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1 Division of Structural Biology, German Research Centre for Biotechnology, D-38124, Braunschweig, Germany
2 Division of Molecular Microbiology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland
Reprint requests to: Dirk W. Heinz, Division of Structural Biology, German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124, Braunschweig, Germany; e-mail: dirk.heinz{at}gbf.de; fax: +49-531-6181-763.
(RECEIVED March 23, 2005; FINAL REVISION May 24, 2005; ACCEPTED May 24, 2005)
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Abstract |
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-sheet flanked by three 
-helices, SycT lacks the dimerization -helix and has an additional -strand capable of undergoing a conformational change. The dimer interface consists of two -strands and the connecting loops. Two hydrophobic patches involved in effector binding in other TTS effector chaperones are also found in SycT. The structural similarity of SycT to other chaperones and the spatial conservation of effector-binding sites support the idea that TTS effector chaperones form a single functional and structural group. Keywords: chaperone; effector; SycT; type III secretion; Yersinia; YopT
Abbreviations: #1 and #2, crystal form 1 (space group P212121) and crystal form 2 (P62) of SycT122, respectively • CBD, chaperone-binding domain • ORF, open reading frame • TTS, Type III secretion • Yop, Yersinia outer protein • SAD, single-wavelength anomalous dispersion
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051474605.
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Introduction |
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B signalling pathways, thereby impeding the release of proinflammatory cytokines and inducing apoptosis in macrophages. The substrate of the serine/threonine kinase YopO (YpkA in Y. pseudotuberculosis and Y. pestis) is still unknown, as well as the targets of YopM, which locates to the nucleus of the host cell. Effective translocation of some of the TTS effectors and components is regulated by a family of specialized chaperones, which form specific complexes with their cognate substrate in the bacterial cytoplasm. These TTS chaperones, in contrast to other chaperones such as GroEL or heat shock proteins, do not bind or hydrolyze nucleotides (Wattiau et al. 1996). Despite several common properties, such as a low molecular mass (Mr~15,000) and an acidic isoelectric point, they have low amino acid sequence similarity (Wattiau et al. 1994). Functions proposed for TTS chaperones include preventing effector agglomeration by binding the aggregation-prone N-terminal region of the effector; keeping the effector in a partially unfolded, secretion-competent state; and helping to regulate the TTS system (for reviews, see Francis et al. 2002; Feldman and Cornelis 2003; and Parsot et al. 2003). Based on their substrate, TTS chaperones can be grouped into three categories. The first class guides the transfer of one or more effector proteins, and several members have been characterized structurally. A second class promotes the transport of TTS components that form a pore in the host cell membrane (translocators). The third category comprises chaperones binding to other proteins of the injection apparatus. The first structure of a member of the third category of chaperones has recently been solved (Yip et al. 2005). Yop effectors are modular proteins. They bear an N-terminal secretion/translocation signal of ~20 amino acids (Sory et al. 1995) not cleaved after translocation. The chaperone-binding domain in chaperone-complexed effectors generally lies within residues 50–150 (Woestyn et al. 1996; Tampakaki et al. 2004). In Yersiniae three of the translocated effectors (YopE, YopH, and YopT) possess a specific chaperone (Wattiau and Cornelis 1993; Wattiau et al. 1994; Iriarte and Cornelis 1998), whereas no chaperones have as yet been identified for YopM, YopP, or YopO (Trülzsch et al. 2003).
The effector YopT (322 amino acids, 36 kDa) (Iriarte and Cornelis 1998) is a member of a new family of papain-like cysteine proteases with the conserved catalytic triad comprising Cys139, His258, and Asp274 (Shao et al. 2002; Zhu et al. 2004). Residues 75–318 are required for proteolytic activity (Sorg et al. 2003). After translocation into the host cell, YopT cleaves the C-terminal, prenylated cysteine of membrane-anchored Rho-GTPases, thereby releasing the GTPase into the host cytosol (Zumbihl et al. 1999; Shao et al. 2002; Äpfelbacher et al. 2003; Sorg et al. 2003). The lack of Rho-GTPase–induced actin polymerization signal leads to the disintegration of the host actin stress fibres (Iriarte and Cornelis 1998) and contributes to the yersinial resistance to phagocytosis by inhibiting the formation of the actin-rich phagocytotic cup (Äpfelbacher et al. 2003). The specific effector chaperone for YopT is SycT (Mr~15,000; pI 4.6) (Iriarte and Cornelis 1998).
