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J. Biol. Chem., Vol. 282, Issue 9, 6136-6142, March 2, 2007

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

Zhida Zheng, Guozhou Chen, Shreyas Joshi, Evan D. Brutinel, Timothy L. Yahr, and Lingling Chen^¶1

From the Department of Biology and the ^¶Interdisciplinary Biochemistry Program, Indiana University, Bloomington, Indiana 47405 and the Department of Microbiology, University of Iowa, Iowa City, Iowa 52242

Received for publication, December 20, 2006

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 (K_d 1 nM) and ExsC-ExsD (K_d 18 nM) complexes supports a model in which ExsC preferentially binds cytoplasmic ExsE, resulting in the inhibition of T3SS gene transcription.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 secretion 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 Ca²⁺-limiting growth conditions, induce ExsA-dependent transcription of the T3SS (6, 7). Although the mechanism of induction by host cell contact is unclear, Ca²⁺ 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 Ca²⁺ depletion. Under Ca²⁺ 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 Ca²⁺ 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 translocation 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-anti-activator by sequestering the ExsD anti-activator (9). A recent study found that purified ExsC and ExsD individually form self-associated 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 (LEIKRASQPELAPEDPEDVEHHHHHH, 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 fragment 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.

Proteins were expressed in Escherichia coli BL21(DE3) codon-plus (Novagen) grown in LB medium at 37 °C to an optical density at 600 nm (A₆₀₀) of 0.6. The growth temperature was reduced to 23 °C before induction with 0.1 mM isopropyl 1-thio--D-galactopyranoside, and cell growth continued for 5–8 h. Cells were lysed at 4 °C in 50 mM sodium phosphate (pH 8.0), 300 mM NaCl, and 5 mM imidazole using a continuous flow microfluidizer (Microfluidics). The soluble fraction was loaded onto a Ni²⁺ chelating column (Amersham Biosciences), and bound protein was eluted over a linear imidazole gradient (5 mM to 500 mM) in 50 mM sodium phosphate (pH 8.0) and 300 mM NaCl. Protein fractions were combined, concentrated by Centriprep YM10 (Millipore), dialyzed against 50 mM imidazole (pH 7.0), 100 mM NaCl, 0.5 mM EDTA, and 1 mM -mercaptoethanol, and loaded onto a FastQ column (Amersham Biosciences). The protein was eluted over a linear NaCl gradient (100–1000 mM) in 50 mM imidazole (pH 7.0), 0.5 mM EDTA, and 1 mM -mercaptoethanol. Peak protein fractions were concentrated by Centriprep YM10 (Millipore), dialyzed against buffer (50 mM imidazole (pH 7.0), 200 mM NaCl, 0.5 mM EDTA, and 1 mM -mercaptoethanol), and loaded onto a Superdex 200 gel filtration column (Amersham Biosciences). The purified protein was assessed by SDS-PAGE to be at least 95% pure.

To obtain the native form of ExsC, purified ExsC_Tev-His6 was incubated with His₆-tagged Tev protease at 4 °C for 72 h. The mixture was passed through Ni²⁺ 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 Ni²⁺ 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).

Analytical Ultracentrifugation (AUC)—Equilibrium sedimentation experiments were carried out at 25 °C using a Beckman XL-A Ultracentrifuge with an AN-Ti-60 rotor. The buffer for all the AUC experiments contained 50 mM sodium phosphate (pH 7.0), 200 mM NaCl, and 0.5 mM EDTA. Data were collected at three protein concentrations and with different rotor speeds optimized according to the expected molecular weight: ExsC_His6 (0.75, 0.37, 0.19 mg/ml; 7900, 9500, 12,000, 14,000, 18,000 rpm); ExsD_His6 (0.2, 0.1, 0.05 mg/ml; 6500, 7800, 11,500 rpm); ExsE_C (1.2, 0.86, 0.51 mg/ml; 13,000, 16,000, 23,000 rpm); ExsC_His6-short-ExsE (0.75, 0.38, 0.19 mg/ml; 9000, 11,000, 16,000 rpm); ExsC_His6-ExsD_His6 (0.3, 0.15, 0.075 mg/ml; 6000, 7500, 10,500 rpm); ExsC_His6-ExsE_C (0.77, 0.38, 0.19 mg/ml; 8300, 10,000, 14,500 rpm). The scanning wavelength was 280 nm. The partial specific volume of each protein or protein complex was calculated from the amino acid sequence: ExsC_His6 (0.732 cm³ g^–1), ExsD_His6 (0.723 cm³ g^–1), ExsE_C (0.711 cm³ g^–1), ExsC_His6-ExsD_His6 (0.727 cm³ g^–1), ExsC_His6-short-ExsE (0.723 cm³ g^–1), and ExsC_His6-ExsEc (0.7279 cm³ g^–1).

