Evidence for alternative quaternary structure in a bacterial Type III secretion system chaperone
© Barta et al; licensee BioMed Central Ltd. 2010
Received: 27 January 2010
Accepted: 15 July 2010
Published: 15 July 2010
Type III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg) C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators.
In this study, we present the 3.3 Å crystal structure of an amino-terminally truncated form (residues 10-155, denoted IpgC10-155) of the class II chaperone IpgC from Shigella flexneri. Our structure demonstrates an alternative quaternary arrangement to that previously described for a carboxy-terminally truncated variant of IpgC (IpgC1-151). Specifically, we observe a rotationally-symmetric "head-to- head" dimerization interface that is far more similar to that previously described for SycD from Yersinia enterocolitica than to IpgC1-151. The IpgC structure presented here displays major differences in the amino terminal region, where extended coil-like structures are seen, as opposed to the short, ordered alpha helices and asymmetric dimerization interface seen within IpgC1-151. Despite these differences, however, both modes of dimerization support chaperone activity, as judged by a copurification assay with a recombinant form of the translocator protein, IpaB.
From primary to quaternary structure, these results presented here suggest that a symmetric dimerization interface is conserved across bacterial class II chaperones. In light of previous data which have described the structure and function of asymmetric dimerization, our results raise the possibility that class II chaperones may transition between asymmetric and symmetric dimers in response to changes in either biochemical modifications (e.g. proteolytic cleavage) or other biological cues. Such transitions may contribute to the broad range of protein-protein interactions and functions attributed to class II chaperones.
Type III secretion systems (TTSSs) use a conserved apparatus (TTSA) to provide an energy-driven conduit from a bacterium to the cell membrane and cytoplasm of targeted eukaryotic cells. A hallmark of TTSSs is the presence of two secreted translocators that assume a position at the tip of the TTSA needle to form a pore in the host cell membrane. Once the mature tip complex has formed, the conduit is completed and vectorial transfer of a pathogen-specific repertoire of secreted effector proteins can occur. Ultimately, it is these effectors that allow for subversion of normal host cellular functions to the benefit of the bacterium.
Within each TTSS a conserved, class II chaperone protein is required to prevent premature association of translocator proteins while maintaining them in a secretion competent state, ready to be presented in a temporal fashion to the TTSA. In the enteric pathogen Shigella flexneri, invasion plasmid gene (Ipg) C is the class II chaperone for invasion plasmid antigens (Ipa) B and C. Owing to this central role in supporting translocator function, IpgC is essential for Shigella virulence as an ipgC null strain is noninvasive. Furthermore, upon release of the IpaB and IpaC effectors, IpgC binds to the AraC-like transcription factor, MxiE to promote expression of late-effector genes. In this manner, IpgC appears to provide a critical link between TTSS induction and those events which occur immediately following bacterial contact with the host cell.
Two reports describing high-resolution crystal structures of two class II chaperones have provided a great deal of insight into the structure and function of these all alpha-helical, tetratricopeptide repeat (TPR) proteins. First, the crystal structure of an amino-terminally truncated form of SycD (SycD21-163) from Yersinia enterocolitica, was reported. In this work, Büttner et al. proposed that the biologically-relevant unit of the SycD chaperone is a dimer comprised of a "head-to-head" arrangement of equivalent monomers. This observation was supported by analytical gel filtration chromatography studies on targeted mutants in two residues, A61 and L65, whose disruption ablated the homomeric contacts that stabilize the dimerization interface. Moreover, a sycD null strain complemented with the dimerization disrupting double mutant (A61E/L65E) was unable to secrete either Yop translocator protein and exhibited characteristics typical of a sycD null mutant. Separately, Lunelli et al. recently described the crystal structure of a carboxy-terminally truncated form of S. flexneri IpgC (IpgC1-151). Though they share only 26% sequence identity, the IpgC polypeptide displays a great deal of structural homology with SycD. However, the biological unit of this class II chaperone appeared to arise from an asymmetric dimer involving the first three alpha helices of each unique monomer. In particular, helix 1 (abbreviated hereafter as H1) and the loop connecting H1 to H2 adopted distinct arrangements in both subunits of the dimer. Deletion of the amino-terminal 21 amino acids of IpgC, which includes the entirety of H1, destroyed the dimerization ability of IpgC, and subsequent complementation studies demonstrated that this deleted chaperone was unable to complement an ipgC null strain of Shigella in HeLa cell invasion assays.
