Structural and Functional Studies on the Interaction of GspC and GspD in the Type II Secretion System

Type II secretion systems (T2SSs) are critical for secretion of many proteins from Gram-negative bacteria. In the T2SS, the outer membrane secretin GspD forms a multimeric pore for translocation of secreted proteins. GspD and the inner membrane protein GspC interact with each other via periplasmic domains. Three different crystal structures of the homology region domain of GspC (GspCHR) in complex with either two or three domains of the N-terminal region of GspD from enterotoxigenic Escherichia coli show that GspCHR adopts an all-β topology. N-terminal β-strands of GspC and the N0 domain of GspD are major components of the interface between these inner and outer membrane proteins from the T2SS. The biological relevance of the observed GspC–GspD interface is shown by analysis of variant proteins in two-hybrid studies and by the effect of mutations in homologous genes on extracellular secretion and subcellular distribution of GspC in Vibrio cholerae. Substitutions of interface residues of GspD have a dramatic effect on the focal distribution of GspC in V. cholerae. These studies indicate that the GspCHR–GspDN0 interactions observed in the crystal structure are essential for T2SS function. Possible implications of our structures for the stoichiometry of the T2SS and exoprotein secretion are discussed.


Introduction
Many Gram-negative bacteria use a multi-protein type II secretion system (T2SS) to secrete a wide variety of exoproteins from the periplasm into the extra-cellular milieu [1,2,3,4]. In Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC), cholera toxin and the closely related heat-labile enterotoxin, in addition to other virulence factors, are secreted in their folded state across the outer membrane by the T2SS [5,6,7]. The T2SSs are composed of 12 to 15 different proteins that form three distinct subassemblies: (i) the inner membrane platform consisting of multiple copies each of GspC, GspF, GspL and GspM with an associated cytoplasmic secretion ATPase; (ii) the pseudopilus, a filamentous arrangement of multiple copies of five different pseudopilins; and (iii) a large, pore-forming outer membrane complex, mainly consisting of the secretin GspD [8,9].
Secretins are multimeric outer membrane proteins composed of 50-70 kDa subunits and are among the largest outer membrane proteins known. The secretin superfamily has representatives in several other multi-protein complexes engaged in transport of large macromolecular substrates across the outer membrane [10] including the T2SS, the filamentous phage extrusion machinery [11], the type IV pilus system (T4PS) [12,13,14], and the type III secretion system (T3SS) [15,16]. Of these systems, the T2SS is most closely related to the T4PS which assembles and disassembles long filamentous fibers on bacterial surfaces and is responsible for diverse functions including attachment to host cells, biofilm formation, DNA uptake and twitching motility [17,18].
The T2SS secretin GspD forms a dodecameric assembly according to electron microscopy studies [19,20]. The C-terminal 300 to 400 residues of GspD contain the most conserved segments of the secretin superfamily, which form the actual outer membrane pore [21,22,23]. The N-terminal part of GspD consists of four domains: N0-N1-N2-N3 ( Figure 1A) [19,24]. The crystal structure of the N0-N1-N2 domains of the ETEC secretin GspD has been solved previously with the assistance of a single-domain llama antibody fragment or nanobody [24]. Nanobodies are the antigenbinding fragments (VHH) of heavy-chain-only camelid antibodies, which have been proven as effective crystallization chaperones for challenging targets, e.g. the T2SS pseudopilins complex [25], a trypanosomal editosome protein [26], and activated G-protein coupled receptor [27]. In the case of the secretin GspD N0-N1-N2 structure, nanobody Nb7 provided new crystal contacts and stabilized the N0-N1 domains lobe with respect to the N2 domain. The N0 domain is structurally related to domains from several proteins in bacterial multi-protein membrane complexes [28,29,30,31], and to a domain of protein gp27 from T4-related bacteriophages [32]. As expected from sequence homology, the repeat N1 and N2 domains have the same fold, whereas the N3 domain is predicted to have a similar structure [24]. The fold of the N1 domain is different from that of the N0 domain and is structurally related to the eukaryotic type I KH (hnRNP K homology) domain [33]. By combining crystallographic and cryoelectron microscopy studies, it has been proposed that the N0, N1, N2 and N3 domains form the large periplasmic vestibule of the GspD dodecamer [20]. According to a number of biochemical studies, the outer membrane protein GspD has also been reported to interact with exoproteins [20,34].
The inner membrane protein GspC consists of several domains: a short N-terminal cytoplasmic domain that is followed by the single transmembrane helix, a Pro-rich linker, the so-called homology region (HR) domain in the periplasm, a second linker and a C-terminal domain ( Figure 1A) [35]. Most frequently, this C-terminal domain is a PDZ domain, but in some cases it is a coiled-coil domain [36,37]. Crystal structures of the GspC PDZ domain showed that this domain can adopt open and closed conformations [38].
It has been shown in vivo in V. cholerae that GspC and GspD interact [39]. The interaction between GspC and GspD appears critical for the function, and possibly even for the assembly, of the T2SS [39]. Besides providing a physical link between the two membranes, either or both of these proteins or their interaction could also be important for exoprotein recognition, pseudopilus formation and release of the exoprotein through the GspD pore. Biochemically, we showed that the HR domain of GspC is the key part of GspC that interacts with the periplasmic GspD N0-N1-N2 [38]. This interaction was confirmed and further investigated recently in the plant pathogen Dickeya dadantii, a species previously called Erwinia chrysanthemi [40]. The interaction between GspC and GspD of Xanthomonas campestris has also been observed in vitro [37].
We report three structures of GspC HR in complex with Nterminal domains of GspD that provide a structural basis to understand the functional interplay between the inner membrane platform and the outer membrane secretin of the T2SS. The observed interface led to the design of experiments to probe the importance of specific amino acids by biochemical and in vivo studies. Altering interface residues disabled the interaction of GspC and GspD in a bacterial two-hybrid system. It also abrogated protease secretion and had a dramatic effect on the localization of GspC in the cell envelope in V. cholerae. Together these results show the physiological importance of the molecular interactions observed between the inner and the outer platform. In addition, the resultant structure of the HR domain of GspC means that the structures of essentially all globular domains of the major T2SS proteins are presently known. The structures of ETEC GspC HR in complex with N-terminal domains of GspD reported here are the first to reveal critical interactions between the inner membrane platform and the outer membrane complex of the T2SS at the atomic level.