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 µl of ExsC_His6 (150 µM) were titrated to 1.4 ml of ExsD_His6 (15 µM). A second experiment was done by reversing the injectant and the titratant: 30 injections of ExsD_His6 (110 µM) into 1.4 ml of ExsC_His6 (10 µM). 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 Ni²⁺ 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 >95% 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 self-associated 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 ± 2.0 and 101.1 ± 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 ± 1.7 kDa, consistent with a homotrimeric state (predicted molecular mass = 97.3 kDa). Thus, the presence of the carboxyl-terminal His tag (3.1 kDa) did not affect the aggregation state of ExsD.

View larger version (44K): [in this window] [in a new window]   FIGURE 1. A, gel filtration profiles of ExsEC (), ExsCHis6-ExsEC (), ExsCHis6 (dashed line), ExsDHis6 (dotted line), and ExsCHis6-ExsDHis6 (dashed-dotted line). Also shown are the elution volumes of protein calibration standards with known molecular mass sizes: 1, ferritin, 440 kDa; 2, catalase, 232 kDa; 3, aldolase, 158 kDa; 4, albumin, 67 kDa; 5, ovalbumin, 43 kDa; 6, chymotrypsinogen A, 25 kDa; 7, ribonuclease A, 13.7 kDa. B, Coomassie-stained SDS-polyacrylamide gel showing purified ExsDHis6 (lane 1), ExsCHis6-ExsDHis6 complex (lane 2), ExsCHis6 (lane 3), ExsCHis6-ExsEC complex (lane 4), and ExsEC (lane 5). Molecular mass standards are shown on the right-hand side of the gel. C, mixture of ExsDHis6 with untagged ExsD over the Ni2+ chelating column on an imidazole gradient. Lanes 1 and 4, control samples of ExsD and ExsDHis6, respectively, over 24 h; lane 2, the unbound fraction; lane 3, the fraction corresponding to the elution peak; lane 5, molecular mass standards.

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 Ni²⁺ 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 Ni²⁺ 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 Ni²⁺ resin in forms of (ExsD_His6)_n(ExsD)_{3 – n} (n = 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 Ni²⁺ 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 ± 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 = 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 ± 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 Ni²⁺ affinity chromatography. This purified ExsC_His6-short-ExsE complex migrates as a 63.4-kDa species by gel filtration, and its molecular mass was determined by AUC to be 45.7 ± 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).

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Sedimentation equilibrium analyses of the ExsCHis6-ExsDHis6 complex (A) and the ExsCHis6-ExsEC complex (B). The experimental conditions are described under "Experimental Procedures." Nine data sets were simultaneously fitted into a single species model; the data fitting is shown in the lower panels and the fitting residual in the upper panels.

Binding Affinity of ExsC for ExsD and ExsE—A previous study employing ITC found formation of the (ExsC_His6)₂·(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·ExsE_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.

View larger version (20K): [in this window] [in a new window]   FIGURE 3. A, binding ratio within the ExsC-ExsE complex. ExsCHis6 and ExsEC were incubated at various molar ratios (1:0.5, solid line; 1:1, dashed line; 1:2, dashed-dotted line; 1:4, dotted line) in 50 mM imidazole (pH 7.0), 200 mM NaCl, 0.5 mM EDTA, and 0.5 mM -mercaptoethanol, and the mixture was applied to a Superdex 200 gel filtration column. The chromatograms are normalized to the amount of ExsEC input. The first elution peak, centered at 74.6 ml, is the ExsCHis6-ExsEC complex as it resolves into ExsCHis6 and ExsEC by SDS-PAGE; the second peak, centered at 88.8 ml, corresponds to isolated ExsEC as confirmed by SDS-PAGE. B, ITC data for the interaction of ExsCHis6 and ExsEC. Shown here is the titration of ExsCHis6 into ExsEC. The upper panel shows the heat exchange during each injection, and the lower panel shows the corresponding integrated enthalpies (after background correction).

