Biochemical Characterization of a Regulatory Cascade Controlling Transcription of the Pseudomonas aeruginosa Type III Secretion System*

Many Gram-negative pathogens utilize type III secretion systems (T3SS) to translocate effector proteins into eukaryotic host cells. Expression of T3SS genes is highly regulated and is often coupled to type III secretory activity. Transcription of the Pseudomonas aeruginosa T3SS genes is coupled to secretion by a cascade of interacting regulatory proteins (ExsA, ExsD, ExsC, and ExsE). ExsA is an activator of type III gene transcription, ExsD binds ExsA to inhibit transcription, ExsC inhibits ExsD activity, and ExsE inhibits ExsC activity. The entire process is coupled to secretion by virtue of the fact that ExsE is a secreted substrate of the T3SS. Changes in the intracellular concentration of ExsE are thought to govern formation of the ExsC-ExsE, ExsC-ExsD, and ExsD-ExsA complexes. Whereas formation of the ExsC-ExsE complex allows ExsD to bind ExsA and transcription of the T3SS is repressed, formation of the ExsC-ExsD complex sequesters ExsD from ExsA and transcription of the T3SS is induced. In this study, we characterized the self-association states of ExsC, ExsD, and ExsE and the binding interactions of ExsC with ExsE and ExsD. ExsC exists as a homodimer and binds one molecule of ExsE substrate. Dimeric ExsC also interacts directly with ExsD to form a heterotetrameric complex. The difference in binding affinities between the ExsC-ExsE (Kd 1 nm) and ExsC-ExsD (Kd 18 nm) complexes supports a model in which ExsC preferentially binds cytoplasmic ExsE, resulting in the inhibition of T3SS gene transcription.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a primary cause of pneumonia and urinary tract infections in intensive care units (1,2). Among the most vulnerable individuals are those with immunodeficiency, cystic fibrosis, or severe burns or who require mechanical ventilation. A major virulence determinant of P. aeruginosa is a type III secre-tion system (T3SS). 2 The T3SS encodes a multiprotein complex, called an injectisome, which functions by injecting or translocating effector proteins directly into the cytoplasm of eukaryotic host cells. The T3SS of P. aeruginosa is used to translocate four known effectors that subvert signal transduction pathways to inhibit phagocytosis and elicit cytotoxicity toward host cells (3,4).
Expression of the P. aeruginosa T3SS is highly regulated and under the direct transcriptional control of ExsA, a member of the AraC family of transcriptional activators (5). Environmental signals, including contact of P. aeruginosa with host cells and Ca 2ϩ -limiting growth conditions, induce ExsA-dependent transcription of the T3SS (6,7). Although the mechanism of induction by host cell contact is unclear, Ca 2ϩ depletion regulates T3SS gene transcription by inducing type III secretory activity (8). Transcription of T3SS genes is intimately coupled to type III secretory activity through a regulatory cascade that governs ExsA activity. This regulatory cascade consists of three interacting proteins (ExsD, ExsC, and ExsE). ExsD functions as an anti-activator by directly binding to and inhibiting ExsA-dependent transcription (8). ExsC functions as an anti-anti-activator by binding to and antagonizing ExsD activity (9). Finally, ExsE is an inhibitor of ExsC activity and a secreted substrate of the T3SS (10,11). Dasgupta et al. (9) have proposed the following model to account for transcriptional induction of T3SS genes by Ca 2ϩ depletion. Under Ca 2ϩ replete conditions, ExsE is retained in the cytoplasm because of the lack of type III secretory activity and forms a complex with ExsC. The sequestration of ExsC by ExsE allows for the binding of the anti-activator ExsD to ExsA, thereby blocking transcription. In response to Ca 2ϩ depletion, however, ExsE is secreted into the extracellular milieu. The corresponding decrease in intracellular ExsE levels releases ExsC, which then sequesters ExsD and thereby makes ExsA available to activate transcription of T3SS genes.