Structures of Three GspC-GspD Complexes from ETEC
A complex of ETEC GspC HR and GspD N0-N1-N2 could be obtained but yielded only poorly diffracting crystals. To improve the quality of these crystals, we screened the same set of GspD specific nanobodies that had been used previously to solve the structure of GspD N0-N1-N2 [24] as crystallization chaperones for the GspC HR -GspD N0-N1-N2 complex. Using nanobody Nb3, we obtained crystals of a ternary ETEC GspC HR -GspD N0-N1-N2 -Nb3 complex, which diffracted initially only to ,5.5 Å resolution. Nevertheless, a partial molecular replacement structure revealed that the HR domain of GspC interacts with the lobe formed by the N0-N1 domains of GspD. To better characterize this interaction we also crystallized smaller complexes of GspC HR -GspD N0-N1 with or without nanobodies. To assist in crystallographic phasing, we also engineered a lanthanide-binding tag (LBT) into the N0 domain of GspD N0-N1 [41]. The LBT to GspD N0-N1 facilitated crystal growth and the resultant crystals of the binary GspC HR -GspD N0-N1 complex diffracted to better than 2.7 Å resolution, with the LBT engaged in multiple crystal contacts ( Figure S1). The structure of this binary GspC HR -GspD N0-N1 complex was solved by molecular replacement and refined with good crystallographic and stereochemical statistics (Table 1). In parallel, we also obtained crystals and solved the 4 Å resolution structure of a ternary GspC HR -GspD N0-N1 -Nb3 complex, and also improved the diffraction of crystals of the GspC HR -GspD N0-N1-N2 -Nb3 complex to ,4 Å resolution (Table 1, Figure S2).
The three multiprotein structures obtained from different crystal forms allow a detailed description of the interactions between GspC and GpsD. In all three structures, the N0 domain of GspD interacts exclusively with the HR domain of GspC. In the 2.63 Å resolution binary complex, the LBT introduced into GspD N0 faces away from the interface with GspC ( Figure 1B). In the two lowresolution ternary complex structures, the nanobody Nb3 binds the N0 domain of GspD, opposite to the binding site of the HR domain of GspC ( Figure 1C and Figure S3). In all three structures the HR domain binds in very similar orientation to GspD, relative to its N0 domain. Hence, neither the LBT nor the nanobody appears to affect the binding mode of GspC to GspD. Because the structure of the binary GspC HR -GspD N0-N1 complex has the

Author Summary
Many bacterial pathogens affecting humans, animals and plants export diverse proteins across the cell membranes into the medium surrounding the bacteria. Some of these secreted proteins are involved in pathogenesis. One example is cholera toxin secreted by the bacterium Vibrio cholerae, a causative agent of cholera. The sophisticated type II secretion system is responsible for moving this toxin, and several other proteins, across the outer membrane. Here, we studied the interaction between the outer membrane pore of the type II secretion system, the secretin GspD, and the inner membrane protein GspC. We have solved three crystal structures of complexes between the interacting domains and identified critical contacts in the GspC-GspD interface. We also showed the importance of these contacts for assembly of the secretion system and for secretion of proteins by V. cholerae. Our studies provide a major piece in the puzzle of how the type II secretion system is assembled and how it functions. One day this knowledge might allow us to design compounds which interfere with this secretion process. Such compounds would be useful in the battle against bacteria affecting human health.
highest resolution, this structure will be used below to analyze the specific features of the GspC-GspD interaction.

