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J. Biol. Chem., Vol. 282, Issue 15, 11562-11572, April 13, 2007

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Structural and Functional Characterization of Partner Switching Regulating the Environmental Stress Response in Bacillus subtilis^*

Steven W. Hardwick, Jan Pané-Farré, Olivier Delumeau, Jon Marles-Wright, James W. Murray¹, Michael Hecker, and Richard J. Lewis²

From the Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom and Institute for Microbiology, Ernst-Moritz-Arndt-University, Greifswald D-17487, Germany

Received for publication, October 16, 2006 , and in revised form, February 6, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The general stress response of Bacillus subtilis and close relatives provides the cell with protection from a variety of stresses. The upstream component of the environmental stress signal transduction cascade is activated by the RsbT kinase that switches binding partners from a 25 S macromolecular complex, the stressosome, to the RsbU phosphatase. Once the RsbU phosphatase is activated by interacting with RsbT, the alternative sigma factor, ^B, directs transcription of the general stress regulon. Previously, we demonstrated that the N-terminal domain of RsbU mediates the binding of RsbT. We now describe residues in N-RsbU that are crucial to this interaction by experimentation both in vitro and in vivo. Furthermore, crystal structures of the N-RsbU mutants provide a molecular explanation for the loss of interaction. Finally, we also characterize mutants in RsbT that affect binding to both RsbU and a simplified, binary model of the stressosome and thus identify overlapping binding surfaces on the RsbT "switch."

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In Gram-positive bacteria such as Bacillus subtilis, and the human pathogens Listeria monocytogenes and Staphylococcus aureus, exposure to physical or energy stress results in the transcription of the general stress regulon (1, 2). In B. subtilis the general stress regulon comprises 150 genes (3, 4), the protein products of which confer multiple resistances onto the cell. The general stress regulon is controlled by the alternative sigma factor, ^B (5-9). The availability of ^B for forming transcriptionally competent complexes with RNA polymerase is regulated by a set of signal transduction proteins termed regulators of sigma B (Rsb). In unstressed cells ^B is held in a complex with its anti-sigma factor RsbW and is unable to direct RNA polymerase to the promoters of the general stress genes. RsbV is the anti-anti-sigma factor specific to RsbW, and the RsbV-driven release of ^B from the RsbW-^B complex allows ^B to displace the housekeeping sigma factor, ^A, from RNA polymerase holoenzyme (10). RsbW is also a protein kinase that is specific for RsbV, and phosphorylated RsbV (RsbV-P), which cannot form a complex with RsbW, accumulates in unstressed cells (11). Hence, it is the phosphorylation state of RsbV that determines the amount of freely available ^B for directing the transcription of the general stress regulon.

To activate ^B during stress, RsbV-P must be dephosphorylated. In B. subtilis this is achieved by one of two phosphatases depending on the type of stress that is being experienced by the cell. A decrease in ATP concentration leads to the activation of RsbP (12, 13), either via an interaction with RsbQ or as a direct consequence of the product of the RsbQ hydrolase activity on RsbP. Environmental stresses such as heat or salt shock or ethanol treatment (14-16) result instead in the activation of RsbU. Like RsbP, RsbU belongs to the type 2C serine/threonine phosphatase family, and RsbU is activated by a physical interaction with its partner protein, RsbT (16, 17). RsbT is a member of the GHKL family of kinases/ATPases (18), as is RsbW, but unlike RsbW, RsbT does not bind to factors. In an unstressed cell the RsbU activator, RsbT, is believed to be sequestered in a supramolecular complex termed the stressosome, composed of the RsbT substrates RsbR and RsbS (19) and the RsbR paralogues YojH, YqhA, and YkoB (19-23). Environmental stress results in the stimulation of the kinase activity of RsbT toward its substrates leading to the dissociation of RsbT from the stressosome to activate RsbU and consequently ^B (17).