In this work, we present a structural and functional characterization of the chaperone SycT from Y. enterocolitica, including complex formation studies with its effector YopT.
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Results and Discussion |
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Structure determination
Native full-length SycT crystallized in 5% isopropanol, 0.1 M sodium citrate (pH 5.6), and 21% polyethylene glycol 4000 at 4°C as rhombic plates (100 µm) in the monoclinic space group P21. Attempts to solve the structure of SycT by molecular replacement using the structures of other TTS chaperones failed. Soaking crystals with mercury, platinum, osmium, or bromide caused crystal cracking and loss of diffraction or crystals did not incorporate the heavy atom. Seleno-methionine (SeMet)–substituted SycT122 was crystallized in 1.6 M ammonium sulfate, 0.1 M CAPS (pH 10.5), and 0.15 M lithium sulfate (plate-like crystal form #1, space group P212121). An unrelated crystal form #2 (P62 ) was obtained from 1.8 M ammonium sulfate, 0.1 M sodium bicarbonate (pH 10.5), and 0.05 M magnesium chloride with hexagonal rod-like crystals growing to a size of 200 x 150 µm. The structure of SycT122 was determined in crystal form #2 by selenium single-wavelength anomalous dispersion (SAD) and refined to a resolution of 1.9 Å. SycT122 crystal form #1 and the structure of native SycT were then solved by molecular replacement to a resolution of 2.0 Å and 1.8 Å, respectively. In all three crystal forms, the asymmetric unit bears one homodimer (Table 1
; Fig. 1A). The three crystal structures are very similar; e.g., the C-atoms of SycT122 monomers have a root mean square deviation (RMSD) of 0.6 Å in #1 and 0.7 Å in #2. Differences are confined to the flexible N- and C-terminal parts with the exception of residues 20–28 of one of the native SycT monomers. Well-defined electron density is observed for residues 3–130 (A), 2–117, and 125–130 (B) of native SycT; residues 1–114 of both monomers in SycT122 crystal form #1; and residues 3–122 (A) and 1–113 (B) in #2.
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-helices packed against one side of a twisted six-stranded, anti-parallel -sheet (SycT122 #2) (Fig. 1A
). It thereby shares most characteristic features of the globular fold common to effector-binding TTS chaperones. Despite low sequence identity of 9%–21%, the RMSD for common C-positions (79–109 aligned residues) is <2.7 Å compared with other TTS chaperones such as SicP and SigE from Salmonella sp. (Luo et al. 2001; Stebbins and Galan 2001), SycE and SycH from Yersinia sp. (Birtalan and Ghosh 2001; Evdokimov et al. 2002; Trame and McKay 2003; Phan et al. 2004), CesT from enterohemorrhagic E. coli (Luo et al. 2001), and AvrPphF Orf1 from Pseudomonas syringae (Singer et al. 2004). SycT shares the highest sequence identity with the structurally uncharacterized Orf1 from Pseudomonas aeruginosa (abbreviated as SpcS) believed to be the chaperone of ExoS (Frithz-Lindsten et al. 1997). Apart from the overall similarity, SycT displays some unique and distinct features. The depression between the N-terminal -helices 1 and the adjoining loops L4 and L6, typical for other effector chaperones, is less pronounced in the SycT dimer (Fig. 1A). Instead, SycT exhibits a pronounced groove between loops L5 on the opposite dimer face. Overall, SycT is thus quite compact compared with the heart-shaped outline of other chaperones (Fig. 2; Fig. 4A, below).
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-helices packing against the -sheet. Of these, 2 is extensively involved in dimerization (Fig. 2) (Parsot et al. 2003). Strikingly, -helix 2 is consistently absent in all three crystal forms of SycT. Instead, dimerization involves hydrophobic and polar residues exclusively located in loops and -strands (Fig. 1B). The SycT dimerization interface involves some residues of the region between residues 43 and 90. In particular, residues from strands 4 and 5 are involved as are loops L4, L6, and the extended loop L5 (Fig. 1). The accessible surface area buried per monomer is 1100 Å2 or ~15% of the total surface area. This is similar to the buried surface of other TTS effector chaperones ranging from 900 to 1200 Å2.