A common feature of proteins secreted by the T3SS is the requirement for chaperone activity (12). ExsC has biochemical characteristics of a type III-specific chaperone and is required for ExsE stability within the cytoplasm and for ExsE secretion (9,10). T3SS-specific chaperones fall into one of three classes based on substrate specificity. Class I, II, and III chaperones facilitate secretion of effectors, components of the transloca-tion channel, and components of the injectisome, respectively (13). Members of the Class I family are generally small in size (100 -150 amino acids), acidic, and usually encoded adjacent to their cognate effector gene. Based on these characteristics, ExsC is likely a member of the class I chaperone family. The solved three-dimensional structures of several class I chaperones reveals a dimeric binding configuration with each monomer possessing a conserved fold consisting of five ␤-strands and three ␣-helices. Although the precise role of the T3SS chaperones is unclear, proposed functions include targeting of substrates to the secretion machinery, maintaining substrates in a secretion competent conformation, preventing substrate aggregation prior to secretion, and establishing a secretion hierarchy among different substrates (12). In the case of ExsC, the primary role appears to be protection of ExsE from proteolysis in the cytoplasm (10).
In addition to the role in secretion, many type III-specific chaperones are also involved in transcriptional regulation of T3SS gene expression (14 -16). ExsC functions as an anti-antiactivator by sequestering the ExsD anti-activator (9). A recent study found that purified ExsC and ExsD individually form selfassociated oligomeric complexes as judged by gel filtration chromatography (17). In addition, incubation of purified ExsC with ExsD resulted in the formation of a large molecular weight complex. Neither the subunit composition of the self-associated ExsC or ExsD complexes nor the binding stoichiometry of the ExsC-ExsD complex, however, could be accurately determined by gel filtration.
In the present study the oligomeric states of ExsE, ExsC, ExsD, as well as the complexes of ExsC-ExsD and ExsC-ExsE were characterized by analytical ultracentrifugation. By this technique purified ExsE exists as monomers, ExsC forms homodimers, and ExsD self-associates into homotrimers. In addition, the subunit stoichiometry of the ExsC-ExsD and ExsC-ExsE complexes was resolved to 2:2 and 2:1, respectively. Thus, it appears that the trimeric ExsD dissociates in forming the heterotetrameric ExsC-ExsD complex, whereas the dimeric ExsC maintains during complex formation. The binding affinities for the ExsC-ExsD and ExsC-ExsE complexes were studied by isothermal calorimetry. The tighter interaction of the ExsC-ExsE (1 nM) complex when compared with the ExsC-ExsD (18 nM) complex is supportive of a model in which preferential binding of ExsC to ExsE results in inhibition of T3SS gene expression in the absence of type III secretory activity.

EXPERIMENTAL PROCEDURES
Protein Expression and Purification-The genes encoding ExsC and ExsD were amplified by PCR and individually cloned into pET25b (Novagen) with NdeI/XhoI restriction sites resulting in carboxyl-terminal histidine-tagged (LEIKRASQPELA-PEDPEDVEHHHHHH, 3088 Da) fusion proteins (termed ExsC His6 and ExsD His6 ). exsC with a Tev protease site before the carboxyl-terminal hexahistidine tag was also cloned into pET24a (Novagen) using NdeI/XhoI restriction sites (termed ExsC Tev-His6 ). For the ExsC-ExsD and ExsC-ExsE co-expression studies, the genes were cloned into the pCOLADuet-1 (Novagen) expression vector. The gene encoding ExsC was PCR-amplified and cloned as an NcoI/NotI restriction frag-ment into the corresponding sites of MCS1 in pCOLA-Duet-1. ExsC expressed from this vector (ExsC His6-short ) carries a carboxyl-terminal hexahistidine tag (LEHHHHHH, 1083 Da) encoded by the PCR primer. The genes encoding ExsD and ExsE were cloned into MSC2 as NdeI/XhoI restriction fragments.
To obtain the native form of ExsC, purified ExsC Tev-His6 was incubated with His 6 -tagged Tev protease at 4°C for 72 h. The mixture was passed through Ni 2ϩ chelating resin (Amersham Biosciences) to remove the digested His6-tag, the undigested ExsC Tev-His6 , and the Tev protease. The unbound material was loaded onto a Superdex 200 gel filtration column for further purification. The native form of ExsD was generated as follows. Purified ExsC His6-short -ExsD complex was incubated with an excess amount of purified ExsE C (defined below) at 4°C for 24 h, and the reaction mixture was loaded on a Ni 2ϩ cheating column to remove ExsC His6-short -ExsE and the residual ExsC His6-short -ExsD. The unbound fraction was further purified by gel filtration via a Superdex 200 column.