The apparent importance of asymmetric dimerization in the face of previous results that argued similarly in favor of the "head-to-head" motif suggests that both modes of dimerization may be functionally relevant. For this to be the case, however, evidence must be provided to suggest that a single class II chaperone can adopt both the asymmetric and "head-to-head" dimer arrangements. Here we present the crystal structure of an amino-terminally truncated variant (IpgC10-155) of the Shigella class II secretion chaperone IpgC. Like its full-length counterpart, we find that IpgC10-155 is a dimer in solution. However, the dimerization interface of IpgC10-155 observed in our crystal structure is characterized by a rotationally symmetric "head-to-head" arrangement of identical polypeptides chains. Surprisingly, this mode of quaternary structure is far more similar to that reported for SycD rather than for the longer form of IpgC (IpgC1-151). The potential implications for these observations to class II chaperone activity and TTSSs function are discussed.
Results and Discussion
Crystallization and Structure Determination of an Amino-terminally Truncated form of IpgC
X-ray Diffraction Data and Refinement Statistics
APS 22 - BM
P 31 or P 32
a = 140.50
a = 86.11
b = 71.47
b = 86.11
c = 171.01
c = 476.27
ß = 93.86°
B factor (Å2)
Bond Length (Å)
Bond Angle (°)
Dihedral Angle (°)
Evidence for Alternative Quaternary Structures in IpgC
Superposition Analysis for Selected Monomers and Dimers as Determined by Local-Global Alignment
Sequence Identity (%)
IpgC1-151 (chain A)
IpgC1-151 (chain B)
IpgC10-155 (chains A&B)
IpgC10-155 (chains A&B)
Examination of the refined structure indicates that the IpgC10-155 crystals consist of an ordered lattice of protein dimers. However, much like the structures of SycD21-163, IpgC1-151, and PcrH21-160, more than one plausible dimerization arrangement are observed. The first class of dimer is present in nine crystallographically unique copies and is characterized by a "head-to-head" orientation, where each monomer is related by a single axis of rotational symmetry (Figure 1C and Additional file 1, Figure S1). This "head-to-head" dimer buries an average of 1262.5 Å2 of surface area upon formation, which compares favorably to the 1381.9 Å2 interface previously described for IpgC1-151. A distinct class of rotationally-symmetric arrangement occurs only once within the asymmetric unit, though it can be generated for all other IpgC10-155 chains by application of crystallographic operators (Additional file 2, Figure S2). It is worth noting, however, that this "tail-to-tail" dimer buries only an average of 756.5 Å2 upon formation, or approximately 60% of the surface masked by the "head-to-head" structure. Furthermore, the contacts present in the "head-to-head" dimer are more extensive and conserved to a far greater extent (21 of 40 residues, or roughly 53%) than are those found in the "tail-to-tail" structure (6 of 18 residues, or roughly 33%). When considered together, these data strongly suggest that the "head-to-head" dimer is the relevant structure for IpgC10-155, and that the other dimerization mode most likely arises from crystallization.
The identification of a rotationally symmetric dimer within the IpgC10-155 crystal raised questions about its relationship to the dimers previously described for TTSS class II chaperones (Figure 1D, E and Table 2). In this regard, the "head-to-head" arrangement of IpgC10-155 superimposes relatively poorly with the asymmetric dimer of IpgC1-151; overall, only 135 of 274 corresponding Cα positions lie within 4.0 Å distance and an RMSD of 1.35 Å. By contrast, the IpgC10-155dimer overlays surprisingly well with the dimer previously described for SycD21-163; here, 199 of 274 corresponding Cα positions lie within 4.0 Å distance with an RMSD of 2.34 Å. It is important to note that the lower RMSD for the first superposition arises because Cα positions that lie outside of the 4.0 Å distance cutoff are omitted from RMSD calculation. Thus, even though the two IpgC structures share a substantially higher level of identity in terms of sequence and overall monomer structure (Figure 2 and Table 2), the far greater number of aligned residues between the IpgC10-155 and SycD21-163 dimers indicates that their "head-to-head" quaternary structures are closely related.