The precise mechanism of the interaction and activation of RsbU by RsbT remains unclear. It is known that RsbU is not a substrate for the kinase activity of RsbT (24) and that the binding of RsbT to RsbU is entirely localized to the N-terminal domain of RsbU (17). The crystal structure of this domain, a dimeric arrangement of two four-helix bundles, has revealed the identities of surface-exposed residues that may be key in the binding of RsbU to RsbT (17). In some Gram-positive microbes, for instance the genus Staphylococcus, the regulation of RsbU differs from the B. subtilis paradigm (25) as these bacteria do not encode rsbT in their genomes. We report here an investigation into the interaction between RsbU and RsbT, and we demonstrate both in vitro and in vivo the precise region on the surface of RsbU crucial for the binding of RsbT. In complementary studies, we also identify residues in RsbT that are critical for the interaction with RsbU and the RsbR-RsbS complex. These data establish that RsbT utilizes a highly overlapped set of amino acids for interacting with RsbU and stressosomes. This mutually exclusive binding characteristic is ideal for the propagation of a signal transduction cascade.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
B. subtilis Strains and Growth Conditions—The bacterial strains and plasmids used in this study are listed in Table 1. Experiments to analyze the influence of various amino acid substitutions in RsbU on ^B activity were performed in derivatives of the B. subtilis wild type strain 168 (26). The B. subtilis 168 derivative BSM154 (27) that carries an internal deletion in rsbU and a ^B-dependent ctc::lacZ transcriptional reporter gene fusion was transformed with plasmids expressing variants of rsbU. The plasmids used to transform BSM154 were derived from the B. subtilis nonintegrative, self-replicating vector pDG148 (28) in which the different alleles of rsbU are under control of the isopropyl 1-thio--D-galactopyranoside (IPTG)³-regulated P_spac promoter.

To generate plasmids expressing wild type rsbU, and mutated versions thereof, the rsbU gene was amplified by PCR using primers RsbU^WT_f and RsbU^WT_r (supplemental Table 1a) with chromosomal DNA from B. subtilis 168 as template. The DNA fragment was digested with BsaI generating HindIII- and SalI-compatible ends and ligated into HindIII- and SalI-digested pDG148 to produce plasmid pJPF01. Four rsbU mutants were generated with pJPF01 as template with the respective primer pairs (supplemental Table 1a) using the Gene-Tailor mutagenesis system (Invitrogen). The resulting plasmids harbored mutated versions of rsbU with the following amino acid substitutions (in parentheses) in the RsbU protein: pJPF02 (E24K), pJPF03 (Y28I), pJPF05 (I74Q), and pJPF06 (I78K). For the construction of pJPF07 (S61A), plasmid pETRsbUS61A was used as a template for amplification and the PCR product cloned into pDG148 using the same restriction enzymes as for pJPF01. The correct sequence of all rsbU inserts was confirmed by sequencing prior to transformation into B. subtilis BSM154 generating strains BSGH01, BSGH02, BSGH03, BSGH05, BSGH06, and BSGH07, respectively. Selection of B. subtilis transformants was performed on LB agar plates supplemented with phleomycin at a concentration of 5 µgml^-1.

B. subtilis cells were routinely grown at 37 °C under vigorous shaking in a synthetic medium (29) supplemented with 0.05% (w/v) glucose as a carbon source and L-tryptophan (0.78 mM). The cultures for the stress experiments were inoculated from overnight cultures propagated in synthetic medium to an absorbance at 500 nm of 0.05. If appropriate, the overnight culture was supplemented with kanamycin (20 µgml^-1) and phleomycin (0.2 µgml^-1). Expression of the plasmid-encoded variants of rsbU was induced by the addition of IPTG to a final concentration of 1 mM. Addition of IPTG did not influence the growth of B. subtilis; a sample growth curve is shown for strain BSM151 (gray line, Fig. 2). To activate ^B via the RsbU-dependent environmental stress pathway, ethanol (4% v/v final concentration) was added to exponentially growing cells (A₅₀₀ of 0.3, t = 0). To determine the -galactosidase activity of the ctc::lacZ transcriptional reporter gene fusion, and hence infer ^B activity, 1-ml aliquots of cell cultures were harvested at various time points and assayed for -galactosidase enzyme activity as described previously (14, 30). To exclude the possibility that a lack of ^B activity in response to ethanol could be correlated to the absence of RsbU in these B. subtilis strains, the expression of RsbU in vivo was monitored by Western blotting cell extracts with an anti-RsbU antibody (31). The blot confirmed that there was no difference in the cellular levels of plasmid-expressed RsbU wild type and mutant proteins (data not shown).

