Structure, Binding, and Activity of Syd, a SecY-interacting Protein

• DOI: 10.1074/jbc.m808305200
• Publication Date: 2009-01-13T01:45:03Z
• Authors: Kush Dalal; Nham Nguyen; Meriem Alami; Jennifer Tan; Trevor F. Moraes; Woo Cheol Lee; Robert Maurus; Stephen S. Sligar; Gary D. Brayer; Franck Duong
• Keywords: 0301 basic medicine; 0303 health sciences; Escherichia coli Proteins; Cell Membrane; Static Electricity; Membrane Proteins; Crystallography, X-Ray; Protein Structure, Secondary; Protein Subunits; Protein Transport; 03 medical and health sciences; Escherichia coli; Protein Structure, Quaternary; SEC Translocation Channels; Protein Binding

The Syd protein has been implicated in the Sec-dependent transport of polypeptides across bacterial inner membrane. Using Nanodiscs, we here provide direct evidence that binds SecY complex, and demonstrate interaction involves two electropositive cytosolic loops subunit. We solve crystal structure together with cysteine cross-link analysis, show a conserved concave electronegative groove constitutes SecY-binding site. At membrane, decreases activity translocon containing loosely associated SecY-SecE subunits, whereas detergent solution disrupts SecYEG heterotrimeric associations. These results support role proofreading complex biogenesis point to electrostatic nature Sec channel its partners. membrane-embedded heterotrimer, also called core or is protein-conducting essential for most secretory integral membrane proteins (1Rapoport T.A. Nature. 2007; 450: 663-669Crossref PubMed Scopus (704) Google Scholar). passive conduit thus cooperates membranous partners drive efficient polypeptide into During co-translational transport, associates ribosome elements signal recognition particle (2Menetret J.F. Hegde R.S. Aguiar M. Gygi S.P. Park E. Rapoport Akey C.W. Structure (Lond.). 2008; 16: 1126-1137Abstract Full Text PDF (89) Scholar, 3Menetret Schaletzky J. Clemons Jr., W.M. Osborne A.R. Skanland S.S. Denison C. Kirkpatrick D.S. Ludtke S.J. Mol. Cell. 28: 1083-1092Abstract (82) post-translational translocation bacteria, interacts SecA ATPase push (4Schiebel Driessen A.J. Hartl F.U. Wickner W. 1991; 64: 927-939Abstract (374) In itself form oligomers other components such as SecFDyajC insertase YidC (5Duong F. EMBO 1997; 2756-2768Crossref (231) 6Samuelson J.C. Chen Jiang Moller I. Wiedmann Kuhn A. Phillips G.J. Dalbey R.E. 2000; 406: 637-641Crossref (428) 7Bessonneau P. Besson V. Collinson Duong 2002; 21: 995-1003Crossref (121) reconstituted functional vitro system, channel, SecA, determined at atomic level (8Cate J.H. Yusupov M.M. Yusupova G.Z. Earnest T.N. Noller H.F. Science. 1999; 285: 2095-2104Crossref (524) 9Clemons May J.L. Wimberly B.T. McCutcheon J.P. Capel M.S. Ramakrishnan 400: 833-840Crossref (313) 10Van den Berg B. Modis Y. Hartmann Harrison S.C. 2004; 427: 36-44Crossref (995) 11Papanikolau Papadovasilaki Ravelli R.B. McCarthy A.A. Cusack S. Economou Petratos K. Biol. 366: 1545-1557Crossref (118) interactions between these components, along stoichiometry dynamics their associations, are now crucial complete understanding reaction. Much remains be learned about involved biogenesis, regulation, modulation activity. nonessential hydrophilic 181 amino acid residues. Ito co-workers (12Shimoike T. Taura Kihara Yoshihisa Akiyama Cannon Chem. 1995; 270: 5519-5526Abstract (54) 13Matsuo Gen. Genet. 1998; 258: 240-249Crossref (9) Scholar) originally identified gene multicopy suppressor dominant-negative secY-d1 mutation (and termed SecYdominance). Biochemical analysis then provided first but indirect an (14Matsuo Mori H. Shimoike 273: 18835-18840Abstract (25) For example, was shown interfere translocation, only cells which SecE interact weakly. physiological uncertain (the deletion causes no obvious phenotype, see Ref. 12Shimoike Scholar), it anticipated should new clues report complex. chart association investigate consequence binding on both stability channel. This comprehensive provides framework interactome. Plasmids Biological Reagents-The open reading frame PCR-amplified from Escherichia coli genome cloned expression vector pET23a (Clontech) using restriction sites NdeI XhoI. plasmid pBAD22-hisEYG purification were previously described (15Collinson Breyton Tziatzios Schubert D. Or