Although the biological unit of the IpgC1-151 structure has been defined as an asymmetric dimer of structurally unique subunits, examination of IpgC1-151 chains related by crystallographic symmetry reveals two separate contacts reminiscent of the IpgC10-155 quaternary structure (Additional file 3, Figure S3). The first of these contacts is found in the IpaB peptide-bound form of IpgC1-151, and buries 666.9 Å2 of surface area upon formation (Additional file 3, Figure S3A). When compared carefully to the IpgC10-155 dimer, it is apparent that the symmetry-related IpgC1-151 chain in this contact is rotated to a greater extent relative to the monomer found within the asymmetric unit; as a result, only 130 of 264 Cα positions superimpose within 4.0 Å distance. Separately, a "head-to-head" dimer is also observed within the lattice contacts of the unbound IpgC1-151 structure (Additional file 3, Figure S3B). This arrangement buries 950.4 Å2 of surface area upon formation and, aside from the previously mentioned difference in the amino terminal region, shares a higher level of homology to the IpgC10-155 dimer. In this case, 176 of 264 possible Cα positions superimpose within 4.0 Å distance. Most importantly, the aromatic residues which line this interface are identical to those found in the IpgC10-155 dimer (Additional file 3, Figure S3C), as described below.
Residues that Comprise the 'Head-to-Head' Dimerization Interface in IpgC are Conserved Across TTSS Class II Chaperones
While the IpgC10-155 dimer buries approximately 1260 Å2 of surface area upon formation, closer inspection reveals that this interaction is comprised largely of two separate regions from each respective monomer. The first of these gives rise to the SycD-like interface (~680 Å2), and involves an intricate array of almost exclusively hydrophobic interactions between the α2 and α3 helices of opposing IpgC10-155 chains. Chief among these is a network of homophilic contacts between Phe residues at positions 46, 58, and 61, whose sidechains nearly intercalate with one another (Figure 1B, C). Hydrophobic interactions aside, a single hydrogen bond between the sidechains of Tyr42 and Glu53 is also found within this interface. Separately, a distinct region of contact that masks nearly 580 Å2 of surface area is likewise observed in the IpgC10-155 dimer. This interface arises from packing of nearly the entire extended amino terminus of one IpgC10-155 chain against primarily the α5 helix of its counterpart polypeptide (Additional file 4, Figure S4). Intriguingly, this region includes residues Ala94 and Val95, two residues whose concerted mutation has been shown to disrupt the dimerization of IpgC1-151. Analytical gel filtration chromatography was used to analyze the effect of this double mutant on the oligomeric state of both IpgC1-151 and IpgC10-155 (Additional file 5, Figure S5). However, both proteins migrated as a single species with an observed molecular weight of approximately 40 kDa in this assay (Additional file 6, Figures S6). In contrast to previous data, these results suggest that dimerization may not be fully ablated by simultaneous mutation of both Ala94 and Val95.
The 'Head-to-Head' Dimer of IpgC Supports Chaperone Activity
In the last year, two separate studies have reported the crystal structures of class II chaperones bound to peptide fragments of TTSS translocator proteins. Even though the structures of IpgC1-151-IpaB and PcrH21-160-YopD are meant to mimic recognition of separate classes of full-length translocator proteins from two distinct organisms, both structures reveal that the translocator peptide lies within a groove found on the concave TPR "hand" of the chaperone. This indicates that their mechanism of translocator/ligand recognition is similar, despite the fact that the quaternary structures appear to differ considerably between IpgC1-151 and PcrH21-160. The significant differences between the "head-to-head" dimer observed in both SycD21-163 and PcrH21-160 with the asymmetric structure of IpgC1-151 raise important questions regarding the precise nature of class II chaperone dimers in the physiological setting. Further complicating this issue are the cogent biophysical, biochemical, and/or functional data which support each of these crystal structures.
Our observation that a single TTSS chaperone can adopt two distinct quaternary arrangements suggests that both the asymmetric and head-to-head dimers may have important physiological roles in bacterial TTSSs. As stated earlier, IpgC has the ability to bind two separate translocator proteins, IpaB and IpaC, as well as the AraC-family transcription factor, MxiE. The ability of IpgC to bind each of these proteins is regulated by the secretion state of the S. flexneri cell. Secretion of both IpaB and IpaC through the TTSA needle liberates IpgC, and allows it to interact with MxiE; this culminates in the expression of the late effectors. It is believed that an amino terminal secretion signal targets effectors to the secretion system and that chaperones may also be involved in guidance of their complexes to the base of the TTSA needle. The potential switch in quaternary structure of IpgC may therefore be involved with its ability to effectively bind or deliver translocators to the secretion system. For example, though both types of chaperone dimer are competent to bind peptide mimics of their translocator targets, a change in dimerization state might alter the stoichiometry of various chaperone/ligand complexes within the context of full-length proteins. Addressing this possibility will require more thorough characterization of translocator proteins, such as IpaB and IpaC, for which little tertiary structural information is currently available. Along these lines, IpgC could also transition between asymmetric and symmetric dimerization modes to accommodate its broad range of interaction partners. Because the change in dimerization appears to correlate with a loss of amino acids at the amino terminus of IpgC, an ordered proteolytic event in this region might trigger a change in quaternary structure that affects IpgC function. Whether such a transition could result in a change in the role of IpgC from secretion chaperone to transcriptional coactivator remains to be determined. In any case, additional study will be needed to explore the potential roles of both modes of dimerization in the Shigella TTSS as well as that from other pathogens.