Site-directed Mutagenesis—Plasmids containing N-rsbU, rsbU, and rsbT, the construction of which has been described previously (17, 32), served as templates for site-directed mutagenesis by the QuikChange procedure (Stratagene). Individual amino acid mutations were introduced into rsbU, N-rsbU, and rsbT by the addition of specific complementary oligonucleotide primers to the mutagenizing PCR mixture (supplemental Table 1b). After amplification by PCR the template DNA was removed by digestion with DpnI, and the remaining DNA was used to transform Escherichia coli DH5. Correct mutations were confirmed by DNA sequencing. In the N-rsbU construct, the following amino acids substitutions were made: E24K, Y28I, R35D, I74Q, I78K, M82Y, and A83S. These substitutions were selected because these amino acids are conserved in RsbU in those bacteria that code for RsbT and are not necessarily conserved in bacteria that do not encode for RsbT. These residues are also clustered together on the surface of the structure of N-RsbU and are therefore available for potential interactions with RsbT. Three other conserved amino acids in the immediate vicinity of the original target list were each converted to alanine in N-RsbU as follows: Tyr¹⁸, Gln³¹, and Ser³⁴. In addition, one surface-exposed residue that is remote from this region, Ser⁶¹, was also converted to alanine. Only mutations E24K, Y28I, Q31A, S61A, I74Q, and I78K were also mutated in full-length rsbU. The mutations that were made in RsbT were based on the work of Woodbury et al. (33), who had previously mutated polar and charged amino acids in RsbT to alanine in assessing the roles that these amino acids played in the induction of ^B activity. Glu¹², Asp¹⁴, Arg¹⁹, Gln²⁰, Asp³⁵, and Gln³⁶ were each mutated to alanine by QuikChange procedures in addition to a double alanine substitution of R23A/R24A and a triple alanine replacement of V107A/K108A/R109A.

Protein Expression and Purification—Plasmids directing overexpression of wild type and mutant RsbU and RsbT proteins were transformed into E. coli strain BL21 (DE3), and the resulting E. coli strains were grown in 1 liter of LB media to an absorbance at 600 nm of 0.4. Expression of recombinant proteins was induced by the addition of IPTG to the culture to a final concentration of 1 mM. Cells were harvested 3 h after induction by centrifugation for 20 min at 4000 rpm, and the cell pellets were resuspended in 20 ml of a lysis buffer containing 20 mM Tris·HCl, pH 8.0, 1 mM DTT and lysed by sonication. Soluble proteins were separated from cell debris by centrifugation at 15,000 rpm for 1 h. For RsbU and N-RsbU proteins, filtered supernatant was loaded onto a 10-ml Q-Sepharose anion exchange column (GE Healthcare) pre-equilibrated with lysis buffer. Bound proteins were eluted over a 50-ml linear gradient of lysis buffer supplemented with 1 M NaCl. The fractions containing RsbU proteins were identified by SDS-PAGE and concentrated for loading onto Superdex 75 (N-RsbU) or Superdex 200 (RsbU) gel filtration columns (GE Healthcare). Fractions containing RsbU proteins were concentrated to 5 mg ml^-1, supplemented with glycerol to a final concentration of 20%, and stored at -80 °C in 50-µl aliquots.