A closed cavity at the dimer interface
The dimerization interface of SycT harbors a cavity of 150 Å3 (Fig. 3). Five (native SycT, SycT122 #2) or six (#1) water molecules trapped within the cavity form H-bonds to polar residues. Symmetrically positioned Ser88 residues from both monomers restrict entry to the cavity and are mutually H-bonded in the native structure and #1. Both Val90 residues in loop L6 shield the cavity from the outside (Fig. 3). A polar cavity located at the dimerization interface of TTS effector chaperones was first described for SycE (Evdokimov et al. 2002). Fully enclosed cavities, as in SycT, are also present in Spa15 and AvrPphF Orf1 dimers (Singer et al. 2004; Van Eerde et al. 2004). The dimerization interfaces of other TTS effector chaperones, e.g., SycE, YscB/SycN (Schubot et al. 2005), or SycH (Phan et al. 2004), reveal similar yet open, solvent-accessible cavities (Fig. 3, right). Compared with other solvated intersubunit cavities (Hubbard and Argos 1994) that of SycT is quite large, with ~100 Å2 or 9% of the dimerization interface per monomer not being involved in direct protein –protein interactions. Conservation of the cavity, open or closed, among TTS effector-binding chaperones is strongly suggestive of a biological role. In SycE, SycH, and SycN/YscB, the three chaperones from Y. pestis for which structural information is available, two arginines create a positively charged patch inside the cavity (Evdokimov et al. 2002; Phan et al. 2004; Schubot et al. 2005). These arginines are conserved among many of the effector chaperones and were therefore proposed to be functionally relevant, possibly for interaction with the type III secretion machinery (Phan et al. 2004; Schubot et al. 2005). The lack of charged residues in the cavity of SycT means that they are unlikely to be a general point of interaction between TTS effector chaperones and the secretion machinery.
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-helices. A third hydrophobic patch (patch 3) stretches between both N-terminal helices 1 (residues Met0, Leu8, Leu89, Val90, Gly91, Leu92, and Ile94) (Fig. 4A, left). Patches 1 and 2 are present at roughly the same position in other effector chaperones and were initially predicted to be involved in effector binding (Birtalan and Ghosh 2001). This hypothesis was later confirmed by the crystal structures of several effector/chaperone complexes such as YopE23-78/SycE, SptP36-139/SicP, and YopN23-273/SycN-YscB complexes (Stebbins and Galan 2001; Birtalan et al. 2002; Schubot et al. 2005). In these structures, the N-terminal region of the effector wraps around the chaperones, displaying only secondary but no tertiary structure. In the YopE/SycE complex, both patches 1 bind -helices, while both patches 2 bind -strands of the YopE chaperone-binding domain (Fig. 4A, right). The conserved position of patches 1 and 2 in SycT suggests a similar mode of effector binding. The details of YopT/SycT interaction will presumably differ from those of other effector–chaperone pairs. The third hydrophobic patch of SycT may indicate an enlarged contact area and the effector YopT may follow a different path wrapping around SycT.
Significant differences between SycT and the other effector- binding chaperones (except possibly Spa15) are observed in the vicinity of patch 2. In most chaperones studied to date, patch 2 consists of a pocket, formed by conserved hydrophobic residues from the -helices 1 and 3 and a shallow groove between -strand 1 and the loop connecting the N-terminal -helix 1 with 1. The effectors YopE, SptP, and YopN bind to patch 2 of their cognate chaperone, filling this shallow groove and extending the chaperone -sheet by an additional strand along side strand 1 (Fig. 4B, left). In five of the six crystallographically independent monomers of SycT, the residues connecting 1 and 1 form an additional -strand 0 anti-parallel to 1 that occupies the shallow groove (Fig. 4B, left). This strongly resembles the situation in Spa15 (Van Eerde et al. 2004) (Fig. 4B, right). In one monomer of the native SycT dimer, these connecting residues do not form 0. Instead they form a loop flipped toward 1, thereby opening the abovementioned groove. This conformational rearrangement of 0 may be required for binding of YopT, although no conformational changes are observed upon effector binding in SycE (Birtalan et al. 2002).
Interaction of the flexible C terminus with a hydrophobic patch
In full-length SycT, the hydrophobic peptide SPILFI (residues 125–130) of both monomers interacts with one hydrophobic patch 1 of the primary dimer and of a symmetry-related dimer at the same time, thereby mediating extensive crystal contacts (Fig. 4A, left). Hydrophobic patch 1 harbors a groove (Phe65 in loop L5 from one monomer; Val27 from 2; Leu39, Ala41, and Tyr43 from 3; Gln49 and Phe51 from 4 in the other monomer). In crystal form #2 of truncated SycT122, this hydrophobic groove of one monomer is occupied by Phe119 from the C-terminal peptide of a neighboring monomer in the crystal (Figs. 1, 5), sustaining an important crystal contact. These C-terminal residues (115–122) are disordered in monomer B (#2) and in both monomers of #1. Residues 115–122 thus seem to be intrinsically flexible. Depending on the crystal packing environment, these C-terminal stretches in SycT are ordered by fortuitously binding to patch 1 of a neighboring molecule. This resembles the situation in one of the SycE structures where the flexible C terminus similarly binds to the hydrophobic patch 1 of an adjacent dimer (Trame and McKay 2003).