Cloning, overexpression and purification of recombinant forms of IpgC
A designer gene fragment encoding residues 10-155 of IpgC was amplified from the virulence plasmid of Shigella flexneri via PCR and subcloned into pT7HMT. Following confirmation of its DNA sequence, this expression vector was transformed into BL21 (DE3) Escherichia coli cells and cultured in Terrific Broth at 37°C to an A600 nm of 0.8. Protein expression was induced overnight at 18°C by adding IPTG to 1 mM final concentration. Cells were harvested by centrifugation, resuspended in lysis buffer (20 mM Tris pH 8.0, 500 mM NaCl, and10 mM imidazole), and then lysed in a microfluidizer. The soluble target protein was collected in the supernatant following centrifugation of the cell homogenate and purified on a Ni2+-NTA Sepharose column according to standard protocols. Recombinant TEV protease was used to digest the fusion affinity tag from the target protein. After desalting into 20 mM Tris (pH 8.0), final purification was achieved by Resource Q anion-exchange chromatography (GE Biosciences). Following this, the purified protein was concentrated to 10 mg/mL and exchanged into H2O for further use. A similar protocol was used to subclone, overexpress, and purify full-length IpgC, a further truncated form that consisted of residues 21-155 (IpgC21-155), and the IpgC1-151 variant described by Lunelli et al.. Expression vectors encoding the Ala94Glu/Val95Gln double mutant of both IpgC10-155 and IpgC1-151 were generated by PCR using the two-step megaprimer method; the corresponding proteins were overexpressed and purified as described above.
IpgC10-155 was crystallized by vapor diffusion of hanging drops at 20°C. Specifically, 1 μL of protein solution (10 mg/mL in ddH2O) was mixed with 1 μL of reservoir solution containing 0.2 M magnesium chloride hexahydrate, 0.1 M Bis-Tris (pH 6.5) and 25% (w/v) PEG 3350, and the drops were equilibrated over 500 μL of reservoir solution. Clusters of needle-shaped crystals appeared overnight and continued to grow in size for approximately 7 days. Mechanical disruption of these clusters was used to obtain single, diffraction quality samples for diffraction analysis. Crystals were flash cooled in a cryoprotectant solution consisting of reservoir buffer with an additional 5% (w/v) PEG 3350. Crystals of IpgC21-155 were also produced using an analogous approach. Briefly, 1 μL of protein solution (10 mg/mL in ddH2O) was mixed with 1 μL of reservoir solution containing 0.1 M HEPES (pH 7.5) and 2.0 M ammonium formate, and the drops were equilibrated over 500 μL of reservoir solution. Single diamond shaped crystals appeared overnight and continued to grow for 2-3 days. Crystals were flash cooled in a cryoprotectant solution consisting of reservoir buffer with 30% (v/v) glycerol. Diffraction quality crystals were not obtained for full-length IpgC.
Structure determination, refinement and analysis
Monochromatic X-ray diffraction data (γ = 1.000 Å) were collected from single IpgC10-155 and IpgC21-155crystals at 100 K using beamlines 22-BM and 22-ID, respectively, of the Advanced Photon Source, Argonne National Laboratory (Table 1). Following data collection, individual reflections were indexed, integrated, and scaled using HKL2000. Initial phase information was obtained for the IpgC10-155 data by maximum-likelihood molecular replacement using PHASER. Residues 30-151 of a single copy of the refined IpgC1-151 structure were used as a search model. The single most highly scored solution contained 18 unique IpgC10-155 polypeptides in the asymmetric unit, which corresponded to a solvent content of 56.8%.