For the ease of purification, RsbT was overproduced as a glutathione S-transferase fusion with rsbT cloned immediately downstream of gst in pGEX6p2 (GE Healthcare). Filtered supernatants of GST-RsbT proteins were loaded onto a 10-ml glutathione-Sepharose column (GE Healthcare) equilibrated with buffer A (20 mM Tris·HCl, pH 8.0, 300 mM NaCl, 1 mM DTT, and 0.1 mM ATP). After extensive washing with buffer A, bound proteins were eluted in buffer A supplemented with 50 mM reduced glutathione. Glutathione S-transferase was cleaved from the RsbT fusion protein by incubation with 3C protease at 4 °C overnight. RsbT was purified further by gel filtration chromatography. Pure RsbT was concentrated to 1 mg ml^-1, supplemented with 20% glycerol, and stored at -80 °C in 50-µl aliquots.

Native Gel Electrophoresis—N-RsbU-RsbT binding experiments were performed in a buffer of 20 mM Tris·HCl, pH 8.0, 150 mM NaCl, and 0.1 mM ATP. RsbT and N-RsbU proteins were incubated at 30 °C for 10 min prior to analysis by nondenaturing PAGE and subsequent visualization by Coomassie Blue staining.

Measurement of Phosphatase Activity—RsbV-P, the phosphatase substrate of RsbU, was prepared as described previously (19). The dephosphorylation reactions were performed in a buffer of 50 mM Tris·HCl, pH 7.5, 50 mM KCl, 10 mM MgCl₂, 1 mM MnCl₂, 1 mM DTT, and 100 µM ATP. 30 µM RsbV-P was incubated with 1 µM RsbU and 1 µM RsbT proteins. The rate of dephosphorylation of RsbV-P was measured by removing 20-µl samples at various time intervals and by stopping the reaction by the addition of 10 µl of stop buffer (50% glycerol, 300 mM DTT, 200 mM EDTA and 0.1% bromphenol blue) and subsequent storage on ice until all samples were ready for analysis by native gel electrophoresis and Coomassie Blue staining. RsbV-P and RsbV bands are separated easily on a 12% acrylamide native gel (11). The gels were scanned, and intensities of the bands corresponding to the appearance of RsbV were measured with Scion Image software (Scion Corp.). The phosphatase activities of the RsbU mutants and wild type RsbU in the presence of mutants of RsbT were then compared with that of wild type RsbU in the presence of wild type RsbT.

Surface Plasmon Resonance—Surface plasmon resonance studies were carried out with the BIAcore 2000 system (BIAcore, Uppsala, Sweden). Mutant N-RsbU proteins were immobilized on CM5 gold surfaces by amine coupling using the protocol provided by the manufacturer. Wild type N-RsbU was immobilized on each chip as a positive control. Briefly, N-RsbU proteins were diluted into a buffer of 50 mM sodium acetate at pH 5.0 to a final concentration of 25 µgml^-1, and then injected onto the activated CM5 surface at 5 µl min^-1 in phosphatebuffered saline running buffer until a final resonance value of 500 response units was achieved. The sensor surface was then deactivated by blocking with 1 M ethanolamine. A range of concentrations of RsbT analyte in running buffer of 50 mM Tris·HCl, pH 7.5, 50 mM KCl, 10 mM MgCl₂, 1 mM MnCl₂, 1 mM DTT, and 100 µM ATP (the same buffer that is used in the RsbU phosphatase dephosphorylation reaction) was injected onto the biosensor chip for 1 min at a rate of 30 µl min^-1. Each experiment was replicated on at least two separate occasions. The surface plasmon resonance data for all N-RsbU proteins were analyzed using the BIAevaluation software (BIAcore, Uppsala, Sweden) and SigmaPlot (Systat). The data were plotted as a binding curve to which a 1:1 Langmuir binding interaction, describing 1:1 binding between an analyte A and a ligand B, A + B AB (34), could be modeled. The reliability of the fit of the model to the data is such that R² values of no less than 0.985 were obtained for all the individual data sets.