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To determine the chaperone-binding site in YopT, the YopT/SycT complex was subjected to a protease-protection assay and digested with different proteases (trypsin, thermolysin, subtilisin, papain, chymotrypsin, and endoproteinase lys-C). Under the conditions used, SycT was stable except for the eight C-terminal residues. YopT was cleaved, and 28 degradation fragments detectable on SDS gels were N-terminally sequenced. Mapping of the protease cleavage sites revealed numerous sites in the region N-terminal of residue 52 and C-terminal of residue 140 with a single site at residue 104 (Fig. 6). Protection of residues 52–103 of YopT from proteolytic digestion in complex with SycT suggests this region to be protected by binding to the chaperone. This is supported by similar experiments defining the chaperone-binding domains of YopE and SptP (Stebbins and Galan 2001; Birtalan et al. 2002). The chaperone-binding domain of YopT most likely does not include residues N-terminal of amino acid 52, while the C-terminal boundary may be located after residue 104. Based on our findings, we propose a more detailed domain organization for YopT: The N-terminal ~20 amino acids of the effector comprise the secretion/translocation signal. The chaperone- binding domain of YopT covers at least residues 52– 103. The minimal YopT fragment capable of substrate binding and proteolytic cleavage includes residues 75–318 (Fig. 6) (Sorg et al. 2003).
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-helix set SycT apart from other chaperones. Although the location of two hydrophobic patches involved in effector binding in other chaperones is conserved in SycT, the precise mode of interaction may be different, as SycT possesses an additional strand 0 that extends the -sheet alongside 1. Other chaperones bind their effectors (YopE, SptP, and YopN) by allowing these to extend the chaperone -sheet by a -strand adjacent to strand 1. In one crystal packing of SycT 0 undergoes a conformational change and opens a groove. This may indicate a mechanism for effector binding.
Despite low sequence conservation, TTS effector-binding chaperones share a common fold and effector binding mode. In addition to binding multiple substrates (Parsot et al. 2003), Spa15 is structurally distinct from other effector-binding chaperones because of the relative orientation of the monomers in the dimer and the presence of an additional -strand (Van Eerde et al. 2004). SycT shares the additional strand 0 with Spa15 yet binds a single effector. This indicates that chaperones such as Spa15 that bind multiple effectors are structurally not set apart from other TTS chaperones. Instead effector-binding chaperones form a single functional and structural group.
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Protein expression and purification
YopT and full-length GST-tagged SycT were produced by coexpression in E. coli BL21(DE3) codon plus RIL (Stratagene). Native, full-length SycT was produced as a GST-fusion protein in the same strain. SeMet-substituted GST-tagged SycT122 protein was produced as described using the strain from above (Guerrero et al. 2001). Cells were grown at 37°C to an OD600 of 0.7. Recombinant gene expression was induced with 1 mM isopropyl--D-thiogalactoside overnight at 20°C. Cells were resuspended in ice-cold lysis buffer (50 mM phosphate [pH 8.0], 150 mM NaCl, 10 mM -mercaptoethanol) and lysed using a French press (SLM Aminco). Clarified lysates were applied to glutathione-loaded Sepharose resin (Amersham Biosciences) and washed with 20 mM Tris-HCl (pH 8.0), 150 NaCl, and 5 mM dithiothreitol (DTT). The GST-tag was cleaved on column at 16°C overnight using 1:60 TEV protease. His-tagged TEV protease was removed by Ni2+ loaded agarose (Qiagen). The TEV protease cleavage site (Glu-Asn-Leu-Tyr-Phe-Gln | Gly-Ala) introduces two additional amino acids (Gly-Ala) at the N terminus of the cleaved protein. The YopT/SycT complex was further purified by gel permeation chromatography (Superdex-75, Amersham Biosciences) with 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1 mM DTT as running buffer. Native SycT and SycT122 were purified by anion exchange chromatography (MonoQ, Amersham Biosciences) using a linear NaCl gradient in 20 mM Tris-HCl (pH 8.0) and gel permeation chromatography as above. Native SycT and SycT122 were dialyzed against 20 mM Tris-HCl (pH 8.0), 10 mM NaCl, and 1 mM DTT; subjected to MALDI-TOF analysis to check the protein size and complete incorporation of SeMets (three per molecule), respectively; and concentrated to ~12 mg/mL for crystallization trials.