Structure refinement was carried out using the protocols implemented in phenix.refine. First, three rounds of individual coordinate and isotropic atomic-displacement factor refinement were conducted and the refined model was used to calculate both 2Fo-Fc and Fo-Fc maps. These maps were used to manually build residues 18-29 and 152-154 of the master polypeptide chain, which is denoted chain A in the PDB file. This intermediate model was subjected further to an identical series of refinement steps prior to a final, single round of TLS refinement in phenix.refine; each individual polypeptide chain was treated as its own unique TLS group. The final model displays Rwork/Rfree values of 25.9/29.6%, respectively, and consists of residues 18-154 for all 18 copies of IpgC10-155 present in the asymmetric unit. RAMPAGE analysis of the final model revealed that 91.5% and 5.8% of the 2,430 residues modeled occupied either favored or allowed regions of the Ramachandran plot, respectively. Additional electron density that corresponded to N terminally directed residues were visible in both 2Fo-Fc and Fo-Fc maps calculated from the final model. Side chain features were poor in these areas, however, and this precluded accurate modeling of these residues in the final structure. The coordinates of the crystal structure described here have been deposited in the RCSB database under the accession code 3KS2.
Analytical gel filtration chromatography
Purified protein samples (5 mg/mL) were separated on a Tricorn Superdex 200 10/300 analytical gel filtration chromatography column (GE Biosciences) that had been previously equilibrated in a buffer of 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM DTT at 4°C. Estimates of molecular weight and oligomerization were made by comparing the retention time of individual samples to those of globular protein standards (Bio-Rad).
Bis(Sulfosuccinimidyl) suberate (BS3; 80 μL of a 250 μM solution in ddH2O) was added to 20 μL samples (2 mg/mL) of purified IpgC1-151 and IpgC10-155 at 20°C. 5 μL aliquots from each reaction were withdrawn at various time points over the course of 240 min and excess BS3 was quenched by adding 0.75 μL of 25 mM Tris (pH 8.0) for 30 min. Samples were analyzed under reducing conditions by electrophoresis (10% SDS-PAGE) using a Tris-Tricine buffer system.
Copurification assay for chaperone activity
Chaperone activity of full-length IpgC and various deletion proteins was monitored by chromatographic copurification. Specifically, a designer gene fragment encoding a protease-stable domain of S. flexneri IpaB (residues 58-357; denoted IpaB58-357) was generated by PCR, subcloned in the expression vector pACYC-Duet (Novagen), and sequenced; this vector provides for expression of IpaB58-357 without any fusion tag, and IpaB does not bind significantly to Ni2+-NTA Sepharose on its own accord. The resulting plasmid was co-transformed with various pT7HMT-IpgC expression vectors (described above) into E. coli BL21(DE3) cells. Cotransformants were identified by antibiotic selection with both chloramphenicol and kanamycin. Cells harboring both expression vectors were cultured and protein expression was induced according to standard methods. Homogenates of induced cells (250 mL total culture volume) were prepared by microfluidization, clarified by centrifugation, and subjected to Ni2+-NTA Sepharose chromatography as described above. Following this, the crude eluate was further separated on a Superdex 75 26/60 preparative gel-filtration column (GE Biosciences). Samples were analyzed under reducing conditions by 4-10% gradient SDS-PAGE using a Tris-Tricine buffer system.
Multiple sequence alignments were carried out using CLUSTALW and aligned with secondary structure elements using ESPRIPT. Sequences used in alignment, along with their respect accession numbers, were as follows: Shigella flexneri IpgC (GI:32307022), Burkholderia pseudomallei BicA (GI:126447932), Salmonella typhimurium SicA (GI:975294), Pseudomonas aeruginosa PcrH (GI: 29826004) and Yersinia enterocolitica SycD (GI:23630571). Three-dimensional structures were analyzed using the Protein Interfaces, Surfaces, and Assemblies server (PISA)  and superimposed using the Local-Global Alignment method (LGA). Representations of all structures were generated using PyMol.
Invasion plasmid gene C
Specific Yersinia chaperone D
Type III Secretion System
Type III Secretion Apparatus
Invasion plasmid antigen B
Invasion plasmid antigen C
Membrane expression of ipa E
Liquid Chromatography Mass Spectrometry
Tobacco etch virus
Salmonella invasion chaperone A
Burkholderia invasion chaperone A.
This work was supported by grants from the National Institutes of Health (AI071028 to B.V.G. and AI067858 to W.L.P.) and the Missouri Life Sciences Research Board (13238 to B.V.G.). Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, and Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. Data were collected at Southeast Regional Collaborative Access Team (SER-CAT) beamlines at the Advanced Photon Source, Argonne National Laboratory. A list of supporting member institutions may be found at http://www.ser-cat.org/members.html.
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