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Biochemical Characterization of Protein Complexes from the Helicobacter pylori Protein Interaction Map

2004; Elsevier BV; Volume: 3; Issue: 8 Linguagem: Inglês

10.1074/mcp.m400048-mcp200

1535-9484

Laurent Terradot, Nathan Durnell, Min Li, Ming Li, Jeramia Ory, Agnès Labigne, Pierre Legrain, Frédéric Colland, Gabriel Waksman,

Galectins and Cancer Biology

We have investigated a large set of interactions from the Helicobacter pylori protein interaction map previously identified by high-throughput yeast two-hybrid (htY2H)-based methods. This study had two aims: i) to validate htY2H as a source of protein-protein interaction complexes for high-throughput biochemical and structural studies of the H. pylori interactome; and ii) to validate biochemically interactions shown by htY2H to involve components of the H. pylori type IV secretion systems. Thus, 17 interactions involving 31 proteins and protein fragments were studied, and a general strategy was designed to produce protein-interacting partners for biochemical and structural characterization. We show that htY2H is a valid source of protein-protein complexes for high-throughput proteome-scale characterization of the H. pylori interactome, because 76% of the interactions tested were confirmed biochemically. Of the interactions involving type IV secretion proteins, three could be confirmed. One interaction is between two components of the type IV secretion apparatus, ComB10 and ComB4, which are VirB10 and VirB4 homologs, respectively. Another interaction is between a type IV component (HP0525, a VirB11 homolog) and a non-type IV secretion protein (HP01451), indicating that proteins other than the core VirB (1-11)-VirD4 proteins may play a role in type IV secretion. Finally, a third interaction was biochemically confirmed between CagA, a virulence factor secreted by the type IV secretion system encoded by the Cag pathogenicity island, and a non-type IV secretion protein, HP0496. We have investigated a large set of interactions from the Helicobacter pylori protein interaction map previously identified by high-throughput yeast two-hybrid (htY2H)-based methods. This study had two aims: i) to validate htY2H as a source of protein-protein interaction complexes for high-throughput biochemical and structural studies of the H. pylori interactome; and ii) to validate biochemically interactions shown by htY2H to involve components of the H. pylori type IV secretion systems. Thus, 17 interactions involving 31 proteins and protein fragments were studied, and a general strategy was designed to produce protein-interacting partners for biochemical and structural characterization. We show that htY2H is a valid source of protein-protein complexes for high-throughput proteome-scale characterization of the H. pylori interactome, because 76% of the interactions tested were confirmed biochemically. Of the interactions involving type IV secretion proteins, three could be confirmed. One interaction is between two components of the type IV secretion apparatus, ComB10 and ComB4, which are VirB10 and VirB4 homologs, respectively. Another interaction is between a type IV component (HP0525, a VirB11 homolog) and a non-type IV secretion protein (HP01451), indicating that proteins other than the core VirB (1-11)-VirD4 proteins may play a role in type IV secretion. Finally, a third interaction was biochemically confirmed between CagA, a virulence factor secreted by the type IV secretion system encoded by the Cag pathogenicity island, and a non-type IV secretion protein, HP0496. Proteomics aims at studying proteins on the scale of an entire pathway or a whole cell. Proteomic analyses encompass the characterization of protein expression profile, the identification of post-translational modification, and the detection of protein-protein interactions (see for review Ref. 1Pandey A. Mann M. Proteomics to study genes and genomes..Nature. 2000; 405: 837-846Google Scholar). The study of protein-protein interactions has benefited from the development of large-scale high-throughput (ht) 1The abbreviations used are: ht, high-thoughput; Y2H, yeast two-hybrid system; MBP, maltose-binding protein; SID, selected interacting domain; FC, functional category. 