionsa = b, c (Å)109.8, 107.0142.0, 251.8α, β, γ (°)90, 90, 9090, 90, 90Unique reflections5,906 (1,627)2,281 (220)Completeness (%)99.8 (100.0)95.9 (96.9)Redundancy5.8 (6.0)6.8 (7)RmergebRmerge = Σhkl Σi |Ii(hkl) − [I(hkl)]|/Σhkl ΣiIi(hkl), where Ii(hkl) is the ith observation of reflection hkl and 〈I(hkl)〉 is the weighted average intensity for all observations i of reflection hkl.0.127 (0.918)0.048 (0.79)Mean I/σ(I)6.6 (1.7)22.45 (2.70)RefinementReflections: work/free5,885/5672,164/98Rwork/RfreecR = Σ(| Fobs − Fcalc |)/Σ| Fobs |, where Fobs and Fcalc are observed and calculated structure factors amplitudes, respectively.0.288/0.3230.344/0.429Number of protein atoms1,8193,608Protein B factors (Å2)247.76474.5RMSD bond lengths (Å)0.0030.004RMSD bond angles (°)0.8820.95Ramachandran outliers (%)0.44a Values in parentheses are for the highest resolution shell.b Rmerge = Σhkl Σi |Ii(hkl) − [I(hkl)]|/Σhkl ΣiIi(hkl), where Ii(hkl) is the ith observation of reflection hkl and 〈I(hkl)〉 is the weighted average intensity for all observations i of reflection hkl.c R = Σ(| Fobs − Fcalc |)/Σ| Fobs |, where Fobs and Fcalc are observed and calculated structure factors amplitudes, respectively. Open table in a new tab The majority of TatC homologs are expected to have six membrane-spanning helices (Behrendt et al., 2004Behrendt J. Standar K. Lindenstrauss U. Brüser T. Topological studies on the twin-arginine translocase component TatC.FEMS Microbiol. Lett. 2004; 234: 303-308Crossref PubMed Google Scholar; Ki et al., 2004Ki J.J. Kawarasaki Y. Gam J. Harvey B.R. Iverson B.L. Georgiou G. A periplasmic fluorescent reporter protein and its application in high-throughput membrane protein topology analysis.J. Mol. Biol. 2004; 341: 901-909Crossref PubMed Scopus (36) Google Scholar; Punginelli et al., 2007Punginelli C. Maldonado B. Grahl S. Jack R. Alami M. Schröder J. Berks B.C. Palmer T. Cysteine scanning mutagenesis and topological mapping of the Escherichia coli twin-arginine translocase TatC Component.J. Bacteriol. 2007; 189: 5482-5494Crossref PubMed Scopus (38) Google Scholar) consistent with the structure, where all the TMs are visible (Figure 1A). TatC spans the membrane with the longest dimension, ∼55 Å, essentially the same as the width of the bilayer, resulting in very little of the protein exposed outside the membrane. Viewed from the cytoplasm (Figure 1B), the longest dimensions result in a length and width of approximately 35 × 20 Å. For reference, residues will be noted using E. coli numbering signified by Ec followed by the equivalent A. aeolicus number in italics unless noted (see Figure 1H for reference). Starting with the N terminus in the cytoplasm, TM1 is roughly perpendicular to the membrane, ending in a sharp turn that continues as an amphipathic helix (H1A) curling under TM2 to form a large part of the periplasmic face of TatC (Figures 1A–1C). The rest of the first periplasmic loop (Per1) continues as a structured loop. This is followed by TM2, which starts angled relative to TM1 until a conserved proline (Ec85/78) generates a kink, and the remaining cytoplasmic half of the helix forms a parallel interface with TM1. TM3 is connected to TM2 by a short loop (Cyt1) and then forms a long, steeply angled helix that makes contacts across the back to TM2, TM4, and TM6. This is reminiscent of the TM of SecE in the SecY translocon, suggesting a role in stabilization of the overall complex (van den Berg et al., 2004van den Berg B. Clemons Jr., W.M. Collinson I. Modis Y. Hartmann E. Harrison S.C. Rapoport T.A. X-ray structure of a protein-conducting channel.Nature. 2004; 427: 36-44Crossref PubMed Scopus (984) Google Scholar). The following periplasmic loop (Per2) runs below TM5 and TM6 and is partially disordered in the structure. TM4 has similar features and is parallel to TM2, including a highly conserved proline kink (Ec172/167). In addition, a highly conserved glycine (Ec166/161) forms a tight interface with a reciprocal conserved glycine (Ec121/114) in TM3. This is connected via a short loop (Cyt2) to TM5 that begins with a steep angle out from the core of the protein and then sharply kinks, making contacts to TM4. TM5 ends with a highly conserved proline turn (Per3) that lies just within the hydrophobic core of the bilayer. TM6 has a fairly shallow angle on the backside of the protein, making contacts to TM3, TM4, and TM5 with the C terminus of the protein ending in the cytoplasm. Overall, the most noticeable feature of TatC is that the kinked helices are arranged in a manner where they are perpendicular to the membrane in the cytoplasmic leaflet and then kinked into an angle in the periplasmic leaflet. This results in a membrane-exposed concave surface reminiscent of a baseball glove (Figures 1A–1C). TMs 1, 2, 4, and 5 line the pocket with the base made by the angled parts of TM2 and TM4. H1A, TM3, and TM6 line the back of the glove, forming contacts to multiple helices. The bulk of this pocket lies deep within the lipid bilayer. For many membrane proteins, mapping the hydrophobic parts of the surface helps to estimate the orientation of the protein in the membrane. As expected, much of the surface buried in the membrane is hydrophobic (Figure S1B); however, there are a number of polar groups within the hydrophobic part of the membrane, including in the pocket. This is further illustrated when the electrostatic surface potential is mapped to the exposed surface. Here, we solved the Poisson-Boltzmann equation using the dielectric value of water (Figure 1D; Figure S1C). This highlights a number of charged patches that would be buried in the membrane, including a negative charge deep within the pocket. Another useful visualization is to look at the conservation of residues exposed on the surface. Pfam is a curated database that clusters proteins into family groups based on homology across all of the available genomes (Punta et al., 2012Punta M. Coggill P.C. Eberhardt R.Y. Mistry J. Tate J. Boursnell C. Pang N. Forslund K. Ceric G. Clements J. et al.The Pfam protein families database.Nucleic Acids Res. 2012; 40: D290-D301Crossref PubMed Scopus (2904) Google Scholar). For TatC, Pfam PF00902, 2560 TatC homologs have been identified across bacteria, archaea, and eukaryotes (chloroplast and a few mitochondria) with a generated seed group (144 homologs) representative of sequence diversity. Taking the seed sequences, along with the sequence for AaTatC, an alignment was performed using ClustalX (Larkin et al., 2007Larkin M.A. Blackshields G. Brown N.P. Chenna R. McGettigan P.A. McWilliam H. Valentin F. Wallace I.M. Wilm A. Lopez R. et al.Clustal W and Clustal X version 2.0.Bioinformatics. 2007; 23: 2947-2948Crossref PubMed Scopus (22586) Google Scholar) that was then mapped on to the surface of TatC (Figures 1E and 1F; Figure S1D). Despite many of the" @default.