Structure of the HR Domain of GspC
The HR domain folds into a b-sandwich formed by six consecutive b-strands arranged as two three-stranded anti-parallel b-sheets ( Figure 1B). The residues between strands b3 and b4 adopt an approximately one-turn helical conformation. In its folded structure as seen in the complex with GspD N0-N1 (Figure 2), the distribution of charges on the surface of GspC HR is quite uneven with the main hydrophobic surface interacting with GspD N0 . Part of the remaining HR surface that is not involved in the GspD interaction (upper panel Figure 2) has a preponderance of negative charges and a deep pocket defined by residues Val127, Ile142 and Leu157. The other side of the HR domain (lower panel Figure 2) displays a mix of positive, negative and . Nanobody Nb3 is colored in orange. The N2 subdomain of GspD is statistically disordered in the crystal lattice ( Figure S2). The structure of the GspC HR -GspD N0-N1 -Nb3 ternary complex is essentially the same, despite the differences in GspD constructs, crystallization conditions and crystal forms ( Figure S3). doi:10.1371/journal.ppat.1002228.g001 hydrophobic patches. The functions of these features during assembly and action of the T2SS, if any, remain to be determined.
The Comparison of the HR Domain of GspC from the T2SS and PilP from the T4PS The closest known structural homolog of the HR domain of ETEC GspC appears to be Neisseria meningitidis lipoprotein PilP (NmPilP) which interacts with the secretin of the T4PS [42]. The HR domain of GspC and the core domain of NmPilP superimpose with an r.m.s. deviation of 1.6 Å and 25% sequence identity over 59 residues (Figure 3). The structure of NmPilP has been described as a b-sandwich composed of 7 b-strands [42]. Whereas residues 154-156 of GspC, corresponding to strand b4 of NmPilP, make some main chain hydrogen bonds to residues in strand b4 of GspC (corresponding to b5 of NmPilP), the secondary structure assignment algorithm of DSSP [43] does not classify these residues as b-structure.
A potential binding site has been described for the core NmPilP domain [42]. It consists of a hydrophobic crevice on the open end of the b-sandwich. The residues that create this hydrophobic groove appear to be conserved between these two proteins from the T2SS and the T4PS when they are superimposed ( Figure 3C). However, the area equivalent to the NmPilP pocket is covered by residues N-terminal to strand b1 in ETEC GspC and, therefore, the NmPilP pocket is absent in GspC ( Figure 3B). These differences do not appear to stem from crystal contacts in the GspC HR -GspD N0-N1 structure. Moreover, these residues are well conserved ( Figure S4A) and contribute to the hydrophobic core of the HR domain. The full implications of the global structural similarity between the core PilP domain of the T4PS and the HR domain of GspC from the T2SS remain to be established, but it is in line with several known similarities between the T2SS and T4PS [17,18].

The GspC-GspD Interface
The interface between GspC HR and GspD N0 buries 1280 Å 2 of accessible surface area with a calculated DG of interaction of 25.4 kcal6mol 21 as assessed by the PISA server ( Figure 4) [44]. The overall shape of the interface is relatively flat with a small concave area on the GspD surface. A total of 18 residues from GspC HR Table 1. Data collection and refinement statistics.  and 19 residues from GspD N0 engage in a combination of hydrophobic interactions and hydrogen bonds. The first three b strands of GspC HR and the first b strand plus the subsequent helix a1 of GspD N0 are the major contributors to the interface. The majority of the hydrogen bonds are formed by an antiparallel arrangement of strand b1 of GspC HR and strand b1 of GspD N0 ( Figure 4C). This b-strand augmentation is frequently observed in protein-protein interfaces [45]. Several nonpolar residues are engaged in intermolecular hydrophobic interactions, e.g. Ala133/ Val141 from GspC, and Phe5/Phe9 from GspD. The hydrophobic nature of these interacting residues is well conserved, with GspC residue 133 being Ala, Leu, Val or Met; GspC residue 141 either a Val or Ile; GspD residue 5 a Phe or Tyr; and GspD residue 9 a Phe according to a family sequence alignment ( Figure  S4). Nonetheless, the GspC-GspD interface provides a speciesspecific connection point between outer and inner membrane assemblies of the T2SS as has been observed in genetic complementation studies [46,47].  Based on the GspC HR -GspD N0-N1 structure, we selected several well-conserved interface residues for subsequent substitutional analysis. Ala133 and Val141 from ETEC GspC (equivalent to Val118 and Val129 from V. cholerae GspC, respectively; Figure 4E) and Thr20 from ETEC GspD (equivalent to Ile18 of V. cholerae GspD) are completely buried upon complex formation and are located in the center of the interacting surfaces ( Figure 4D). Asn24 from ETEC GspD (equivalent to Asn22 of V. cholerae GspD) makes a hydrogen bond with the main chain oxygen of ETEC GspC Arg137. We evaluated the role of these residues on complex formation of truncated forms of GspC and GspD in a bacterial two-hybrid system and in a functional V. cholerae secretion assay in vivo. We also assessed the effect of interface substitutions on the distribution of GspC in the cell envelope of V. cholerae.

Tests of GspC-GspD Interactions in the Bacterial Two-hybrid System
The effect of several interface substitutions on the ability of GspD to associate with GspC was assayed in a bacterial twohybrid system based on reconstitution of activity of the catalytic domain of Bordetella pertussis adenylate cyclase when T18 and T25 fragments are fused to interacting proteins (see Methods) [48]. VcGspD-T18 with a conservative Asn22Gln substitution retained the ability to interact with T25-VcGspC and formed dark red colonies on indicator agar. In contrast, VcGspD-T18 with either an Asn22Arg substitution or an Ile18Asp substitution lost the ability to interact with T25-VcGspC and formed colorless colonies ( Table 2). Two variants of T25-VcGspC, with either Val118Arg or a Val129Arg substitution, also lost the ability to interact with VcGspD-T18 and formed colorless colonies in the bacterial twohybrid system.

Mutations in the GspC-GspD Interface Interfere with Protease Secretion in V. cholerae
The functional importance of residues involved in the GspC-GspD interface was also assessed in vivo by monitoring the effect of the Ile18Arg and Asn22Tyr mutations in VcGspD on the extracellular secretion of protease by V. cholerae. No protease secretion was observed when plasmid-encoded VcGspD Ile18Arg/Asn22Tyr was produced in a V. cholerae mutant strain lacking the gene encoding VcGspD ( Figure 5A), indicating that the simultaneous exchange of these two amino acids prevents protein secretion by the T2SS. The singly substituted variants, however, remained functional ( Figure 5A). Immunoblot analysis of cell extracts from the DgspD mutant strain producing plasmid-encoded wild type and mutant VcGspD showed that the double VcGspD mutant protein was made at levels similar to that of wild-type VcGspD ( Figure 5B).