Crystallographic Methods—Crystals of mutants of N-RsbU suitable for x-ray diffraction were grown by hanging drop vapor diffusion. Crystals of 200 µm in diameter appeared in a few days for N-RsbU^E24K, N-RsbU^Y28I, and N-RsbU^I78K in a mother liquor of 10% PEG 20000, 100 mM MES, pH 6.5, conditions similar to that of wild type N-RsbU (35). Crystals of N-RsbU^I74Q were not obtained from our crystallization screening procedures. Single crystals were soaked in a cryoprotectant of mother liquor supplemented with 30% PEG 400 for 10 s before flashfreezing in liquid nitrogen in preparation for diffraction analysis.

Analysis of the diffraction data from crystals of N-RsbU^Y28I and N-RsbU^I78K revealed that they had retained the N-RsbU^WT space group and unit cell dimensions with one protein molecule in the asymmetric unit (Table 2). N-RsbU^E24K crystallized in space group C2 with unit cell dimensions a = 100.2 Å, b = 47.8 Å, c = 94.7 Å, = 104.4° with five copies of N-RsbU^E24K in the asymmetric unit. All diffraction data sets were integrated and scaled with MOSFLM and SCALA, and all subsequent analyses were performed with programs from the CCP4 suite (36). After rigid body refinement in REFMAC (37), inspection of electron density maps of N-RsbU^Y28I and N-RsbU^I78K confirmed the amino acid substitutions. Rounds of manual correction to the models with COOT (38) were interspersed with refinement in REFMAC until refinement converged (Table 2).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Native PAGE Analysis of N-RsbU Mutants—The interaction between RsbU and RsbT at a 1:2 molar ratio results in a 40-fold increase in RsbU phosphatase activity in vitro (17). This stimulation is an absolute requirement of the induction of ^B activity and therefore the response of B. subtilis to environmental stress. Mutation of residues in RsbU that result in a disruption of the interaction with RsbT should therefore inhibit ^B induction. We predicted previously that residues located in two contiguous patches on either side of the N-RsbU dimer interface may be involved in the interaction between N-RsbU and RsbT (17). Many of these amino acids are conserved across bacterial species that encode RsbT (e.g. members of the genera Bacillus and Listeria) but are not necessarily conserved in those bacteria that do not encode RsbT (e.g. the genus Staphylococcus), in which the regulation of RsbU differs from that in B. subtilis.To assess the influence of each residue on RsbT binding, 12 new constructs of N-RsbU were created by site-directed mutagenesis. Wherever possible the B. subtilis RsbU amino acid sequence was replaced with the equivalent from S. aureus. The purified N-RsbU mutant proteins were assessed for RsbT binding by a standard nondenaturing PAGE assay (11, 17).

View larger version (39K): [in this window] [in a new window]   FIGURE 1. RsbT interaction with N-RsbU analyzed by native PAGE. RsbT localizes in the well of a 12% acrylamide native gel (lane A); N-RsbUWT forms a distinct band toward the bottom of the well (lane B); a mixture of N-RsbUWT and RsbT results in a protein-protein complex being formed with a different migration rate to N-RsbU and RsbT (lane C). The remaining panels show the results of mixing wild type RsbT with mutant N-RsbU proteins.

Surface Plasmon Resonance Analysis—Surface plasmon resonance was used to quantify the binding of RsbT (as the analyte) to N-RsbU as the ligand. Consistent with the native gel binding assay, the N-RsbU mutants E24K, Y28I, I74Q, and I78K immobilized on the BIAcore chip did not respond to the RsbT-analyte regardless of the analyte concentration used. The remaining mutants of N-RsbU, including a mutation of S61A (Ser⁶¹ is surface-exposed but remote from the projected RsbT-binding site), all responded to RsbT similar to wild type N-RsbU in both the magnitude and the kinetics of response.

