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

The Syd protein has been implicated in the Sec-dependent transport of polypeptides across the bacterial inner membrane. Using Nanodiscs, we here provide direct evidence that Syd binds the SecY complex, and we demonstrate that interaction involves the two electropositive and cytosolic loops of the SecY subunit. We solve the crystal structure of Syd and together with cysteine cross-link analysis, we show that a conserved concave and electronegative groove constitutes the SecY-binding site. At the membrane, Syd decreases the activity of the translocon containing loosely associated SecY-SecE subunits, whereas in detergent solution Syd disrupts the SecYEG heterotrimeric associations. These results support the role of Syd in proofreading the SecY complex biogenesis and point to the electrostatic nature of the Sec channel interaction with its cytosolic partners. The Syd protein has been implicated in the Sec-dependent transport of polypeptides across the bacterial inner membrane. Using Nanodiscs, we here provide direct evidence that Syd binds the SecY complex, and we demonstrate that interaction involves the two electropositive and cytosolic loops of the SecY subunit. We solve the crystal structure of Syd and together with cysteine cross-link analysis, we show that a conserved concave and electronegative groove constitutes the SecY-binding site. At the membrane, Syd decreases the activity of the translocon containing loosely associated SecY-SecE subunits, whereas in detergent solution Syd disrupts the SecYEG heterotrimeric associations. These results support the role of Syd in proofreading the SecY complex biogenesis and point to the electrostatic nature of the Sec channel interaction with its cytosolic partners. The membrane-embedded SecYEG heterotrimer, also called core translocon or SecY complex, is a conserved protein-conducting channel essential for the biogenesis of most of the secretory and integral membrane proteins (1Rapoport T.A. Nature. 2007; 450: 663-669Crossref PubMed Scopus (704) Google Scholar). The SecY complex is a passive conduit and thus cooperates with cytosolic and membranous partners to drive efficient polypeptide transport into and across membrane. During co-translational transport, the SecY complex associates with the ribosome and elements of the signal recognition particle (2Menetret J.F. Hegde R.S. Aguiar M. Gygi S.P. Park E. Rapoport T.A. Akey C.W. Structure (Lond.). 2008; 16: 1126-1137Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 3Menetret J.F. Schaletzky J. Clemons Jr., W.M. Osborne A.R. Skanland S.S. Denison C. Gygi S.P. Kirkpatrick D.S. Park E. Ludtke S.J. Rapoport T.A. Akey C.W. Mol. Cell. 2007; 28: 1083-1092Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). During post-translational translocation in bacteria, the SecY complex interacts with the SecA ATPase to push secretory proteins across the channel (4Schiebel E. Driessen A.J. Hartl F.U. Wickner W. Cell. 1991; 64: 927-939Abstract Full Text PDF PubMed Scopus (374) Google Scholar). In the membrane, the SecY complex interacts with itself to form oligomers and with other membrane-embedded components such as the SecFDyajC complex and the insertase YidC (5Duong F. Wickner W. EMBO J. 1997; 16: 2756-2768Crossref PubMed Scopus (231) Google Scholar, 6Samuelson J.C. Chen M. Jiang F. Moller I. Wiedmann M. Kuhn A. Phillips G.J. Dalbey R.E. Nature. 2000; 406: 637-641Crossref PubMed Scopus (428) Google Scholar, 7Bessonneau P. Besson V. Collinson I. Duong F. EMBO J. 2002; 21: 995-1003Crossref PubMed Scopus (121) Google Scholar). The core translocon has been reconstituted into a functional in vitro system, and the structure of the SecY channel, SecA, and the ribosome has been determined at the atomic level (8Cate J.H. Yusupov M.M. Yusupova G.Z. Earnest T.N. Noller H.F. Science. 1999; 285: 2095-2104Crossref PubMed Scopus (524) Google Scholar, 9Clemons Jr., W.M. May J.L. Wimberly B.T. McCutcheon J.P. Capel M.S. Ramakrishnan V. Nature. 1999; 400: 833-840Crossref PubMed Scopus (313) Google Scholar, 10Van den Berg B. Clemons Jr., W.M. Collinson I. Modis Y. Hartmann E. Harrison S.C. Rapoport T.A. Nature. 