Distribution of GFP-VcGspC in V. cholerae Cells
Using V. cholerae strains producing chromosomally encoded VcGspC fused to the green fluorescent protein (GFP), we visually examined the effects of substitutions in the GspC-GspD interface on subcellular localization of GspC. GFP-VcGspC forms fluorescent foci in the V. cholerae cell envelope, which disperse upon deletion of the gene encoding VcGspD and reappear when the deletion strain is complemented with plasmid-encoded VcGspD ( Figure 6, first and second panels) [39]. The substitution of wildtype VcGspD with VcGspD Ile18Arg/Asn22Tyr resulted in loss of fluorescent foci and dispersal of the fluorescence in a manner indistinguishable from cells that do not have the gene encoding VcGspD at all (Figure 6, fourth panel). This result suggests that residues Ile18 and Asn22 of VcGspD are critical for the incorporation of GFP-VcGspC fusion protein into fluorescent foci, and supports the suggestion that the interaction between GspC and GspD observed in the crystal structure of GspC HR in complex with GspD N0-N1 (Figure 4) is physiologically relevant. Based on these results, it appears that VcGspD has to interact directly with VcGspC in order to support its focal distribution in V. cholerae.

Discussion
The current paper reveals for the first time key structural features of critical interactions between the outer membrane secretin GspD and the inner membrane protein GspC of the T2SS. The crystallographic studies benefited from the set of nanobodies against the N0-N1-N2 domains of GspD from ETEC [24] and from the incorporation of a lanthanide-binding tag (LBT) into ETEC GspD N0 . The three GspC-GspD crystal structures elucidated reveal the same 1280 A 2 interface involving the HR domain of GspC and the N0 domain of GspD. The crucial role of this interface was tested and confirmed by subsequent biochemical and functional studies. These results have interesting implications for our understanding of the T2SS and related secretion systems in many bacteria as discussed below.

The Mutual Orientation of the N0 and N1 Domains of GspD
The structures of the first two domains of related secretins have been determined in two prior studies: ETEC GspD from the T2SS and EPEC EscC from the T3SS [24,49]. The relative orientations of the N0 and N1 domains in these two studies appeared to be remarkably different: when the N1 domains of the T2SS and T3SS secretins are superimposed, the N0 domains are rotated by not less than 143 degrees [10]. This raises an important question as to the actual orientation of these two domains in the T2SS and T3SS secretins.
Regarding the T2SS, the relative orientations of the N0 and N1 GspD domains can now be compared in two high resolution structures, i.e. in the current structure of the binary complex of ETEC GspC HR and GspD N0-N1 , and in the previously determined binary complex of ETEC GspD N0-N1-N2 in complex with Nb7 [24]. The linker between the N0 and N1 subdomains is disordered in both these high resolution structures. The interface and relative orientation of the N0 and N1 subdomains, however, is essentially the same in the two structures despite the binding of either Nb7 or the presence of the LBT insertion into the N0 domain: the superposition of the two N0 domains results in an r.m.s. deviation of 0.49 Å for 72 Ca pairs ( Figure S5). Taking also into account the two new low resolution structures of the ternary complexes of GspC HR -GspD N0-N1 -Nb3 and GspC HR -GspD N0-N1-N2 -Nb3 ( Figure S3), then the N0-N1 lobe in the T2SS secretin GspD is observed as the same compact unit in four different crystal structures, independent of the presence or absence of a GspC HR domain, Nb molecules or crystal contacts. The available data suggest that the N0-N1 orientation in GspD is a characteristic feature in the T2SS. However, we cannot exclude the possibility that the relative orientation of the N0 and N1 domains may change as the secretin oligomerizes. Only high resolution structures of the dodecameric secretin will resolve this question.
Since the N0-N1 lobe of the T2SS secretin fits well into the cryo-electron microscopy reconstruction of VcGspD [20], and the N0 and N1 domains of the T3SS secretin fit well into a cryoelectron microscopy density of the Salmonella typhimurium needle complex [16], it might be that the N0 and N1 domains of these related secretins adopt different mutual orientations in the assembled T2SS and T3SS in vivo as observed in crystals. Obviously further studies are required to confirm this hypothesis where it also should be kept in mind that secretins are dynamic proteins and multiple orientations of N-terminal secretin domains might transiently occur during the secretion process [10].