The SPR data and calculated K_d values between RsbT and all N-RsbU proteins are summarized in Table 3. For the wild type N-RsbU-RsbT interaction, a simple 1:1 Langmuir binding model yielded a K_d of 2.1 µM. The weak interaction is ideal for a transient signaling system dependent upon alternative proteinprotein interactions for signal transduction. Where dissociation constants could be calculated for the mutant N-RsbURsbT interactions, the K_d values differ only by approximately ±2-fold from the wild type. For instance, the mutation to alanine of Gln³¹ and Ser³⁴ both resulted in a slight reduction in the K_d value of the N-RsbU-RsbT interaction, to 0.98 and 0.91 µM respectively. These values indicate that the complex formed with the mutant N-RsbU proteins is marginally more stable than for wild type, but the 2-fold difference is unlikely to have a significant physiological effect.

View larger version (24K): [in this window] [in a new window]   FIGURE 2. Effect of mutations in RsbU on B activity in vivo. Various strains of B. subtilis (Table 1) were grown in the absence (a) and presence (b) of 1 mM IPTG. The B. subtilis strains in both panels are as follows: , BSM151 (wild type); , BSM154 (rsbU::kan); , BSGH00 (rsbU::kan, Pspac); , BSGH01 (rsbU::kan, Pspac::rsbU); +, BSGH02 (rsbU::kan, Pspac::rsbUE24K); x, BSGH03 (rsbU::kan, Pspac::rsbUY28I); *, BSGH05 (rsbU::kan, Pspac::rsbUI74Q); , BSGH06 (rsbU::kan, Pspac::rsbUI78K); and , BSGH07 (rsbU::kan, Pspac::rsbUS61A). The -galactosidase activities are the mean values of three independent experiments.

Only the B. subtilis strain carrying the lacZ reporter and a chromosomal copy of rsbU^WT displayed good -galactosidase, and hence ^B, activity in the absence of IPTG on the imposition of environmental stress (Fig. 2a). The -galactosidase activity peaked within 20 min of the addition of ethanol to the growth medium, and the transient stress response was, in effect, complete after a further 30 min. Other than this strain, only those strains carrying plasmid-encoded RsbU^WT or RsbU^S61A displayed good -galactosidase activity in the presence of IPTG (Fig. 2b). The magnitude and kinetics of the ^B response in these two strains are similar to that of the parental strain with a chromosomal copy of rsbU^WT. The strains carrying the RsbU mutations E24K, Y28I, I74Q, and I78K do not respond to environmental stress in vivo (Fig. 2b). Therefore, these mutations prevent the activation of ^B and highlight the important role that these amino acids play in RsbT-recruitment and RsbU activation. These observations are perfectly consistent with our findings in vitro where it is no longer possible to detect the interaction between RsbU and RsbT and the subsequent stimulation of the phosphatase activity of RsbU with these four mutants of RsbU.

Structural Analysis—To understand further the molecular effect of the mutations E24K, Y28I, I74Q, and I78K in the N-terminal domain of RsbU, we solved the structure of three of these four mutant N-RsbU proteins by x-ray crystallography. Residues Glu²⁴, Tyr²⁸, Ile⁷⁴, and Ile⁷⁸ of RsbU are located in a distinct region on the surface of N-RsbU, a region we will term the RsbT-binding pocket (Fig. 3a). N-RsbU^I74Q did not crystallize despite repeated attempts. The structures of N-RsbU^E24K (Fig. 3b), N-RsbU^Y28I (Fig. 3c), and N-RsbU^I78K (Fig. 3d) were solved by molecular replacement using the wild type N-RsbU as the molecular replacement search model, and refined against diffraction data extending to 1.85 Å for N-RsbU^E24K and 1.95 Å for N-RsbU^Y28I and N-RsbU^I78K. Details of the data collection and refinement statistics are given in Table 2.