1The abbreviations used are: ht, high-thoughput; Y2H, yeast two-hybrid system; MBP, maltose-binding protein; SID, selected interacting domain; FC, functional category. methods (2Ito T. Ota K. Kubota H. Yamaguchi Y. Chiba T. Sakuraba K. Yoshida M. Roles for the two-hybrid system in exploration of the yeast protein interactome..Mol. Cell. Proteomics. 2002; 1: 561-566Google Scholar) based on in vitro and in vivo systems, such as yeast two-hybrid systems (Y2H) (3Boulton S.J. Gartner A. Reboul J. Vaglio P. Dyson N. Hill D.E. Vidal M. Combined functional genomic maps of the C. elegans DNA damage response..Science. 2002; 295: 127-131Google Scholar, 4Fromont-Racine M. Rain J.C. Legrain P. Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens..Nat. Genet. 1997; 16: 277-282Google Scholar, 5Ito T. Chiba T. Ozawa R. Yoshida M. Hattori M. Sakaki Y. A comprehensive two-hybrid analysis to explore the yeast protein interactome..Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4569-4574Google Scholar, 6Uetz P. Giot L. Cagney G. Mansfield T.A. Judson R.S. Knight J.R. Lockshon D. Narayan V. Srinivasan M. Pochart P. Qureshi-Emili A. Li Y. Godwin B. Conover D. Kalbfleisch T. Vijayadamodar G. Yang M. Johnston M. Fields S. Rothberg J.M. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae..Nature. 2000; 403: 623-627Google Scholar), protein chips (7Zhu H. Bilgin M. Bangham R. Hall D. Casamayor A. Bertone P. Lan N. Jansen R. Bidlingmaier S. Houfek T. Mitchell T. Miller P. Dean R.A. Gerstein M. Snyder M. Global analysis of protein activities using proteome chips..Science. 2001; 293: 2101-2105Google Scholar), or systematic analysis of protein complexes by tandem affinity purification and mass spectrometry (8Ho Y. Gruhler A. Heilbut A. Bader G.D. Moore L. Adams S.L. Millar A. Taylor P. Bennett K. Boutilier K. Yang L. Wolting C. Donaldson I. Schandorff S. Shewnarane J. Vo M. Taggart J. Goudreault M. Muskat B. Alfarano C. Dewar D. Lin Z. Michalickova K. Willems A.R. Sassi H. Nielsen P.A. Rasmussen K.J. Andersen J.R. Johansen L.E. Hansen L.H. Jespersen H. Podtelejnikov A. Nielsen E. Crawford J. Poulsen V. Sorensen B.D. Matthiesen J. Hendrickson R.C. Gleeson F. Pawson T. Moran M.F. Durocher D. Mann M. Hogue C.W. Figeys D. Tyers M. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry..Nature. 2002; 415: 180-183Google Scholar, 9Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Functional organization of the yeast proteome by systematic analysis of protein complexes..Nature. 2002; 415: 141-147Google Scholar). Information about interactions, molecular complexes, and pathways can now be classified and examined via databases on the World Wide Web (10Bader G.D. Betel D. Hogue C.W. BIND: The Biomolecular Interaction Network Database..Nucleic Acids Res. 2003; 31: 248-250Google Scholar). Interpretation of these databases provides a framework on which a variety of target discovery strategies can be implemented (11Legrain P. Wojcik J. Gauthier J.M. Protein-protein interaction maps: A lead towards cellular functions..Trends Genet. 2001; 17: 346-352Google Scholar). One fundamental caveat of ht detection of protein-protein-interacting partners is that both Y2H and tandem affinity purification tagging/MS methods generate large numbers of false-positives or -negatives. Thus, methods need to be developed to assess the biochemical and/or biological significance of the interactions (12Sprinzak E. Sattath S. Margalit H. How Reliable are Experimental Protein-Protein Interaction Data?.J. Mol. Biol. 2003; 327: 919-923Google Scholar, 13Deane C.M. Salwinski L. Xenarios I. Eisenberg D. Protein interactions: Two methods for assessment of the reliability of high throughput observations..Mol. Cell. Proteomics. 2002; 1: 349-356Google Scholar). Depending on the methodological approach, some false-negatives might arise because of incorrect folding or inadequate subcellular localization of the proteins under investigation (14Schachter V. Protein-interaction networks: from experiments to analysis..Drug Discov. Today. 2002; 7: S48-S54Google Scholar). This is noteworthy as some genome-wide Y2H projects missed most (as much as 90%) of already characterized interactions (2Ito T. Ota K. Kubota H. Yamaguchi Y. Chiba T. Sakuraba K. Yoshida M. Roles for the two-hybrid system in exploration of the yeast protein interactome..Mol. Cell. Proteomics. 