The GspC-GspD Crystal Structure and Functional Studies
The crystal structure indicates that a number of residues are critical for the interactions of ETEC GspC and GspD (Figure 4).  Moreover, these residues are conserved in the family sequence alignment ( Figure S4). As many mutants and other useful reagents have already been generated and developed for studies of the T2SS in V. cholerae, subsequent probing of the importance of these residues for the interaction was carried out in three different ways using V. cholerae GspC and GspD homologues. The two-hybrid studies showed that substitutions Val118Arg and Val129Arg in VcGspC, and Asn22Arg in VcGspD, abrogated the interaction between GspC HR and GspD N0-N1-N2 from V. cholerae ( Table 2). The secretion of protease by V. cholerae was also greatly diminished by substitutions Ile18Arg/Asn22Tyr in full length VcGspD ( Figure 5). Finally, the same Ile18Arg/Asn22Tyr variant of VcGspD altered the distribution of full-length VcGspC in the inner membrane of V. cholerae, possibly by interfering with normal assembly of the inner membrane platform of the T2SS (Figure 6). Taking all data together, we conclude that the substitutions altering the interface of GspC with GspD in V. cholerae affect the interactions of GspC with GspD as demonstrated both in a bacterial two-hybrid system and by analysis of protease secretion by the T2SS in V. cholerae.
Interactions between GspC and GspD from D. dadantii have been recently investigated [40]. This study confirmed the interactions between GspC HR and the N-terminal domains of GspD reported earlier for V. vulnificus homologs [38]. A GST-fusion of residues 139-158 of DdGspC (corresponding to residues 168-187 in ETEC GspC) co-purified with both DdGspD N0 and DdGspD N1-N2-N3 [40]. The 139-158 residues of DdGspC were therefore designated as secretin interacting peptide (SIP). In a homology model of DdGspC HR -GspD N0-N1 complex, based on our crystal structure, this fragment is located far from the interface ( Figure S6). It appears that this segment forms an anti-parallel pair of b-strands, b5 and b6, in the ETEC GspC HR crystal structure, with b6 at the surface and b5 located between strands b6 and b4 ( Figure S6). Furthermore, the substitutions introduced into the DdGspC 139-158 fragment had no effect on the interaction with DdGspD N0 , whereas one substitution, Val143Ser, prevented DdGspC interaction with DdGspD N1-N2-N3 [40]. The same substitution, when introduced into full length DdGspC, also interfered with secretion in D. dadantii. We also mapped these substitutions onto the homology model of the DdGspC-GspD complex and it is clear that none of them are buried in the GspC-GspD interface ( Figure S6). The only substitution that had an effect on secretion, Val143Ser, replaces a buried hydrophobic residue in the core of DdGspC HR with a polar residue that would likely be detrimental to the HR domain stability. This is in agreement with the finding that this substitution in GST-DdGspC 128-272 resulted in a protein that is degraded in the cells [40]. A more conservative Val143Ala substitution in full length DdGspC appeared to largely support secretion of pectinases, in agreement with the less drastic change of the nature of the side chain, which could result in a larger proportion of properly folded protein than in the case of the Val143Ser variant. Therefore, the ETEC GspC HR -GspD N0-N1 structure explains several experimental results of the studies on DdGspC-GspD interactions [40]. The observations that a GSTfusion of the DdGspC 139-158 fragment is capable of interacting with fragments of the secretin in the absence of both the rest of the HR domain and the rest of the secretin, and of interfering with pectinase secretion when over-expressed in wild type D. dadantii, are difficult to interpret precisely. Additional studies are required to show that such interactions are not the result of non-specific interactions, possibly due to exposed hydrophobic residues of the peptide which are normally buried in the complete HR domain.

T2SS Stoichiometry
The implications of the GspC HR -GspD N0 interactions unraveled by our studies for the architecture of the T2SS are intriguing.
The three new structures in the current paper show that one GspC HR domain interacts with one GspD N0 domain, which suggests a 1:1 ratio of GspC and GspD in the assembled T2SS. Since the stoichiometry of full length GspC and GspD has not been established yet in the context of a functional T2SS, it is of interest to see if the current complex of GspC HR -GspD N0 is compatible with the dodecameric ring of GspD N0-N1 derived recently by a combination of crystallographic and electron microscopy studies [20,24]. Superimposing the GspC HR -GspD N0 complex twelve times onto the N0-domains of the GspD N0-N1 ring results in a double ring structure where the GspC HR subunits added do not interfere with the formation of the GspD N0-N1 ring. Although this procedure does result in some clashes between the subunits of the GspC HR ring, specifically between residues of the b2-b3 loop of one subunit and residues just prior to b6 in a neighboring subunit, small conformational changes in these loops, or minor adjustments in the mutual orientation of domains in the GspD N0 ring, or both, might alleviate these close contacts. If this would be the case, the GspD dodecamer would interact with twelve GspC subunits in the assembled T2SS ( Figure 7A). Alternatively, only alternating GspD subunits of the dodecameric secretin might interact with GspC HR , obviously removing close contacts between the then well separated GspC HR subunits. In this case, the GspD dodecamer would interact with six GspC subunits ( Figure 7B).
These two alternatives for the interface of the outer membrane complex and the inner membrane platform can be combined with previous studies on the T2SS even though the ratio between GspC and the other components of the inner-membrane platform complex is currently unknown. Yet, the following observations are of interest for the T2SS stoichiometry puzzle: (i) the secretion ATPase GspE of the T2SS has been suggested to be a hexamer [50,51]; (ii) the cytoplasmic domain of the inner membrane T2SS protein GspL forms a 1:1 complex with GspE [52]; (iii) homologs of GspM and of the cytoplasmic domain of GspL from the T4PS have been reported to form heterodimers [53,54]; (iv) there are a few cases of gene fusion of the T4PS proteins PilP and PilO (e.g. Pseudomonas putida PilO-PilP, Uniprot entry Q88CU9) in the T4PS. PilP is a GspC HR homolog ( Figure 3) and PilO is proposed to be a homolog of the inner membrane protein GspM from the T2SS [54,55]. The presence of PilO-PilP fusions may imply a 1:1 stoichiometry of these proteins in the T4PS and, in view of the homology between the T4PS and the T2SS, a GspM:GspC ratio of 1:1 in the T2SS as well.
These four observations suggest that GspE, GspL, GspM and GspC might be present in an equimolar ratio in the inner membrane platform. In view of the likely hexameric nature of GspE, this implies the presence of six subunits of each of these proteins in the assembled T2SS. If the GspD dodecamer would interact with six GspC subunits ( Figure 7B), then this arrangement would agree well with six subunits each of GspC, GspL, GspM and GspE in the inner membrane platform. If a GspD dodecamer, however, would interact with twelve GspC subunits in the assembled T2SS ( Figure 7A), then, a stoichiometry mismatch is likely to occur somewhere along the GspC-GspL-GspM-GspE chain of interactions in the inner membrane platform. This could be possible in spite of the evidence in points (i) to (iv) above for an equimolar ratio of these four proteins in the T2SS since e. g. points (iii) and (iv) are rather indirect and derived from observations on T4PS proteins. Clearly, the stoichiometry of the T2SS remains a fascinating topic for further studies, where the number of GspF subunits, the only T2SS protein which spans the inner membrane multiple times, also remains to be determined.