The gene encoding ExsE was PCR-amplified and cloned into pTWIN1 (New England Biolabs) as an NdeI/EcoRI restriction fragment resulting in an ExsE-chitin-binding fusion protein separated by an Mxe intein. Following cleavage of the intein, seven amino acid residues (EFLEGSS, 768 Da) remain at the carboxyl terminus of ExsE (designated ExsE C ). Expression of ExsE C was induced with 0.4 mM isopropyl 1-thio-␤-D-galactopyranoside for 4 h at 37°C. Cells were lysed in buffer A (20 mM Tris-Cl (pH 7.0), 500 mM NaCl, and 1 mM EDTA), and the cleared lysate was loaded onto a chitin column (New England Biolabs). The column was washed extensively with buffer A and then incubated with buffer B (buffer A plus 40 mM dithiothreitol (pH 8.5)) overnight at 4°C to remove the intein fusion partner from ExsE. ExsE C was further purified using a Superdex 75 column (Amersham Biosciences).
Isothermal Titration Calorimetry (ITC)-ITC experiments were carried at 25°C using a VP-ITC instrument (MicroCal). Protein samples were dialyzed overnight to a buffer containing 50 mM sodium phosphate (pH 7.5), 200 mM NaCl, 0.5 mM EDTA. Two experiments were performed for each study. For example, to study the interaction between ExsC His6 and ExsD His6 , 30 injections of 10 l of ExsC His6 (150 M) were titrated to 1.4 ml of ExsD His6 (15 M). A second experiment was done by reversing the injectant and the titratant: 30 injections of ExsD His6 (110 M) into 1.4 ml of ExsC His6 (10 M). Thermodynamic parameters of the binding process were derived using ORIGIN ITC software (OriginLab) by fitting the corrected binding isotherm to a single-site binding model.

RESULTS
Protein Expression and Purification-To characterize the oligomeric state and binding interactions of ExsC, ExsE, and ExsD, each of the proteins was individually expressed in E. coli. ExsC and ExsD were expressed as carboxyl-terminally tagged hexahistidine fusion proteins (ExsC His6 and ExsD His6 ) and sequentially purified by Ni 2ϩ affinity, ion exchange, and gel filtration chromatography. ExsE was expressed as a chitin-binding domain fusion separated by an intein and purified by chitin affinity chromatography. Following intein cleavage and removal of the chitin binding domain, ExsE contained an additional seven amino acids at the carboxyl terminus (ExsE C ). ExsE C was further purified by gel filtration chromatography (Fig. 1A). Each of the purified proteins was judged by SDS-PAGE to be Ͼ95% pure (Fig. 1B). Complexes of ExsC-ExsD and ExsC-ExsE were formed by incubating ExsD His6 with a 2-fold molar excess of purified ExsC His6 or ExsC His6 with a 2-fold molar excess of ExsE C , respectively, and purified by gel filtration chromatography (Fig. 1A). The presence of individual components within each complex was verified by SDS-PAGE (Fig. 1B).
Oligomeric State of ExsC, ExsD, and ExsE-The respective molecular mass sizes of monomeric ExsC His6 and ExsD His6 are 19.342 and 33.941 kDa as determined by mass spectrometry. A previous study found that purified ExsC His6 and ExsD His6 migrate as 79-and 128-kDa molecular species, respectively, as determined by gel filtration chromatography (Fig. 1A) (17). These findings indicated that purified ExsC and ExsD form selfassociated oligomeric complexes, potentially tetrameric in state. To further assess the molecular weight of the self-associated complexes, we employed AUC. Using this technique the molecular weights of the ExsC His6 and ExsD His6 self-associated complexes was determined to be 47.2 Ϯ 2.0 and 101.1 Ϯ 1.6 kDa, respectively. These data suggest that ExsC His6 exists as a dimer, whereas ExsD His6 exists as a trimer. We also carried out AUC experiments on purified untagged ExsD (native form) and derived its molecular weight to be 96.6 Ϯ 1.7 kDa, consistent with a homotrimeric state (predicted molecular mass ϭ 97.3 kDa). Thus, the presence of the carboxyl-terminal His tag (3.1 kDa) did not affect the aggregation state of ExsD.