2004; 427: 36-44Crossref PubMed Scopus (995) Google Scholar, 11Papanikolau Y. Papadovasilaki M. Ravelli R.B. McCarthy A.A. Cusack S. Economou A. Petratos K. J. Mol. Biol. 2007; 366: 1545-1557Crossref PubMed Scopus (118) Google Scholar). The interactions between these components, along with the stoichiometry and dynamics of their associations, are now crucial for the complete understanding of the translocation reaction. Much remains to be learned about the other components also involved in the biogenesis, regulation, and modulation of the translocon activity. Syd is a nonessential and hydrophilic protein of 181 amino acid residues. Ito and co-workers (12Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 13Matsuo E. Ito K. Mol. Gen. Genet. 1998; 258: 240-249Crossref PubMed Scopus (9) Google Scholar) originally identified its gene as a multicopy suppressor of the dominant-negative secY-d1 mutation (and thus termed Syd for suppressor of SecYdominance). Biochemical analysis then provided the first but indirect evidence for an interaction between Syd and the translocon (14Matsuo E. Mori H. Shimoike T. Ito K. J. Biol. Chem. 1998; 273: 18835-18840Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). For example, Syd was shown to interfere with protein translocation, but only in cells in which SecY and SecE proteins interact weakly. The physiological role of Syd remains uncertain (the deletion of the gene causes no obvious phenotype, see Ref. 12Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), but it is anticipated that Syd should provide new clues about the nature of SecY interactions with its cytosolic partners. We here report the crystal structure of Syd and provide direct evidence for its interaction with the SecY complex. We also chart the association and investigate the consequence of Syd binding on both activity and stability of the SecY channel. This comprehensive analysis provides a new framework for the understanding of the SecY channel interactome. Plasmids and Biological Reagents-The Syd open reading frame was PCR-amplified from the Escherichia coli genome and cloned into the expression vector pET23a (Clontech) using the restriction sites NdeI and XhoI. The plasmid pBAD22-hisEYG and purification of the SecY complex were previously described (15Collinson I. Breyton C. Duong F. Tziatzios C. Schubert D. Or E. Rapoport T. Kuhlbrandt W. EMBO J. 2001; 20: 2462-2471Crossref PubMed Scopus (111) Google Scholar). The deletion Δ251–258 and Δ354–357 into SecY and Δ7–67 into SecE was obtained by PCR amplification using primers introducing a BglII restriction site on each side of the deletion. Purification of Syd-Plasmid pET23-Syd was transformed in E. coli strain BL21 (DE3). Overproduction of Syd was initiated at A600 nm ∼ 0.5 with 1 mm isopropyl 1-thio-β-d-galactopyranoside for 3 h. Cells were collected in TSG buffer (25 mm Tris-Cl, pH 7.5; 50 mm NaCl; 10% glycerol; no DTT 4The abbreviations used are: DTT, dithiothreitol; DDM, dodecyl maltoside; IMVs, inner membrane vesicles; TMS, transmembrane segment. ) and lysed with a French press (8,000 p.s.i., three passes). After centrifugation (100,000 × g, 1 h at 4 °C), the supernatant was applied onto a Ni2+-chelated Sepharose column (GE Healthcare) equilibrated in TSG buffer. Syd was eluted with 500 mm imidazole and applied onto a 5-ml Q-Sepharose Fast Flow column (GE Healthcare) equilibrated in TS buffer (50 mm Tris-Cl, pH 7.5; 50 mm NaCl; 1 mm DTT). Syd was eluted with 250 mm NaCl and concentrated to 40 mg/ml using an Amicon 5-kDa centrifugation device. For selenomethionine labeling, cells were grown in 9 liters of M9 media. At A600 ∼ 0.3, amino acids were added (l-lysine, l-phenylalanine, l-threonine, l-isoleucine, l-leucine, l-valine, and l-selenomethionine; each 50 mg/liter), and Syd expression was induced with 1.5 mm isopropyl 1-thio-β-d-galactopyranoside during 16 h at 30 °C. 125I labeling was performed using IODO-GEN-coated tubes (Pierce) containing 60 μg of Syd and 25 μCi of Na125I. The reaction was quenched with 5 mm DTT, and the protein was desalted through a G-25 spin column equilibrated in TSG buffer. 