Implications for Exoprotein Secretion
Another major outstanding question is the recognition of exoproteins by the T2SS. Interestingly, the inner diameter of the dodecameric GspC HR -GspD N0-N1 double ring is ,68 Å , which implies that a large exoprotein like the cholera toxin AB 5 heterohexamer [56] just fits into this ring ( Figure 7C). This is in agreement with recent electron microscopy studies which indicate that the B-pentamer of cholera toxin can bind to the entrance of the GspD periplasmic vestibule [57]. The periplasmic domains of GspD and of GspC have been implicated in this crucial exoprotein recognition function [34,46,57,58,59], but the specific details of exoprotein-T2SS interactions remain to be uncovered. The accumulation of recent structural and biochemical data provides a platform for asking increasingly precise questions regarding the many remaining mysteries still pertaining to the architecture and mechanism of the sophisticated T2SS.

Expression and Purification of GspC and GspD for Crystallization
ETEC GspD N0-N1-N2 (residues 1-237; numbering corresponds to mature protein sequence) was expressed and purified as described [24]. The DNA sequence corresponding to residues 1-165 of ETEC GspD was PCR amplified and cloned into the pCDF-NT vector to obtain a GspD N0-N1 expression construct. pCDF-NT is a modified pCDF-Duet1 vector (Novagen) encoding an N-terminal His 6 -tag sequence and a TEV protease cleavage site. The DNA sequence corresponding to residues 122-186 of ETEC GspC was PCR amplified and cloned into a pCDF-NT vector to obtain a GspC HR expression construct.
A lanthanide binding tag (LBT) was introduced into GspD N0-N1 construct in order to assist with crystallographic phase determination and promote crystal formation. In order to decrease the flexibility of the LBT, we introduced it into the loop between two adjacent b-strands rather than at the termini. The design was based on the crystal structure of ubiquitin with the double LBT (PDB 2OJR) [41] where two b-strands flank one of the LBT. The LBT sequence YIDTNNDGYIEGDEL was inserted between residues Met70 and Val74 of GspD N0 ( Figure S4B) using the polymerase incomplete primer extension method [60]. While this manuscript was in preparation, a similar approach for the LBT insertion was successfully applied to a model protein, interleukin-1b [61].
GspD N0-N1 was expressed at 25uC in BL21(DE3) cells (Novagen) in LB medium containing 50 mg6ml -1 streptomycin. Protein production was induced with 0.5 mM IPTG. Cells were harvested 3 h after induction. GspD N0-N1 variants with or without LBT were purified by Ni-NTA agarose (Qiagen) chromatography followed by His 6 -tag cleavage with TEV protease; a second Ni-NTA chromatography to remove His 6 -tag, uncleaved protein and His-tagged TEV protease; and a final size-exclusion chromatography using Superdex 75 column (GE Healthcare). GspC HR was expressed and purified under same conditions as GspD N0-N1 . The proteins were concentrated, flash-frozen [62] and stored at 280uC. Se-Met-labeled proteins were expressed using metabolic inhibition of methionine biosynthesis [63] and purified using the protocols for native proteins.  [20,24] with a dodecameric ring of GspC HR (green) assembled as described in the text. (B) Two perpendicular views of the same dodecameric ring of GspD N0-N1 (blue) shown in (A) with six GspC HR subunits (green) binding to alternating GspD subunits as described in the text. (C) Two perpendicular views of the exoprotein cholera toxin (B pentamer in gold, A subunit in yellow) positioned inside the dodecameric GspC HR -GspD N0-N1 ring depicted in (A). The five-fold axis of the Bpentamer is aligned by hand with the twelve-fold axis of the ring. The orientation of the AB 5 hexamer with respect to the dodecamer is otherwise arbitrary. The cross-section of the double dodecamer of GspD N0-N1 and GspC HR is just sufficient to permit binding of the toxin heterohexamer. Obviously, the alternative assembly shown in (B) would also provide sufficient space for the toxin to bind at this location. doi:10.1371/journal.ppat.1002228.g007