Kuhlbrandt 2001; 20: 2462-2471Crossref (111) Δ251–258 Δ354–357 Δ7–67 obtained by PCR amplification primers introducing BglII site each side deletion. Purification Syd-Plasmid pET23-Syd transformed strain BL21 (DE3). Overproduction initiated A600 nm ∼ 0.5 1 mm isopropyl 1-thio-β-d-galactopyranoside 3 h. Cells collected TSG buffer (25 Tris-Cl, pH 7.5; 50 NaCl; 10% glycerol; DTT 4The abbreviations used are: DTT, dithiothreitol; DDM, dodecyl maltoside; IMVs, vesicles; TMS, transmembrane segment. ) lysed French press (8,000 p.s.i., three passes). After centrifugation (100,000 × g, h 4 °C), supernatant applied onto Ni2+-chelated Sepharose column (GE Healthcare) equilibrated buffer. eluted 500 imidazole 5-ml Q-Sepharose Fast Flow TS (50 DTT). 250 NaCl concentrated 40 mg/ml Amicon 5-kDa device. selenomethionine labeling, grown 9 liters M9 media. 0.3, acids added (l-lysine, l-phenylalanine, l-threonine, l-isoleucine, l-leucine, l-valine, l-selenomethionine; mg/liter), induced 1.5 during 16 30 °C. 125I labeling performed IODO-GEN-coated tubes (Pierce) 60 μg 25 μCi Na125I. reaction quenched 5 desalted through G-25 spin 125I-Syd (∼2 105 cpm/μg) stored -80 °C within month. Crystallization Determination-Native crystallized hanging drop vapor diffusion method 0.8–1.0 m sodium citrate, 0.2 chloride, 0.1 Tris, 7.0. starting concentration 20 mg/ml. Crystals reached dimensions up 0.45 0.40 0.25 mm. Selenomethionine under same conditions, although resultant crystals smaller (up 0.18 mm). All cryo-cooled presence 30% malonate, 7.0, prior x-ray data collection Stanford Synchrotron Radiation Laboratory (Stanford, CA). Details collection, processing, structural refinement statistics given supplemental material. Each dataset integrated scaled programs MOSFLM SCALA, respectively (16Collaborative Computational Project, Number 4Acta Crystallogr. Sect. 1994; 50: 760-763Crossref (19797) single wavelength anomalous diffraction method. Two sites, one molecules asymmetric unit, located refined program SOLVE, phases subsequently improved density modification RESOLVE (17Terwilliger T.C. Berendzen Acta 55: 849-861Crossref (3220) ARP/WARP build initial model (18Perrakis Morris R. Lamzin V.S. Nat. Struct. 6: 458-463Crossref (2565) primary sequence GenBank™ (accession number ABE08615). REFMAC5 refine native dataset. iterative cycles fitting rebuilding COOT (19Emsley Cowtan 60: 2126-2132Crossref (23628) Further conducted CNS (20Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros Grosse-Kunstleve R.W. J.S. Kuszewski Nilges Pannu N.S. Read R.J. Rice L.M. Simonson Warren G.L. 54: 905-921Crossref (16979) Note 6-histidine tag attached molecule unit not observed electron maps therefore included model. Notably, determination clearly indicates disulfide bridge cysteines 147 154 Syd. ideally positioned respect another chain fold this linkage. However, there some may broken ∼20% molecules. present too far removed bridge. Structures visualized PyMol (version 0.99), APBS plug-in 1.0.0). Atomic charges radii generated AMBER option Protein Data Bank code 2PQR on-line service. Analytical Gel Filtration-Analytical gel filtration Superdex 200 HR 10/30 (Amersham Biosciences) connected in-line miniDAWN multiangle light scattering equipment coupled interferometric refractometer (Wyatt Technologies). recorded real time ASTRA software Molecular masses calculated Debye fit Other Methods-The preparation reagents Nd-SecYEG, 125I-proOmpA, urea-stripped well conditions assays native/blue-native electrophoresis (7Bessonneau 21Alami Dalal Lelj-Garolla Sligar S.G. 26: 1995-2004Crossref (125) Detection Na125I-labeled densitometry scanning phosphorimager scanner. A Complex Syd-Nanodiscs permit investigation biochemistry without liposome (21Alami 22Denisov I.G. Grinkova Y.V. Lazarides Am. Soc. 126: 3477-3487Crossref (807) particle, referred Nd-SecYEG SecYEG-Nanodisc, made embedded small patch lipid bilayer supported scaffold proteins. Although homogeneous water-soluble, particles (∼125 kDa) appear smeared native-PAGE (Fig. 1A). When incubated purified protein, however, mobility greatly enhanced, possibly result formation electrophoretic characteristics. On own, migrates bottom dramatically shifts expected high molecular weight position when 1B). subunit carries internal largest loops, C4 C5, respectively, appears reduced structure, extend prominently surface cytoplasmic (3Menetret 23Osborne 129: 97-110Abstract 24Karamanou Bariami Papanikou Kalodimos C.G. Microbiol. 