2002; 1: 561-566Google Scholar). False-positives can be generated because searching for many potential interactions increases the probability of selecting interacting polypeptides of no biological relevance (2Ito T. Ota K. Kubota H. Yamaguchi Y. Chiba T. Sakuraba K. Yoshida M. Roles for the two-hybrid system in exploration of the yeast protein interactome..Mol. Cell. Proteomics. 2002; 1: 561-566Google Scholar). As a result, htY2H does not guarantee that the inferred interactions are of physiological relevance and users of interaction network databases must further evaluate each interaction of interest. One means of alleviating at least partially this problem is to increase the sampling of the genome by using overlapping DNA fragments as preys. Such strategy leads to added redundancy of Y2H data, increasing data accuracy, and decreasing false-positives or -negatives significantly. It has the additional advantage of defining more finely the regions of the proteins involved in protein-protein interaction (selected interacting domains or SIDs) (3Boulton S.J. Gartner A. Reboul J. Vaglio P. Dyson N. Hill D.E. Vidal M. Combined functional genomic maps of the C. elegans DNA damage response..Science. 2002; 295: 127-131Google Scholar, 4Fromont-Racine M. Rain J.C. Legrain P. Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens..Nat. Genet. 1997; 16: 277-282Google Scholar, 11Legrain P. Wojcik J. Gauthier J.M. Protein-protein interaction maps: A lead towards cellular functions..Trends Genet. 2001; 17: 346-352Google Scholar). The first protein interaction map reported for a human pathogen was that of Helicobacter pylori (15Rain J.C. Selig L. De Reuse H. Battaglia V. Reverdy C. Simon S. Lenzen G. Petel F. Wojcik J. Schachter V. Chemama Y. Labigne A. Legrain P. The protein-protein interaction map of Helicobacter pylori..Nature. 2001; 409: 211-215Google Scholar). H. pylori is probably the most common chronic bacterial infection in humans, present in almost half of the population (16Cover T.L. Blaser M.J. Helicobacter pylori infection, a paradigm for chronic mucosal inflammation: Pathogenesis and implications for eradication and prevention..Adv. Intern. Med. 1996; 41: 85-117Google Scholar). It is a Gram-negative, spiral-shaped bacterium that colonizes the gastric mucosa of primates and is known to be responsible for several major diseases such as type B gastritis and peptic ulcer disease (17Blaser M.J. Helicobacter pylori: Its role in disease..Clin. Infect. Dis. 1992; 15: 386-391Google Scholar) and is a risk factor for gastric mucosa (18Nomura A. Stemmermann G.N. Chyou P.H. Kato I. Perez-Perez G.I. Blaser M.J. Helicobacter pylori infection and gastric carcinoma among Japanese Americans in Hawaii..N. Engl. J. Med. 1991; 325: 1132-1136Google Scholar, 19Parsonnet J. Friedman G.D. Vandersteen D.P. Chang Y. Vogelman J.H. Orentreich N. Sibley R.K. Helicobacter pylori infection and the risk of gastric carcinoma..N. Engl. J. Med. 1991; 325: 1127-1131Google Scholar) and mucosa-associated lymphoid tissue lymphoma (20Wotherspoon A.C. Doglioni C. Diss T.C. Pan L. Moschini A. de Boni M. Isaacson P.G. Regression of primary low-grade B-cell gastric lymphoma of mucosa-associated lymphoid tissue type after eradication of Helicobacter pylori..Lancet. 1993; 342: 575-577Google Scholar). H. pylori pathogenicity is at least in part associated with the secretion of the cytotoxin CagA, a protein which is thought to interfere with normal signaling once injected into gastric or duodenal epithelial cells. CagA secretion requires the assembly of a type IV secretion system encoded by genes clustered in the Cag pathogenicity island (21Censini S. Lange C. Xiang Z. Crabtree J.E. Ghiara P. Borodovsky M. Rappuoli R. Covacci A. cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors..Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14648-14653Google Scholar). Typically, a type IV secretion system is composed of at least 12 proteins termed VirB1-11 and VirD4. The core of the secretion machinery is formed by the VirB8, 9, and 10 proteins (22Krall L. Wiedemann U. Unsin G. Weiss S. Domke N. Baron C. Detergent extraction identifies different VirB protein subassemblies of the type IV secretion machinery in the membranes of Agrobacterium tumefaciens..