Crystallization, Data Collection and Structure Determination
ETEC GspC HR , GspD N0-N1-N2 and individual nanobodies were mixed at 1:1:1 molar ratio, concentrated to 4-8 mg6ml 21 total protein concentration and subjected to crystallization conditions screening by the vapor diffusion method at 4 or 21uC. The crystallization conditions were identified using SaltRx (Hampton Research) and JCSG+ (Qiagen) screens. The complex of GspC HR -GspD N0-N1-N2 -Nb3 was crystallized in 1.2 M lithium sulfate, 0.1 M Tris-HCl pH 7 at 4uC. The crystals were gradually transferred to precipitant solution supplemented with 30% glycerol and flash-frozen in liquid nitrogen. Initial crystals diffracted to 5.5 Å resolution and optimized crystals with Se-Met substituted GspD N0-N1-N2 showed improved diffraction to 4.6 Å . Data were processed and scaled using XDS [64]. The structure was solved by molecular replacement using Phaser [65]; the search models included the GspD N0-N1 structure (PDB 3EZJ) [24], a camelid antibody fragment (PDB 1QD0) [66], and a homology model of GspC HR obtained using the I-TASSER server [67] and the N. meningitidis PilP structure as template (PDB 2IVW) [42]. The N2 domain of GspD could not be located in the electron density maps due to statistical disorder ( Figure S2).
The complex of GspC HR -GspD N0-N1 with an engineered LBT in the GspD N0 domain was crystallized in 0.9 M magnesium sulfate, 0.1 M bis-tris propane pH 7.0 at 21uC. The crystals were transferred to precipitant solution supplemented with 20% ethylene glycol and flash-frozen in liquid nitrogen. The structure of the GspC HR -GspD N0-N1 complex was solved by molecular replacement using Phaser and rebuilt using Buccaneer [68] and Coot [69]. The metal binding site of the LBT appears to be occupied by a Ca 2+ ion based on the electron density and the B factor values after refinement ( Figure S1). Most likely, Ca 2+ ions were acquired during E. coli expression, which prevented Tb 3+ ions binding during treatment of purified protein according to a published protocol [41]. The capture of ions during heterologous expression by Ca 2+ binding proteins has been observed previously for the major pseudopilin GspG [70]. The structure was refined with REFMAC [71] using translation, libration and screw-rotation displacement (TLS) groups defined by the TLSMD server [72]. The quality of the structure was assessed using the Molprobity server [73].
Protein-protein interfaces were evaluated using the PISA server [44]; structural homologs were searched for using the DALI server [74]; the electrostatic surface potential was calculated using APBS [75]; figures were prepared using PyMol [76].

Two Hybrid Analysis of VcGspC and VcGspD Domain Interactions
Interaction between protein domains was detected by the ability of fusion proteins containing the enzymatically inactive T18 and T25 fragments of adenylate cyclase toxin from Bordetella pertussis to confer adenylate cyclase activity (and the ability to ferment maltose and form red colonies on maltose-MacConkey plates) to a cyaA mutant E. coli strain as described previously [48]. E. coli DC8F9 is a cyaA::Km R derivative of the strain MM294 (endA1 hsdR17 glnV44 thi-1) with the Tc R F9 lacI q Tn10 from XL1blue (Stratagene). Plasmids pCT25VcGspC (encoding a T25-VcGspC fusion protein) and pAVcGspDT18 (encoding a VcGspD-T18 fusion protein) were separately transformed into E. coli DC8F9, and transformants were selected on LB-Cm or LB-Ap plates, respectively. Each of the resulting transformants formed white colonies when streaked onto maltose MacConkey plates and incubated at 30uC. In contrast, when both plasmids were transformed together into E. coli DC8F9, the resulting transformants formed red colonies when streaked onto maltose MacConkey plates, demonstrating a productive protein-protein interaction between the VcGspC and VcGspD domains of the T25-VcGspC and VcGspD-T18 fusion proteins, bringing together the T18 and T25 fragments to form active cyclase. A positive control also demonstrated a productive protein-protein interaction between CTA1 R7K T18 and CT25ARF6 fusion proteins in E. coli DC8F9 and formation of red colonies on maltose MacConkey agar, as reported previously [48]. Negative controls failed to demonstrate any productive protein-protein interaction between the CTA1 R7K T18 and T25VcGspC fusion proteins or between theVcGspDT18 and CT25ARF6 fusion proteins in E. coli DC8F9.

Cloning of Sequences Encoding Soluble Cytoplasmic Domains of VcGspC and VcGspD into Two Hybrid Vectors
A DNA sequence encoding residues 53-305 of VcGspC (AAA58784.1) was amplified by PCR using the primers EpsCXF and EpsCHIIIR adding XbaI (Leu-Glu frame) and a stop codon-HindIII sites at the 59 and 39 ends respectively. This product was cloned in frame after the T25 domain in place of ARF6 in pXCT25arf6 (pCT25ARF6 from [48] but with a vector XbaI site deleted) to generate pCT25VcGspC. Similarly the coding sequence for residues 25-294 of VcGspD (AAA58785.1) was amplified with primers EpsDNdeIF and EpsDClaR which add NdeI (and Met codon) and ClaI (Ser-Met frame) sites at the 59 and 39 ends respectively; this PCR product was cloned in place of the CTA1 gene in pCTA1 R7K T18 [48] to generate pAVcGspDT18. The primers sequence information is available upon request. Specific mutations in the eps gene domains (encoding GspC or GspD) in pAVcGspDT18 and pCT25VcGspC were generated by SOE-PCR [77] or by subcloning of a PCR fragment performed with a restriction site containing mutagenic primer and a vector primer, followed by recloning into the parental vector. All clones were verified in-frame and correct by DNA sequencing to ensure no additional PCR-generated mutations.

Generation of VcGspD Mutants
The DgpsD strain of V. cholerae, a gfp-gspC DgspD strain, and the complementing pMMB-gspD plasmid were constructed previously [39]. Mutations were introduced in the gspD gene of V. cholerae with the QuikChange II site-directed mutagenesis kit (Stratagene) using pBAD-gspD as a template. Primers used for the site change in gspD I18R and gspD N22Y were 59-GAATT-TATCAATCGTGTGGGACGCAATC-39, 59-GATTGCGTC-CCACACGATTGATAAATTC-39 and their reverse complements, respectively. gspD I18R/N22Y was then constructed using pBAD-gspD I18R as a template and the above primers specific for the gspD N22Y site change. All mutations were verified by sequencing. Following sequencing, the gspD variants of V. cholerae obtained were cloned into the low-copy-vector pMMB67 using restriction enzymes BamHI and SphI.