The molecular weight of monomeric ExsE C is 9.422 kDa by mass spectrometry. Whereas ExsE C migrated as a 28-kDa molecular species by gel filtration (Fig. 1A), its molecular mass was found to be 9.5 Ϯ 0.5 kDa by AUC, indicating that ExsE C exists in a monomeric state. For all three proteins, the larger molecular mass as estimated by gel filtration compared with that by AUC is indicative of an asymmetric molecular shape (18).
Stability of the Oligomeric State of ExsC and ExsD-To examine monomer interactions within the ExsD complex, mixture of ExsD His6 and ExsD (1:1 molar ratio) as well as the individual ExsD His6 and ExsD were kept at 4°C for 24 h. The mixture was loaded onto a Ni 2ϩ chelating column. Following extensive washing, bound protein was eluted over an imidazole gradient, and fractions corresponding to the elution peaks were concentrated and analyzed by SDS-PAGE. Because trimeric ExsD does not bind to the Ni 2ϩ resin (see "Experimental Procedure"), it eluted as the unbound portion (Fig. 1C, lane 2). Nevertheless, a substantial amount of ExsD was also found in the fraction corresponding to the elution peak (Fig. 1C, lane 3). Presumably, this "bound" ExsD might be retained by the Ni 2ϩ resin in forms of (ExsD His6 ) n (ExsD) 3 Ϫ n (n ϭ 1 or 2). These data suggest that trimeric ExsD may dissociate into smaller oligomeric states (monomer and dimer) and reassemble to generate mixed trimeric species. A similar experiment was carried out using ExsC His6 and native ExsC. In contrast to ExsD, native ExsC was not present in the elution fractions from the Ni 2ϩ chelating column (data not shown). Taken together, it appears that the subunit assembly in trimeric ExsD is dynamic, whereas the monomer associations within dimeric ExsC are much stronger.

Molecular Masses of the ExsC-ExsD and ExsC-ExsE
Complexes-A previous study found that purified ExsC His6 and ExsD His6 readily formed a complex when co-incubated for 16 h at 4°C (17). The isolated ExsC His6 -ExsD His6 complex migrates with an apparent molecular mass of 197 kDa by gel filtration chromatography (Fig. 1A). Analysis of the same complex by AUC, however, yielded a much smaller molecular mass of 100.1 Ϯ 1.6 kDa ( Fig. 2A). The AUC data are indicative of a complex consisting of two molecules of both ExsC His6 and ExsD His6 (which has a predicted molecular mass of 106.6 kDa). This conclusion is further supported by previous ITC data, which predicted an equal molar binding ratio of ExsC and ExsD within the complex (n ϭ 0.87) (17). Similar to the experiments described above, incubation of purified ExsC His6 with a molar excess of ExsE C resulted in the formation of an ExsC His6 -ExsE C complex. The complex isolated by gel filtration chromatography migrated as a 96.1-kDa species (Fig. 1A), and the presence of both ExsC His6 and ExsE C were verified by SDS-PAGE (Fig. 1B). By AUC, however, the molecular mass of the ExsC His6 -ExsE C complex was determined to be 46.9 Ϯ 1.2 kDa (Fig. 2B). The ExsC-ExsE complex was also isolated by co-expressing ExsC His6-short and native ExsE in E. coli and subsequent purification by Ni 2ϩ affinity chromatography. This purified ExsC His6-short -ExsE complex migrates as a 63.4-kDa species by gel filtration, and its molecu-lar mass was determined by AUC to be 45.7 Ϯ 1.0 kDa. Both molecular masses derived from AUC are consistent with a complex containing dimeric ExsC bound to one ExsE monomer (predicted molecular mass values: 48.1 kDa for ExsC His6 -ExsE C and 43.7 kDa for ExsC His6-short -ExsE).