125I-Syd (∼2 × 105 cpm/μg) was stored at -80 °C and used within a month. Crystallization and Structure Determination-Native E. coli Syd was crystallized using the hanging drop vapor diffusion method in 0.8–1.0 m sodium citrate, 0.2 m sodium chloride, and 0.1 m Tris, pH 7.0. The starting protein concentration was 20 mg/ml. Crystals reached dimensions of up to 0.45 × 0.40 × 0.25 mm. Selenomethionine E. coli Syd crystallized under the same conditions, although the resultant crystals were smaller (up to 0.2 × 0.18 × 0.1 mm). All crystals were cryo-cooled in the presence of 30% sodium malonate, pH 7.0, prior to x-ray data collection at the Stanford Synchrotron Radiation Laboratory (Stanford, CA). Details of data collection, processing, and structural refinement statistics are given in the supplemental material. Each dataset was integrated and scaled using the programs MOSFLM and SCALA, respectively (16Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D. Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). The structure of Syd was determined by the single wavelength anomalous diffraction method. Two selenomethionine sites, one each from the two Syd molecules in the asymmetric unit, were located and refined using the program SOLVE, and phases were subsequently improved by density modification using the program RESOLVE (17Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D. Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). The program ARP/WARP was used to build the initial model (18Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2565) Google Scholar). The primary sequence used in structure refinement was that from GenBank™ (accession number ABE08615). The program REFMAC5 (16Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D. Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) was used to refine the initial model using the native Syd dataset. This involved iterative cycles of fitting and rebuilding using the COOT program (19Emsley P. Cowtan K. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar). Further structural refinement was conducted using the native Syd dataset and CNS (20Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). Note that the 6-histidine tag attached to each Syd molecule in the asymmetric unit was not observed in electron density maps and therefore was not included in the structural model. Notably, the structure determination clearly indicates the presence of a disulfide bridge between cysteines 147 and 154 in Syd. These two cysteines are ideally positioned with respect to one another within the polypeptide chain fold to form this linkage. However, for one of the molecules of Syd in the asymmetric unit, there is some evidence from electron density maps that this disulfide bridge may be broken in ∼20% of the crystallized molecules. The other two cysteines present in Syd are too far removed from one another to form a disulfide bridge. Structures were visualized using PyMol (version 0.99), and electrostatic maps were obtained using the APBS plug-in (version 1.0.0). Atomic charges and radii were generated with the AMBER option at the Protein Data Bank code 2PQR on-line service. Analytical Gel Filtration-Analytical gel filtration was performed using a Superdex 200 HR 10/30 column (Amersham Biosciences) connected in-line to miniDAWN multiangle light scattering equipment coupled to an interferometric refractometer (Wyatt Technologies). Data analysis was recorded in real time using the ASTRA software (Wyatt Technologies). Molecular masses were calculated using the Debye fit method. Other Methods-The preparation of reagents such as Nd-SecYEG, SecA, 125I-proOmpA, and urea-stripped IMVs, as well as the conditions for in vitro translocation assays and native/blue-native gel electrophoresis were described previously (7Bessonneau P. Besson V. Collinson I. Duong F. EMBO J. 2002; 21: 995-1003Crossref PubMed Scopus (121) Google Scholar, 21Alami M. Dalal K. Lelj-Garolla B. Sligar S.G. Duong F. EMBO J. 2007; 26: 1995-2004Crossref PubMed Scopus (125) Google Scholar). Detection of Na125I-labeled proteins and densitometry scanning were performed using a phosphorimager scanner. A Complex of SecYEG and Syd-Nanodiscs permit investigation