Detection of Secreted Protease Activity
V. cholerae cultures were grown overnight at 37uC in Luria broth supplemented with 100 mg6ml 21 thymine, 200 mg6ml 21 carbenicillin, and 20 mM IPTG and centrifuged to separate the supernatant and cellular material. The supernatants were centrifuged once more, and the protease activity was measured as described previously [78].

Microscopy
Cultures of V. cholerae were grown overnight at 37uC in M9 medium containing 0.4% casamino acids, 0.4% glucose, and 100 mg6ml 21 thymine; diluted 50-fold into fresh medium; and grown to mid-log phase before observation. Plasmids were maintained with 50 and 200 mg6ml 21 carbenicillin in log-phase and overnight cultures, respectively. Plasmid expression was induced with IPTG as described above. For fluorescence microscopy of live cells, cultures were mounted on 1.5% lowmelting temperature agarose pads prepared with M9 glucose medium. All microscopy was performed with a Nikon Eclipse 90i fluorescence microscope equipped with a Nikon Plan Apo VC100 (1.4 numerical aperture) oil immersion objective and a Cool SNAP HQ2 digital camera. Captured images were analyzed with NIS-Elements imaging software (Nikon).

Accession Numbers
Atomic coordinates and structure factors have been deposited in the Protein Data Bank (http://www.pdb.org) with accession code 3OSS.  Figure S3 The GspC HR -GspD N0 interface in three crystal forms. The N0-N1 domains are colored cyan; the HR domains green and magenta; Nb3 nanobodies orange. (A) Two crystallographically related ternary GspC HR -GspD N0-N1-N2 -Nb3 complexes in contact with each other in crystals with space group P6 1 22. (B) Two crystallographically related ternary GspC HR -GspD N0-N1 -Nb3 complexes in contact in crystals with space group P6 4 22. Comparison with (A) above shows that the ternary complexes in these two crystal forms are very similar. The 2-fold crystallographic contacts are essentially the same in the two different crystal forms. (C) The GspC HR chain has the same orientation with respect to GspD N0 in three different crystal forms. The superposition of the three GspC HR -GspD N0 complexes is based on GspD N0-N1 only. The HR domain of GspC HR -GspD N0-N1 is depicted in dark green; the HR domain of GspC HR -GspD N0-N1-N2 -Nb3 in light green; and the HR domain of GspC HR -GspD N0-N1 -Nb3 in magenta. (TIF) Figure S4 Sequence alignments of selected GspC proteins and GspD N0 domains. Residues that make intermolecular Van der Waals contacts and H-bonds in the ETEC GspC HR -GspD N0-N1 complex are labeled by triangles and stars, respectively. (A) Sequence alignment of GspC proteins. The secondary structure elements are shown at the top as determined from the ETEC GspC HR -GspD N0-N1 structure and the V. cholerae GspC PDZ structure (PDB 2I4S) [38]. (B) Sequence alignment of GspD N0 domains. The secondary structure elements are shown at the top as determined from the ETEC GspC HR -GspD N0-N1 structure. The position and sequence of the LBT are shown. (TIF) Figure S5 The structure of GspD N0-N1 is virtually the same in the GspC HR -GspD N0-N1 and GspD N0-N1-N2 -Nb7 structures. A stereoview of a superposition of GspD N0-N1 from the GspC HR -GspD N0-N1 complex (cyan) and the GspD N0-N1-N2 -Nb7 complex (purple, PDB 3EZJ) [24]. The superposition is based on the N0 domain only (r.m.s.d. 0.49 Å for 72 Ca atoms). The mutual orientation of the N0 and N1 domains is very similar indeed. (TIF) Figure S6 Analysis of the Dickeya dadantii GspC HR -GspD N0-N1 complex. (A) A homology model of the D. dadantii (previously Erwinia chrysanthemi) GspC-GspD complex. The structures of the HR domain of DdGspC (light green) and the N0-N1 domains of DdGspD (light blue) were obtained by homology modeling based on our new structure of the ETEC GspC HR -GspD N0-N1 complex (grey) as template, using the SWISS-MODEL server (http:// swissmodel.expasy.org/) [79]. (B) Mutations of DdGspC. The residues in the interface of DdGspC-GspD in the homology model are shown as sticks. A previously suggested interaction region SIP (secretin interacting peptide) that corresponds to residues 139-159 is highlighted in orange [40]. The residues which have been subjected to mutational analysis (R142, V143, V144, R150, E152 and Y157) are shown as sticks and labeled. The mutant DdGspC proteins R142I, V144A, R150L, E152A and Y157A fully supported secretion of pectinases in D. dadantii. Note that, in contrast to the other residues, V143 is completely buried in the model and the substitution V143S leads to decreased secretion [40]. For further discussion see main text. (C) Sequence alignments of GspC HR and GspD N0 from ETEC and D. dadantii. Secondary structure elements are shown above alignment according to ETEC GspC HR -GspD N0-N1 crystal structure. A previously suggested interaction region SIP that corresponds to residues 139-159 is indicated by an orange bar. The residues which have been subjected to mutational analysis (R142, V143, V144, R150, E152 and Y157) are highlighted by circles. (TIF)