Binding Stoichiometry of ExsC-ExsE Complex-Formation of the ExsC-ExsE complex was further examined by mixing purified ExsC His6 and ExsE C in various molar ratios (1:0.5, 1:1, 1:2, and 1:4) and incubating them for 16 h at 4°C. At molar ratios greater than 1:0.5, an excess of ExsE C was detected by gel filtration chromatography, suggesting that ExsC His6 binds ExsE C in a 1:0.5 (or 2:1) molar ratio (Fig. 3A). This binding stoichiometry was further confirmed by ITC, where the binding ratio was determined to be 2.08 (Fig. 3B). These results, combined with the AUC data, are consistent with a complex consisting of one ExsC His6 dimer bound to one monomer of ExsE C .
Binding Affinity of ExsC for ExsD and ExsE-A previous study employing ITC found formation of the (ExsC His6 ) 2 ⅐(ExsD His6 ) 2 complex to be exothermic and energetically favorable, with a dissociation constant (K d ) of 18 nM and an enthalpy release (⌬H) of 7.6 kcal/mol (17). Using the same method, the K d and ⌬H of the (ExsC His6 ) 2 ⅐ExsE C complex were found to 1 nM and 31.5 kcal/mol, respectively (Fig. 3B), indicating that ExsC has a stronger binding affinity for ExsE when compared with ExsD.
Three experiments were conducted to confirm that ExsC has a high binding affinity for ExsE. In the first experiment, samples of ExsC His6 , ExsD His6 , and ExsE C were co-incubated at a molar ratio of 1:1:0.5, respectively, and incubated at 4°C for 16 h. Analysis of the mixture by gel filtration revealed a single peak (Fig. 4A). Because the elution profiles of the (ExsC His6 ) 2 ⅐ExsE C complex and the ExsD His6 self-associated complex demonstrate significant overlap (Fig. 1A), fractions corresponding to the leading shoulder (fraction 1), peak (fraction 2), and trailing shoulder (fractions 3-4) were subjected to SDS-PAGE and Coomassie staining. Fractions 1 and 2 consist primarily of ExsD His6 (which elutes at 71.4 ml), whereas fractions 3 and 4 consist of a mixture of ExsD His6 and the (ExsC His6 ) 2 ⅐ExsE C complex (which elutes at 74.7 ml). Notably, peaks corresponding to monomeric ExsE C (which elutes at 88.6 ml) and the (ExsC His6 ) 2 ⅐(ExsD His6 ) 2 complex (which elutes at 66.4 ml) were not detected. These data suggest that all of the ExsE C exists in a complex with ExsC His6 and that ExsC His6 does not bind to ExsD His6 in the presence of ExsE C .
Lastly, we tested whether a 2-fold molar excess of ExsD His6 could dissociate the (ExsC His6-short ) 2 ⅐ExsE complex by co-incubation overnight. The elution profile consisted of two peaks corresponding to (ExsD His6 ) 2 and the (ExsC His6-short ) 2 ⅐ExsE complex. The absence a peaks corresponding to free ExsE or the (ExsD His6 )⅐(ExsC His6-short ) 2 complex suggests that ExsD His6 cannot competitively remove ExsC His6-short from the (ExsC His6-short ) 2 ⅐ExsE complex under the conditions tested (Fig. 4C). Taken together, these experiments demonstrate that the binding affinity of ExsC is stronger for ExsE when compared with that of ExsD.