70: 311-322Crossref (21) Nd-SecYEG-Syd further assessed ultracentrifugation. floats top sucrose gradient, sediments higher 1C). sedimentation partial either C5 Interestingly, absence salt increases SecYEG-Syd 1C, panel), suggesting forces mediate interaction. oligomeric state analytical filtration. excess Syd, complexed judged shift symmetry elution peak chromatography 2A). Multiangle analyses indicate monodisperse measured mass 152 kDa ±3%. ∼126 ∼23 kDa, 2B). Thus, monomeric forms stable 1:1 Syd-Syd solved dispersion 2.0 Å resolution 3A). There structures include all residues, except C-terminal purify protein. relatively compact globular, being primarily comprised six-stranded antiparallel β-sheet long α-helices. space-filling representation reveals part cavity (or groove) surface. Sequence alignments residues forming highly (supplemental Fig. S1). periphery protruding stalks N terminus (residues 30–34; 45–48) C 138–143) Examination electrostatics distinct negatively charged localized surrounding 3B). similar shows electropositive, because several basic those regions 3C). observations strongly suggest found loops. Interacting Surface Exclusion SecA- Single introduced different positions 4A) tested reactivity IMVs enriched carrying M255A. SecY-255C tip loop C4, (23Osborne None variants formed cysteine-linked dimer (data shown) nor cross-reacted wild-type 4B, top). contrast, strong SecY-Syd detected Syd-115C 4B). specific appeared addition oxidizing agent, engaged corresponding titration experiment bottom). Weaker cross-links residue (90, 97, 135, respectively; top), more distant transient SecY-255. Previous Scatchard does confirm can displace Indeed, almost completely abolished equimolar amount 4C). Nanodiscs resulting consists 4D). seem possess affinity whether oligomer monomer. discussion will require knowledge Kd values, already mutually exclusive. conclusion consistent overlap site, size consideration Effect Translocation Efficiency Stability Complex-Search banks exists Gram-negative bacteria solely where segments (TMS). general, contains TMS Gram- additional coli, cell viability important (25Schatz P.J. Bieker K.L. Ottemann K.M. Silhavy T.J. Beckwith 10: 1749-1757Crossref (109) 26Nishiyama Suzuki Tokuda Biosci. Biotechnol. Biochem. 2121-2127Crossref (8) 5A). indeed critical SecY, any unassembled rapidly eliminated Fts protease (27Kihara Proc. Natl. Acad. Sci. U. 92: 4532-4536Crossref (212) possible evolutive relationship prompted us test effect deleted (deletion SecEΔ7–67). As reported previously, appreciably affected 5B). required achieve half-inhibition ∼5 times lower mutant SecEΔ7–67 inhibition G240D SecY24) interface SecE, reduces SecYE (10Van 28Taura 243: 261-269Crossref (62) Together, strength seems preferentially recognize mis-assembled complexes. stabilize prevent analyzed solution. DDM Blue-Native PAGE analysis; ratio monomers depending equilibrium shifted, dimers dissociated 6A). detergents Triton X-100, labile easily dissociates subunits (29Maillard A.P. Lalani Silva Belin 282: 1281-1287Abstract (57) dissociation enhanced associations weakened micelles, contributes destabilization. acts against mal-formed, unstable, compromised effects proportional failed detect complexes revealed 6A), weaker disruption blue dye electrophoresis. To differentiate possibilities, exclusion chromatography. co-eluted seemingly stoichiometric ratio, later fractions 6B). X-100 together, SecE/SecG alone have capacity seen Concluding Remarks-Syd simplicity makes straightforward comparison ribosome, multiple domains. largely involve electrostatics. likely case cryo-electron microscopy studies acidic phosphate backbone rRNA helices exit tunnel establishing major contact points exact mode needs clarified, few patches would prime candidate dissociate dimeric intramolecular remarkable. It inhibitory certain presenting abnormal (Refs. 14Matsuo Scholar study). Together represents quality control system correct assembly mal-associated recognized upon targeted degradation FtsH possesses unprecedented action, contributing sophistication function translocon.