of the biochemistry of the SecY complex, without using liposome or detergent (21Alami M. Dalal K. Lelj-Garolla B. Sligar S.G. Duong F. EMBO J. 2007; 26: 1995-2004Crossref PubMed Scopus (125) Google Scholar, 22Denisov I.G. Grinkova Y.V. Lazarides A.A. Sligar S.G. J. Am. Chem. Soc. 2004; 126: 3477-3487Crossref PubMed Scopus (807) Google Scholar). Each particle, also referred to as Nd-SecYEG or SecYEG-Nanodisc, is made of a single SecY complex embedded in a small patch of lipid bilayer supported by two membrane scaffold proteins. Although homogeneous and water-soluble, the Nd-SecYEG particles (∼125 kDa) appear smeared on native-PAGE (Fig. 1A). with the Syd the of the particles is as a of complex and modification of the its the 125I-Syd protein at the of the gel but to the with the Nd-SecYEG particles (Fig. the SecY small deletion in one or the other of its cytosolic and the binding of Syd (Fig. In the SecY crystal these loops from the of the membrane and form binding sites for SecA or the ribosome J.F. Schaletzky J. Clemons Jr., W.M. Osborne A.R. Skanland S.S. Denison C. Gygi S.P. Kirkpatrick D.S. Park E. Ludtke S.J. Rapoport T.A. Akey C.W. Mol. Cell. 2007; 28: 1083-1092Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 10Van den Berg B. Clemons Jr., W.M. Collinson I. Modis Y. Hartmann E. Harrison S.C. Rapoport T.A. Nature. 2004; 427: 36-44Crossref PubMed Scopus (995) Google Scholar, A.R. Rapoport T.A. Cell. 2007; Full Text Full Text PDF PubMed Scopus (125) Google Scholar, S. V. E. Economou A. Mol. 2008; PubMed Scopus Google Scholar). The stability of the complex was by The 125I-Syd which at the of the to a density in the presence of Nd-SecYEG (Fig. The is only the SecY the deletion in or loops (Fig. the of in the buffer the stability of the complex (Fig. that electrostatic the The of Syd and the stoichiometry between Syd and Nd-SecYEG was by gel In the presence of an of most of the Nd-SecYEG is as by the and the of the during gel filtration (Fig. light scattering that the complex is with a of their the Nd-SecYEG particles and the Syd protein are also homogeneous but with a of and respectively (Fig. the protein Syd is in solution and a stoichiometry with the SecYEG complex. Atomic Structure of was crystallized and its structure by single wavelength anomalous to (Fig. are two molecules in the asymmetric unit, and their refined for the 6-histidine tag used to the The Syd structure is and of a and two A of Syd that of this structure a concave on the protein show that the the concave of this groove are conserved The of the observed groove is made of two located to the and of the of indicates that Syd has a patch on the concave groove and its (Fig. A electrostatic analysis of the SecY complex that the two SecY loops involved in the binding of Syd are of conserved in (Fig. These that the groove in Syd the binding site for the SecY electropositive between SecY and Syd and of cysteine were at on the concave of Syd (Fig. and for with for the SecY complex the mutation The is located at the of and it is involved in the binding of SecA A.R. Rapoport T.A. Cell. 2007; Full Text Full Text PDF PubMed Scopus (125) Google Scholar). of the Syd in solution not with the (Fig. In a cysteine cross-link was was with the (Fig. This cross-link is and efficient it without the of and it most of the SecY proteins in the (Fig. were observed the cysteine was located at other along the groove and or interactions with the that Syd not interfere with the binding of SecA onto membrane-embedded SecYEG (14Matsuo E. Mori H. Shimoike T. Ito K. J. Biol. Chem. 1998; 273: 18835-18840Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). We here that SecA Syd from the SecY complex. the cysteine cross-link between and is the are with an of SecA and Syd (Fig. the SecYEG complex in is with an of Syd and SecA, most of the complex of Syd only (Fig. Syd and SecA to for the SecY complex it is embedded in the membrane as an or integrated in as a Further of the but the results that the binding of Syd and SecA is The is with the of the interaction along with the of SecA and Syd. of Syd on and of the SecY of genome data that Syd only in and in SecE is of three