• W2140156945 created "2016-06-24" @default.
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• W2140156945 date "2013-05-01" @default.
• W2140156945 modified "2023-10-12" @default.
• W2140156945 title "The Glove-like Structure of the Conserved Membrane Protein TatC Provides Insight into Signal Sequence Recognition in Twin-Arginine Translocation" @default.
• W2140156945 cites W1514781231 @default.
• W2140156945 cites W1563914471 @default.
• W2140156945 cites W1825286507 @default.
• W2140156945 cites W1839045771 @default.
• W2140156945 cites W1955719320 @default.
• W2140156945 cites W1965314886 @default.
• W2140156945 cites W1968612029 @default.
• W2140156945 cites W1974202616 @default.
• W2140156945 cites W1981002074 @default.
• W2140156945 cites W1981397867 @default.
• W2140156945 cites W1985205346 @default.
• W2140156945 cites W1988357511 @default.
• W2140156945 cites W2005632350 @default.
• W2140156945 cites W2006990204 @default.
• W2140156945 cites W2008293899 @default.
• W2140156945 cites W2013524732 @default.
• W2140156945 cites W2013872797 @default.
• W2140156945 cites W2021878227 @default.
• W2140156945 cites W2022086113 @default.
• W2140156945 cites W2029851951 @default.
• W2140156945 cites W2030221133 @default.
• W2140156945 cites W2042620330 @default.
• W2140156945 cites W2045781230 @default.
• W2140156945 cites W2045985595 @default.
• W2140156945 cites W2047624429 @default.
• W2140156945 cites W2049704465 @default.
• W2140156945 cites W2052465711 @default.
• W2140156945 cites W2052470499 @default.
• W2140156945 cites W2053614437 @default.
• W2140156945 cites W2053673239 @default.
• W2140156945 cites W2054525948 @default.
• W2140156945 cites W2055154757 @default.
• W2140156945 cites W2056744168 @default.
• W2140156945 cites W2061385538 @default.
• W2140156945 cites W2061775535 @default.
• W2140156945 cites W2071651020 @default.
• W2140156945 cites W2073630173 @default.
• W2140156945 cites W2073726493 @default.
• W2140156945 cites W2074439439 @default.
• W2140156945 cites W2076876639 @default.
• W2140156945 cites W2080231167 @default.
• W2140156945 cites W2083793715 @default.
• W2140156945 cites W2085779944 @default.
• W2140156945 cites W2089016671 @default.
• W2140156945 cites W2097388832 @default.
• W2140156945 cites W2097634886 @default.
• W2140156945 cites W2105568907 @default.
• W2140156945 cites W2105986552 @default.
• W2140156945 cites W2109802625 @default.
• W2140156945 cites W2110949863 @default.
• W2140156945 cites W2116666954 @default.
• W2140156945 cites W2117847784 @default.
• W2140156945 cites W2118785817 @default.
• W2140156945 cites W2119168667 @default.
• W2140156945 cites W2120875291 @default.
• W2140156945 cites W2121828948 @default.
• W2140156945 cites W2124444587 @default.
• W2140156945 cites W2124701809 @default.
• W2140156945 cites W2125090992 @default.
• W2140156945 cites W2130394977 @default.
• W2140156945 cites W2136767810 @default.
• W2140156945 cites W2137015675 @default.
• W2140156945 cites W2139198526 @default.
• W2140156945 cites W2140891022 @default.
• W2140156945 cites W2141560226 @default.
• W2140156945 cites W2143599839 @default.
• W2140156945 cites W2145066558 @default.
• W2140156945 cites W2148054445 @default.
• W2140156945 cites W2154215718 @default.
• W2140156945 cites W2159071226 @default.
• W2140156945 cites W2161823733 @default.
• W2140156945 cites W2289232069 @default.
• W2140156945 cites W4247548089 @default.
• W2140156945 cites W4320301318 @default.
• W2140156945 cites W70253726 @default.
• W2140156945 doi "https://doi.org/10.1016/j.str.2013.03.004" @default.
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