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11405-11410Google Scholar). Two proteins are extracellular and associated with or are part of a pilus system: VirB2 is the pilus subunit itself, while VirB5 is a minor component of the pilus (23Lai E.-M. Kado C.I. Processed VirB2 is the major subunit of the promiscuous pilus of Agrobacterium tumefaciens..J. Bacteriol. 1998; 180: 2711-2717Google Scholar, 24Schmidt-Eisenlohr H. Domke N. Angerer C. Wanner G. Zambryski P.C. Baron C. Vir proteins stabilize VirB5 and mediate its association with the T pilus of Agrobacterium tumefaciens..J. Bacteriol. 1999; 181: 7485-7492Google Scholar). Finally, one ATPase and two ATP-binding proteins appear to power the entire machinery: VirB11, VirB4, and VirD4, respectively (25Dang T.A. Zhou X.R. Graf B. Christie P.J. Dimerization of the Agrobacterium tumefaciens VirB4 ATPase and the effect of ATP-binding cassette mutations on the assembly and function of the T-DNA transporter..Mol. Microbiol. 1999; 32: 1239-1253Google Scholar, 26Krause S. Barcena M. Pansegrau W. Lurz R. Carazo J.M. Lanka E. Sequence-related protein export NTPases encoded by the conjugative transfer region of RP4 and by the cag pathogenicity island of Helicobacter pylori share similar hexameric ring structures..Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3067-3072Google Scholar, 27Sagulenko E. Sagulenko V. Chen J. Christie P.J. Role of Agrobacterium VirB11 ATPase in T-pilus assembly and substrate selection..J. Bacteriol. 2001; 183: 5813-5825Google Scholar, 28Kumar R.B. Das A. Polar location and functional domains of the Agrobacterium tumefaciens DNA transfer protein VirD4..Mol. Microbiol. 2002; 43: 1523-1532Google Scholar). In H. pylori, homologs for most of these proteins are encoded by the Cag pathogenicity island and are termed HPXXXX (where XXXX denotes the ORF number as defined in Ref. 29). With the exception of proteins possessing significant homology to members of other type IV secretion systems, little is known about the molecular assembly process and the role of each of the Cag proteins in its assembly. Note that the Cag type IV secretion system is not the only type IV secretion system encoded by H. pylori. Two other gene clusters encoding such systems have been recently characterized: the ComB system, which serves as a DNA uptake machinery, and the Tfs3 system, which serves as conjugation system (30Hofreuter D. Odenbreit S. Haas R. Natural transformation competence in Helicobacter pylori is mediated by the basic components of a type IV secretion system..Mol. Microbiol. 2001; 41: 379-391Google Scholar, 31Kersulyte D. Velapatino B. Mukhopadhyay A.K. Cahuayme L. Bussalleu A. Combe J. Gilman R.H. Berg D.E. Cluster of type IV secretion genes in Helicobacter pylori’s plasticity zone..J. Bacteriol. 2003; 185: 3764-3772Google Scholar). The protein interaction map obtained for H. pylori displays a set of 1,524 interactions between 285 baits and an initial prey library of 2 × 106 independent fragments (15Rain J.C. Selig L. De Reuse H. Battaglia V. Reverdy C. Simon S. Lenzen G. Petel F. Wojcik J. Schachter V. Chemama Y. Labigne A. Legrain P. The protein-protein interaction map of Helicobacter pylori..Nature. 2001; 409: 211-215Google Scholar). Surprisingly, no interactions between Cag pathogenicity island-encoded proteins were detected, while several were found to connect them to members of other cell pathways or proteins with unknown functions. Such examples are the interaction between HP0525, a VirB11 component of the type IV secretion system, and HP1451, between HP0527, a VirB10 homolog, with HP0149, and CagA (HP0547) with HP0496. HP1451, HP0149, and HP0496 functions are unknown and are not known components of any type IV secretion systems in H. pylori. Such result suggests that the assembly and function of the Cag-encoded type IV system in H. pylori might depend on previously unidentified proteins. Interactions between proteins of the ComB system were also discovered, notably one that had not been described previously between ComB10 (or HP0042), a VirB10 homolog, and ComB4 (HP0017), a VirB4 homolog (30Hofreuter D. Odenbreit S. Haas R. Natural transformation competence in Helicobacter pylori is mediated by the basic components of a type IV secretion system..Mol. Microbiol. 