transmembrane In SecE a single in about 30% of two are located at the of the In E. the two are not essential for or translocation activity but for the stability of the SecY complex J. EMBO J. 1991; PubMed Scopus Google Scholar, K. H. H. 2000; 64: PubMed Scopus Google Scholar) (Fig. The SecE is for the biogenesis of and SecY is by the A. Akiyama Y. Ito K. S. A. 1995; PubMed Scopus Google Scholar). The between Syd and SecE to the of Syd the two of SecE are from the SecY complex in vitro protein translocation into is not by Syd (14Matsuo E. Mori H. Shimoike T. Ito K. J. Biol. Chem. 1998; 273: 18835-18840Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) (Fig. In the concentration of Syd to of translocation for the SecY complex the deletion (Fig. A of translocation is also observed the SecY complex the mutation (14Matsuo E. Mori H. Shimoike T. Ito K. J. Biol. Chem. 1998; 273: 18835-18840Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The mutation is located at the between SecY and and it the stability of the complex den Berg B. Clemons Jr., W.M. Collinson I. Modis Y. Hartmann E. Harrison S.C. Rapoport T.A. Nature. 2004; 427: 36-44Crossref PubMed Scopus (995) Google Scholar, T. Akiyama Y. Ito K. Mol. Gen. Genet. 1994; PubMed Scopus Google Scholar). the a between the activity of Syd and the of the SecYEG heterotrimeric associations. Syd to SecYEG In the membrane, the two of SecE the SecY complex and may also the binding of Syd. We thus the of Syd on the stability of the SecY complex in The SecY complex in as a during the with on the concentration of detergent (7Bessonneau P. Besson V. Collinson I. Duong F. EMBO J. 2002; 21: 995-1003Crossref PubMed Scopus (121) Google Scholar). However, in the presence of the is and most of the SecYEG are in (Fig. In other such as the SecY is and in single S. F. D. Duong F. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). In the presence of the of the SecY complex is (Fig. the SecYEG are in detergent and Syd to their These are with a model in which Syd a translocation channel in which the SecY-SecE interactions are or (14Matsuo E. Mori H. Shimoike T. Ito K. J. Biol. Chem. 1998; 273: 18835-18840Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Although the observed are to the concentration of we to the presence of this protein in the by (Fig. interaction in detergent solution or the of this interaction by the during the gel the two the SecYEG complex was with Syd in detergent solution and by In the the SecYEG complex and Syd are together in a whereas of Syd is eluted in the (Fig. In the SecY and Syd are also but the proteins clearly appear in the (Fig. Syd and the SecY the to form a complex, as in detergent is a and hydrophilic with a conserved electronegative and concave The of the protein its interaction with the SecY channel in with SecA or the both of which and complex functional For three partners the nature of interactions with the SecY cytosolic loops may This is the with Syd. In the of the that the of the at the ribosome is with the in the SecY loops (2Menetret J.F. Hegde R.S. Aguiar M. Gygi S.P. Park E. Rapoport T.A. Akey C.W. Structure (Lond.). 2008; 16: 1126-1137Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 3Menetret J.F. Schaletzky J. Clemons Jr., W.M. Osborne A.R. Skanland S.S. Denison C. Gygi S.P. Kirkpatrick D.S. Park E. Ludtke S.J. Rapoport T.A. Akey C.W. Mol. Cell. 2007; 28: 1083-1092Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In the of SecA, the binding to be but one of the electronegative at the of the protein be for the The of Syd to the and SecYEG in detergent solution is is with the Syd activity SecY SecY-SecE 12Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, E. Mori H. Shimoike T. Ito K. J. Biol. Chem. 1998; 273: 18835-18840Abstract Full Text Full Text PDF PubMed Scopus (25) Google and this the results support a model in which Syd a for the of the SecY channel. In the membrane, the SecY complex be and binding of Syd. The SecY then be for by the A. Akiyama Y. Ito K. S. A. 1995; PubMed Scopus Google Scholar). The Syd protein an of to an level of to the biogenesis and the of the

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