Molecular mass determination
Gel permeation chromatography as described above, calibrated with standard proteins from low- and high-molecular-mass gel filtration calibration kits (Amersham Biosciences), and dynamic light scattering (DynaPro 801TC system, ProteinSolutions) were used to determine the molecular mass of SycT and the complex YopT/SycT.
Limited proteolysis and domain mapping
Purified YopT/SycT complex (~8 mg/mL) was digested on ice with 1:70 trypsin, chymotrypsin, papain, and endoproteinase Lys-C, as well as with 1:350 subtilisin and thermolysin. SycT mostly resisted proteolytic cleavage. Fragments were separated by SDS-PAGE, blotted onto PVDF membrane, and subjected to N-terminal sequencing.
Crystallization, data collection, and processing
Optimizing birefringent spherulites of native SycT using hanging- drop vapor diffusion at 4°C and a crystallization cocktail of 5% isopropanol, 0.1 M sodium citrate (pH 5.6), and 21% polyethylene glycol 4000 finally yielded orthorhombic crystals. For SeMet-substituted SycT122, initial screening in 96-well format using diverse commercially available screens and nano-drop pipetting resulted in a single promising condition. Optimized SycT122 crystals grew at 4°C using hanging-drop vapor diffusion by mixing equal volumes of SycT122 and 1.6 M ammonium sulfate, 0.1 M CAPS (pH 10.5), and 0.15 M lithium sulfate, revealing crystal form #1. Changing the crystallization condition to 1.8 M ammonium sulfate, 0.1 M sodium bicarbonate (pH 10.5), and 0.05 M magnesium chloride and using a protein/reservoir ratio of 2:1 yielded crystals with a different crystal form (#2). Data of native SycT and SycT122 were collected at 100 K using 19%–25% glycerol in the crystallization mix as cryoprotectant. SAD data of SycT122 at the peak region of the selenium edge were collected at beamline BL14.1 (BESSY). Data of SycT122 were processed with XDS and scaled with XSCALE (Kabsch 1993). Data of native SycT were collected at BW6 (DESY) and processed with DENZO/SCALEPACK (Otwinowski and Minor 1997). Data collection statistics are given in Table 1.
Structure determination
The heavy-atom search, phasing, and solvent flattening programs SHELXD (Uson and Sheldrick 1999) and SHELXE (Sheldrick 2002) run through the graphical user interface HKL2MAP (Pape and Schneider 2004) located all six selenium atoms in the hexagonal packing (#2) and provided interpretable electron density. Phases for #2 as output by SHELXE were used with the same anomalous data set to trace an initial model (80% complete) in ARP/wARP (Perrakis et al. 1999). The final model was obtained by several cycles of manual building with the program O (Jones et al. 1991) and TLS restrained refinement with REFMAC5 (Murshudov et al. 1997). Electron density was visible for SycT122 residues 1–113 in one monomer, and 3–122 in the second. The structure for the orthorhombic (#1) data set was solved by molecular replacement using the #2 structure as search model in the program PHASER (Storoni et al. 2004) and was completed as above. Electron density was not observed for SycT122 residues 115–122 in both monomers of model #1. The native structure was solved using both refined SycT122 structures as search ensemble for molecular replacement in PHASER, and was initially refined with CNS (Brunger et al. 1998) and then with REFMAC5. The refined native SycT model comprises residues 3–130 (A), 2–117, and 125–130 (B). Refinement statistics are given in Table 1. Ribbon diagrams and surfaces were produced with PyMOL (DeLano Scientific) and Swiss-PDBViewer (Guex and Peitsch 1997), secondary structure assignment was calculated using STRIDE (Heinig and Frishman 2004), buried surface areas were calculated with AREAIMOL (Lee and Richards 1971), cavity volumes were calculated with VOIDOO (Kleywegt 1994), pairwise and multiple sequence alignments were produced with EMBOSS (Pearson 1990) and ClustalW (Thompson et al. 1994), and structures were aligned with DALI (Holm and Sander 1993).
Coordinates
Coordinates and structure factors have been deposited in the Protein Data Bank. Accession codes are 2BSH (SycT122 crystal form #2), 2BSI (#1), and 2BSJ (native SycT).
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Acknowledgments |
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References |
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