DISCUSSION
The transcriptional activity of ExsA is controlled through a regulatory cascade consisting of ExsD, ExsC, and ExsE. ExsC is a key player in this regulatory cascade, as it forms a complex with either ExsE or ExsD. Binding of ExsC to ExsE prevents transcription of T3SS genes, whereas binding to ExsD leads to activation of T3SS gene transcription. Here we report that the native states of purified ExsC, ExsD, and ExsE are, respectively, dimeric, trimeric, and monomeric. Our ITC data indicate that the stoichiometry within the ExsC-ExsD complex is 1:1, and the molecular weight of the complex derived from AUC is consistent with a heterotetrameric complex of ExsC 2 ⅐ExsD 2 . Similarly, both ITC and AUC data are in agreement that the binding ratio within the ExsC-ExsE complex is 2:1 and that the complex is a The chromatograms are normalized to the amount of ExsE C input. The first elution peak, centered at 74.6 ml, is the ExsC His6 -ExsE C complex as it resolves into ExsC His6 and ExsE C by SDS-PAGE; the second peak, centered at 88.8 ml, corresponds to isolated ExsE C as confirmed by SDS-PAGE. B, ITC data for the interaction of ExsC His6 and ExsE C . Shown here is the titration of ExsC His6 into ExsE C . The upper panel shows the heat exchange during each injection, and the lower panel shows the corresponding integrated enthalpies (after background correction).
heterotrimer of ExsC 2 ⅐ExsE. Affinity measurements and binding studies show that ExsC binds ExsE stronger than ExsD, supporting a model in which the preferential binding of ExsC to ExsE favors formation of the transcriptionally inactive ExsD-ExsA complex.
Although the carboxyl-terminal tags used for purification of ExsC, ExsD, and ExsE did not affect self-association or complex formation, they did substantially increase the hydrodynamic volume of the proteins (as reflected by the decrease in the elution volume during gel filtration chromatography). For example, the carboxyl-terminal tags in the (ExsC His6 ) 2 ⅐ExsE C complex add an additional 4.4 kDa of mass relative to the (ExsC His6-short ) 2 ⅐ExsE complex. This additional 4.4 kDa resulted in a drastic increase in the apparent molecular mass as estimated by gel filtration (63.4 kDa for the (ExsC His6-short ) 2 ⅐ExsE complex when compared with 96.1 kDa for the (ExsC His6 ) 2 ⅐ExsE C complex). Similarly, the additional 10.1 kDa of mass in the (ExsC His6 ) 2 ⅐(ExsD His6 ) 2 complex when compared with the (ExsC His6-short ) 2 ⅐ExsD 2 complex results in an increase of 35.7 kDa in the molecular mass estimated by gel filtration (197.5 kDa for (ExsC His6 ) 2 ⅐(ExsD His6 ) 2 compared with 161.8 kDa for the (ExsC His6-short ) 2 ⅐ExsD 2 complex). In deriving the apparent molecular mass by gel filtration, a compact globular shape is assumed, and globular proteins are used in the standard calibration. For macromolecules with asymmetric shapes, the apparent molecular mass values from gel filtration tend to be significantly overestimated (18). Thus, the drastic increase in the hydrodynamic volume due to the presence of the carboxyl-terminal tag indicates that these extensions are highly flexible and contribute significantly to the asymmetric shape of the fusion proteins. In contrast to the gel filtration method, the molecular mass values derived from equilibrium sedimentation analytical ultracentrifugation do not rely on model presumption (19); therefore, AUC is an accurate technique to use in assessing the association states of self-associated and in complex. Within the experimental errors, AUC-derived molecular mass values of (ExsC His6 ) 2 ⅐ExsE C and (ExsC His6-short ) 2 ⅐ExsE (46.9 Ϯ 1.2 kDa and 45.7 Ϯ 1.0 kDa, respectively) better reflect the predicted difference of 4.4 kDa and are in good agreements with their respective theoretical values.