2001; 41: 379-391Google Scholar). None of these interactions were confirmed by any other means. Given the abysmal record of htY2H methods in some of the published studies, the interactions recorded by Rain et al. (15Rain J.C. Selig L. De Reuse H. Battaglia V. Reverdy C. Simon S. Lenzen G. Petel F. Wojcik J. Schachter V. Chemama Y. Labigne A. Legrain P. The protein-protein interaction map of Helicobacter pylori..Nature. 2001; 409: 211-215Google Scholar) can only be trusted if other means of investigation are deployed to confirm them. In this report, we describe a protein complex study involving 31 full-length proteins or SIDs identified in H. pylori by htY2H to form interacting binary complexes. One goal of this research was to assess whether the htY2H-derived protein-protein interaction map of H. pylori is reliable enough to make the initiation of a proteome-wide biochemical and structural investigation of the interactome of this important human pathogen worthwhile. A second goal was to characterize biochemically complex formation involving type IV secretion proteins. Here we show that the H. pylori protein-protein interaction map is a valid starting point for the biochemical and structural characterization of protein-protein complexes. We also characterize for the first time interactions between HP0525 and HP1451, between HP547 (CagA) and HP0496, and between ComB10 and ComB4. A panel of 31 targets, i.e. 19 full-length proteins and 12 SIDs, were selected within the protein interaction map (Table I). Note that one protein (HP0522) is involved in interactions with two different proteins (HP1414 and HP0819) and that one interaction (between HP1231 and HP1247) was studied twice using either the full-length HP1231 or its corresponding SID. Also, one protein, HP1338, is self-interacting. Thus, a total of 31 targets engaged in 17 interactions were studied. These targets belong to different functional categories (FC) as defined in the pyloriGen database (genolist.pasteur.fr/pyloriGene/genome.cgi), including FC2 (purines, pyrimidines, nucleosides, and nucleotides), FC7 (transport and binding proteins), FC8 (DNA metabolism), FC9 (transcription), FC11 (protein fate), FC14 (cellular processes), and FC17 (hypothetical, conserved with no known function). Five of the targets under investigation are known to be involved directly in type IV secretion system biology (HP0525, HP0547, HP527, HP0017, HP0042). The size of the targeted proteins ranged from 3 to 69 kDa, and five were predicted to have at least one transmembrane domain using the HMMTOP server (32Tusnady G.E. Simon I. The HMMTOP transmembrane topology prediction server..Bioinformatics. 2001; 17: 849-850Google Scholar). Proteins are named according to the genome annotation (29Tomb J.F. White O. Kerlavage A.R. Clayton R.A. Sutton G.G. Fleischmann R.D. Ketchum K.A. Klenk H.P. Gill S. Dougherty B.A. Nelson K. Quackenbush J. Zhou L. Kirkness E.F. Peterson S. Loftus B. Richardson D. Dodson R. Khalak H.G. Glodek A. McKenney K. Fitzegerald L.M. Lee N. Adams M.D. Venter J.C. The complete genome sequence of the gastric pathogen Helicobacter pylori.Nature. 1997; 388: 539-547Google Scholar). The predicted biological score (PBS) defined as described in the pyloriGen database (see above) is reported in Table I for each putative interaction (15Rain J.C. Selig L. De Reuse H. Battaglia V. Reverdy C. Simon S. Lenzen G. Petel F. Wojcik J. Schachter V. Chemama Y. Labigne A. Legrain P. The protein-protein interaction map of Helicobacter pylori..Nature. 