The second experiment addressed whether ExsE_C could disrupt the preformed (ExsC_His6)₂·(ExsD_His6)₂ complex. ExsE_C was incubated with the purified (ExsC_His6)₂·(ExsD_His6)₂ complex for 16 h at 4 °C, and the mixture was analyzed by gel filtration. Remarkably, peaks corresponding to the input materials, monomeric ExsE_C (88.6 ml) and the (ExsC_His6)₂·(ExsD_His6)₂ complex (66.4 ml), were greatly diminished (Fig. 4B). In place of the input materials was a composite elution profile with a peak centered at 72.2 ml, likely resulting from the overlapping elution profiles of (ExsC_His6)₂·ExsE_C and ExsD_His6. By SDS-PAGE, the leading shoulder (fraction 1) consisted mainly of ExsD_His6, whereas the trailing shoulder contained ExsC_His6 and ExsE_C (fraction 4–5) or the (ExsC_His6)₂·ExsE_C complex. These results suggest that the (ExsC_His6)₂·(ExsD_His6)₂ complex dissociates in the presence of ExsE_C, and that ExsE_C forms a complex with ExsC_His6.

Lastly, we tested whether a 2-fold molar excess of ExsD_His6 could dissociate the (ExsC_His6-short)₂·ExsE complex by co-incubation overnight. The elution profile consisted of two peaks corresponding to (ExsD_His6)₂ and the (ExsC_His6-short)₂·ExsE complex. The absence a peaks corresponding to free ExsE or the (ExsD_His6)·(ExsC_His6-short)₂ complex suggests that ExsD_His6 cannot competitively remove ExsC_His6-short from the (ExsC_His6-short)₂·ExsE 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 ExsC₂·ExsD₂. 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 heterotrimer of ExsC₂·ExsE. 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.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Competitive binding of ExsC to ExsE and ExsD. A, ExsCHis6, ExsDHis6, and ExsEC 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 ExsDHis6 (71.4 ml, which has a broad profile) and ExsCHis6-ExsEC (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, ExsEC was incubated with purified ExsCHis6-ExsDHis6 (solid line) at a 2:1 molar ratio at 4 °C overnight. The elution profiles of the ExsCHis6-ExsDHis6 complex (centered at 66.4 ml, dashed line) and ExsEC (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, ExsDHis6 was added into the purified ExsCHis6-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: ExsCHis6-short-ExsE, 79.5 ml; ExsDHis6, 71.4 ml.

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 = –31.5 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 structure 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 secretion-competent 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 (ExsC₂ + ExsE ExsC₂·ExsE, S =–64.7 cal/mol). In comparison, the entropy change is favorable for ExsC·ExsD complex formation (ExsC₂ + ExsD₃ ExsC₂·ExsD₂, S = +11.3 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 < 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.

	FOOTNOTES

 
^* This work was supported by grants from the Howard Hughes Medical Institute Biomedical Research Support Faculty Start-up Program (to T. L. Y.) and the University of Iowa W. M. Keck Microbial Communities and Cell Signaling Program (to T. L. Y.), National Institutes of Health Grants R01-AI055042 (to T. L. Y.) and R01-GM065260-01A1 (to L. C.), and National Science Foundation Grant MCB-0416447 (to L. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biology, 915 E. 3rd St., Indiana University, Bloomington, IN 47405. Tel.: 812-855-0491; Fax: 812-855-6082; E-mail: linchen{at}indiana.edu.

² The abbreviations used are: T3SS, type III secretion system; Tev, tobacco etch virus; AUC, analytical ultracentrifugation; ITC, isothermal titration calorimetry.

³ Z. Zheng, E. D. Brutinel, T. L. Yahr, and L. Chen, unpublished results.

	ACKNOWLEDGMENTS

 
We thank V. Fiscus, E. Daniel, and M. Afrane for assistance with the protein purification and T. Stone for AUC and ITC measurements.

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