Members of the class I family of type III secretion chaperones, including SycE, SigE, SicP, and CesT (13), are homodimers, and the largely hydrophobic dimeric interface is extensive (20,21), suggesting a strong dimer association. In line with this structural observation, subunit dissociation of the dimeric ExsC reported in this study is minimal. Another structural feature conserved in these secretion chaperones is the exposure of hydrophobic patches on the surface of the homodimer. These hydrophobic regions have been implicated in the interaction with the secreted substrate, and the hydrophobic interaction has been further shown to be the major contributing factor to the energy release resulting from complex formation (20,22). Our observation that a large enthalpy (⌬H ϭ Ϫ31.5 kcal/mol, from ITC experiments) is released accompanying the ExsC-ExsE complex formation supports the idea that the interaction between ExsC and ExsE is also mainly hydrophobic. Like other secretion chaperones (20), the dimeric struc- . Competitive binding of ExsC to ExsE and ExsD. A, ExsC His6 , ExsD His6 , and ExsE C were mixed at a 1:1:0.5 molar ratio and incubated at 4°C overnight. The resulting asymmetric elution peak is the overlap of ExsD His6 (71.4 ml, which has a broad profile) and ExsC His6 -ExsE C (74.7 ml). The compositions of representative fractions before (fraction 1) and after the peak position (fractions 2-4) were resolved by SDS-PAGE as shown in the insert. B, ExsE C was incubated with purified ExsC His6 -ExsD His6 (solid line) at a 2:1 molar ratio at 4°C overnight. The elution profiles of the ExsC His6 -ExsD His6 complex (centered at 66.4 ml, dashed line) and ExsE C (centered at 88.8 ml, dotted line) are included for reference. The compositions of representative fractions were examined by SDS-PAGE as shown in the insert. C, ExsD His6 was added into the purified ExsC His6-short -ExsE complex at a 2:1 molar ratio and incubated at 4°C overnight. No new molecular species was formed, given that the two elution peaks correspond to the input samples: ExsC His6-short -ExsE, 79.5 ml; ExsD His6 , 71.4 ml. ture of ExsC is very likely unaffected upon forming the complex with the secreted substrate ExsE.
The proposed functions of secretion chaperones include protecting the substrate from degradation, preventing the substrate from aggregation or premature interaction with downstream components, maintaining the substrate in a secretioncompetent state, and together with the substrate forming a targeting motif for secretion (23). In Salmonella typhimurium, the type III secretion chaperone SicP is required to stabilize the structure of its substrate SptP. In the absence of SicP mutant, the degradation rate of SptP is elevated (24). Similarly, ExsC has been shown to stabilize ExsE and increase the efficiency of ExsE secretion (10). Although highly soluble, ExsE does not adopt substantial secondary or tertiary structure as suggested by circular dichroism (CD) and nuclear magnetic resonance (NMR) studies. 3 The lack of structural elements suggests that ExsE exists as an assembly of various conformations, i.e. that its structure is highly dynamic. It is conceivable that only certain conformations are compatible for binding ExsC. For ExsE, loss of conformational freedom because of complex formation with ExsC may account for the overall entropy decrease measured by ITC (ExsC 2 ϩ ExsE 3 ExsC 2 ⅐ExsE, ⌬S ϭ Ϫ64.7 cal/mol). In comparison, the entropy change is favorable for ExsC⅐ExsD complex formation (ExsC 2 ϩ ExsD 3 3 ExsC 2 ⅐ExsD 2 , ⌬S ϭ ϩ11.3 cal/mol). The unfavorable entropic decrease in forming the ExsC-ExsE complex is overcome by the large amount of enthalpic release, as discussed above, such that the overall complex formation is thermodynamic favorable (⌬G Ͻ 0). Our data thus far support a model in which by assisting ExsE in adopting certain conformations, ExsC confers structural stability to ExsE and may possibly prime ExsE for secretion.
Besides interacting with ExsE as a secretion chaperone, ExsC also forms complex with the anti-activator ExsD to regulate T3SS gene transcription (9). Our observation of dynamic interaction between subunits in the trimeric ExsD supports a mechanism for ExsC-ExsD complex formation in which one monomer dissociates from the trimeric ExsD, whereas the other two monomers may be rearranged to accommodate the dimeric ExsC. The enthalpy release during this complex formation is modest (7.6 kcal/mol) compared with that of ExsC-ExsE formation, suggesting that the interaction in ExsC-ExsD complex is unlike that in ExsC-ExsE, which is largely hydrophobic. The difference in the nature of these interactions (as reflected by the difference in enthalpy change) suggests that ExsD and ExsE may bind to discrete regions of ExsC. Ultimately, determining the binding sites of ExsD and ExsE on ExsC, as well as the interplay between ExsC and ExsD or ExsC and ExsE, will await structural determination of the ExsD-ExsC and ExsC-ExsE complexes.