2001; 409: 211-215Google Scholar). In brief, the PBS reflects the reliability of the interaction and is defined uniquely on the basis of the htY2H results.Table IProtein targetsProteinLengthTMMrFCFunctionPBSProteinLengthTMMrFCFunctionHP1414113No12.817Unknown◂A▸HP0522481Yes54.914cag3, UnknownHP0819216No247ProV, import system◂A▸HP0522””””UnknownHP0525330No37.414ATPase, virB11◂A▸HP1451 (92–264)173No3017UnknownHP0149194Yes2217Unknown◂A▸HP0527 (1496–1870)375Yes40.514cag7, VirB10 homologueHP0261132No15.617Unknown◂A▸HP0537 (39–365)327No3814CagM, UnknownHP1489510Yes56.817Unknown◂B▸HP0540 (126–381)256No28.614cag19, UnknownHP0496133No15.417Unknown◂A▸HP0547 (392–733)342No38.514CagA, CytotoxinHP1032255No29.39RNA pol. Sigma factor, FliA◂A▸HP1122 (48–76)29No3.317Anti sigma factor FlgMHP1293344No38.39DNA-dependent RNA polymerase, rpoA◂A▸HP1198 (659–1272)614No68.59DNA-dependent RNA polymerase, rpoBHP1231218No25.28DNA polymerase III, holB◂A▸HP1247 (199–340)142No16.417UnknownHP1231 (136–218)83”9.7””◂A▸HP1247 (199–340)”””””HP0650196No22.117Unknown◂A▸HP1245179No19.98DNA helicase, SSBHP1230180No20.817Unknown◂A▸HP1529457No51.58Chromosome replication initiation, DnaAHP1338148No1717Unknown◂A▸HP1338148No1717UnknownHP0042233Yes25.614ComB3, Trbl, virB10 homologue◂A▸HP0017 (34–336)303No35.214VirB4 homologueHP0862223No24.517Unknown◂C▸HP1474 (100–191)92No10.32Thymidylate kinase, tmkHP0608160No17.817Unknown◂A▸HP0175 (43–299)257No29.611Peptidyl-prolyl cis-trans isomerase Open table in a new tab Unless otherwise stated, the SIDs were obtained by PCR amplification using specific primers that inserted the restriction sites BamHI (5′ end) and EcoRI (3′ end). For the SIDs of HP0547 and HP1451, the restriction sites BamHI-PstI and EcoRI-HindIII were used, respectively. Most coding sequences for the full-length proteins were obtained directly by digesting the Y2H bait clones using the BamHI and PstI restriction enzymes (15Rain J.C. Selig L. De Reuse H. Battaglia V. Reverdy C. Simon S. Lenzen G. Petel F. Wojcik J. Schachter V. Chemama Y. Labigne A. Legrain P. The protein-protein interaction map of Helicobacter pylori..Nature. 2001; 409: 211-215Google Scholar). Two full-length proteins, HP0522 and HP0862, were obtained by PCR amplification of genomic DNA using primers that added the restriction sites BamH1 and EcoRI. The digested DNA fragments were visualized on an agarose gel, and correctly sized bands were excised and purified with a gel extraction kit (Concert Life Technologies, Inc., Grand Island, NY). The 31 DNA fragments were each cloned in pPROEXHT (-b or -c) and pMAL (-BE or -G) vectors (see definition below), generating for each a His6- and maltose-binding protein (MBP)-tagged version of the proteins, respectively. The His6 and MBP tags add 6 and 44 kDa to the molecular mass of the proteins, respectively. To generate MBP fusions, two vectors were designed. The pMAL-c2x vector (New England Biolabs, Beverly, MA) that expresses the MBP as a N-terminal fusion domain was modified by insertion of a PreScission protease cleavage site between the XmnI and EcoRI sites (gift of Joe St. Gemes, Washington University, St Louis, MO). The EcoRI and BamHI sites were then inverted to create pMAL-BE. From pMAL-BE, one G was added in front of the BamHI site to create pMAL-G. All SID sequences were cloned between the appropriate restriction sites of pMAL-BE, while all full-length sequences were cloned in pMAL-G. To generate His6 fusions, pPROEXHTb (for the SIDs) and pPROEXHTc (for full-length proteins) were used. One exception to this is HP1451 (a SID), which was cloned in pPROEXHTa. All expression vectors were finally transformed into BL21(DE3)pLysS competent cells (Invitrogen, San Diego, CA). For co-expression studies, HP1451, HP0017, and HP0496 were subcloned into pAlter-Ex2 vector (Promega, Madison, WI) by using NcoI-PstI restriction sites. This vector contains a tetracycline-resistant gene as well as a different origin of replication (p15a), which makes it suitable for co-expressing proteins with pPROEXHT-cloned partners. Freshly transformed recombinants were inoculated into 5 ml of Luria-Bertani broth containing 100 μg/ml ampicillin and 40 μg/ml chloramphenicol, and grown overnight at 37 °C. This preculture was then used to inoculate 500 ml of fresh Luria-Bertani broth containing the same antibiotics. The cells were grown at 37 °C to an OD600 of 0.6 and induced with 1 mm isopropyl β-d-thiogalactoside for 3 h. Solubility of each protein or protein fragment was assessed by harvesting the cells, resuspending them in a lysis buffer (20 mm Tris-HCl, pH 7.6, 150 mm NaCl, 0.2% (v/v) Triton X-100, and 5% glycerol), lyzing the cells by sonication and pelleting cell debris by centrifugation, collecting the supernatant, and analyzing the supernatant using SDS-PAGE for the presence of the induced band visually after Coomassie blue staining for MBP fusions (all MBP fusions were found to be strongly induced and soluble, see “Results”) and using an anti-His6 antibody (see below) to detect His6-tagged fusions. Note that, in our hands, mixing cells of putative interactive partners prior to sonication did not result in detectable changes in solubility of individual proteins. Nor did overnight induction at 20 °C. Several protocols were then devised to form the complexes, depending on the solubility of the His6-tagged interacting partners (all MBP fusions were found to be soluble). Cells corresponding to two soluble interacting partners were mixed together and harvested. The pellet was resuspended into 40 ml of lysis buffer (20 mm Tris-HCl, pH 7.6, 150 mm NaCl, 0.2% (v/v) Triton X-100, and 5% glycerol) and frozen (−80 °C). A protease inhibitor mixture (1 mm PMSF, 1% aprotinin, and 1 mm β-mercaptoethanol) was added to the unfrozen cells prior to sonication. The cells were then disrupted by sonication, and cell debris were separated by centrifugation at 45,000 × g for 20 min. The resulting supernatant was loaded onto an amylose column (fast flow amylose resin, New England Biolabs, 10-ml column volume) equilibrated with buffer A (20 mm Tris-HCl, pH 7.6, 150 mm NaCl). MBP fusion proteins were eluted by applying a short linear gradient (3 column volumes) of buffer B (buffer A containing 10 mm of maltose). The MBP fusion proteins usually eluted around 10–15% of buffer B. The presence of the His6-tagged partner was then detected using SDS-PAGE followed by either Coomassie blue staining or using an anti-His6 antibody (see below). When His6-tagged proteins were found to be insoluble (i.e. in inclusion bodies after sonication and centrifugation), the pellet containing the inclusion bodies was washed with 10 ml of buffer R (8 m urea, 20 mm Tris, pH 7.6, 150 mm NaCl), and then centrifuged. The resulting pellet was then dissolved in 10 ml of buffer R for 3 h at 4 °C under constant stirring. The cell debris were discarded by centrifugation (40,000 × g, 15 min). The resulting supernatant was then diluted three times in buffer U (3 m guanidine, 20 mm Tris, pH 7.6, 150 mm NaCl). The resulting solution was then gently added (0.5 ml/min) to a solution of 300 ml of buffer F (0.5 m l-arginine, 20 mm Tris, pH 7.6, 150 mm NaCl) under stirring at 4 °C. The solution was then centrifuged (40,000 × g, 20 min) to remove any precipitated protein. The solution was finally loaded onto a TALON resin column (10-ml volume, Clontech, Palo Alto, CA) equilibrated with 150 mm NaCl and 20 mm Tris, pH 8. The protein was eluted using a short linear gradient (3 column volumes) of the same buffer containing 1 m imidazole. To assess complex formation, the eluted protein was added to the supernatant resulting from sonication and centrifugation of cells having expressed its binding partner as an MBP fusion. Complex formation was then monitored by elution from an amylose column. The His6-tagged insoluble proteins were solubilized using buffer R as previously described. The unfolded protein was then loaded onto a TALON column equilibrated with 20 mm Tris-HCl, pH 7.6, 200 mm NaCl, and 8 m urea. The protein was eluted with a short linear gradient (3 column volumes) of 1 m imidazole in the same buffer. The fractions were pooled (6–8 ml total) and added to the supernatant containing the soluble MBP fusion partner (final volume of 30 ml). The solution was dialysed overnight against 1 liter of buffer F. The solution was filtered, and complex formation was monitored by elution from the amylose column (as described previously). The same procedure described in the section “Protein Expression” was used for co-expression experiments except that tetracycline was added to the media at a concentration of 12 μg/ml. Complex formation was monitored by elution from an amylose column (as described previously). SDS-PAGE experiments were carried out in 12% polyacrylamide gels using Mini Protean III following the manufacturer’s protocol. Two gels were made per complex, one was stained with Simple Blue (Invitrogen) and the other was used for Western blot. Separated proteins were blotted onto PVDF membranes using liquid transfer