View larger version (58K): [in this window] [in a new window]   FIGURE 3. Crystallographic analysis of N-RsbU mutants. a, molecular surface of the N-RsbU dimer is colored mostly blue. Surfaces colored red correspond to residues targeted in this study as potential RsbT-interacting partners and are also conserved across RsbT-encoding bacterial species; residues colored green are conserved irrespective of the presence of rsbT in the genome, and the orange line delineates the surface region of those amino acids that this study has revealed to mediate RsbT binding in RsbU. The yellow surface represents Ser61, away from the proposed RsbT-binding surface. Primes are used to indicate that the residue in question comes from the 2nd protomer in the N-RsbU dimer. b-d, OMIT electron density maps (each contoured at a level of 1 ) associated with the structures of the N-RsbU mutants E24K (b), Y28I (c), and I78K (d). The final models are shown as solid sticks with blue carbon atoms and with the wild type structure at the mutated position with yellow carbon atoms. For E24K, four of the five independent copies of N-RsbU in the asymmetric unit have very similar side chain conformations at position 24. In all instances, the rotamer of the introduced side chain is one of the two most frequently observed conformations in the COOT rotamer library (38). Ribbon schematics of N-RsbU (e) and N-RsbR (f) colored blue and green, respectively; highlighted in red are the positions of mutations in both proteins that disrupt the binding of RsbT, indicating that a common surface is used.

Within the N-RsbU^E24K asymmetric unit, the five individual monomers can be superimposed with r.m.s.d. in equivalent C positions ranging from 0.24 Å (molecules B and E) to 0.47 Å (molecules A and C). Similar values were obtained for the comparison of N-RsbU^E24K to N-RsbU^WT. The largest differences in structure, shifts of up to 1.5 Å in backbone position in four of the five independent molecules, occur in the immediate vicinity of residue 24. Glu²⁴ is found at the N-terminal end of -helix 2 in N-RsbU^WT. In the N-RsbU^E24K crystal form Lys²⁴ participates in crystal contacts, including a direct hydrogen bond between Lys²⁴ in one molecule and Glu⁷⁵ in another. Therefore, the re-modeling of the local structure to E24K may occur purely as a consequence of crystal contacts in the new crystal form.

The inability to detect an interaction between RsbU and RsbT with the mutations E24K, Y28I, and I78K in RsbU may be explained by comparing the structures of these mutant N-RsbU proteins to that of wild type (Fig. 3, b-d). The mutations E24K, Y28I, and I78K all lead to the mutant side chain conformations protruding into the RsbT-binding pocket, partially blocking the site for RsbT docking. Moreover, potential electrostatic interactions between the acidic residue Glu²⁴ of RsbU and basic residues of RsbT might be disrupted by mutation to the basic amino acid lysine. Similarly, hydrophobic contacts from Ile⁷⁸ of RsbU to RsbT may be affected by the introduction of lysine at this position. Finally, the removal of the tyrosyl hydroxyl group at Tyr²⁸ and the reduction in side chain volume from tyrosine to isoleucine may affect contacts between RsbU and RsbT.

View larger version (74K): [in this window] [in a new window]   FIGURE 4. Partner switching of RsbT. Wild type and mutants of RsbT binding to the RsbR-RsbS complex (a) and RsbU (b). RsbT proteins were each added to pre-purified RsbR-RsbS complex (a) and subjected to gel filtration. Shown in this panel are the first two equivalent fractions across the void volume of the same gel filtration column for each RsbT mutant RsbR/RsbS mixture. RsbT mutants E12A, Q20A and Q36A retain RsbRRsbS binding. The RsbT mutants were also assayed for N-RsbU binding (b) by native PAGE.

Partner Switching of RsbT—Although a complex between RsbU and RsbT can be visualized by native gels and quantified by SPR, the interaction between RsbU and RsbT, in our hands, has proved to be insufficiently tight for the crystallization of RsbU-RsbT complexes for structural studies. To understand how RsbT binds to both RsbU and the RsbR-RsbS complex, it has been necessary to devise an alternative strategy. In a previous study by Woodbury et al. (33), charged residues in RsbT were mutated to alanine, and the response to stress was examined in vivo. From this study several residues were proposed to play roles in the interaction of RsbT with RsbU because of a reduction in ^B activity on ethanol stress. However, these studies could not determine whether the RsbT variants were also affected in "stressosome" binding; RsbT interacts with both the stressosome and RsbU, before and during stress, respectively. To expand further on the study of Woodbury et al. (33), we have made mutations in rsbT that encompass all of the amino acids suggested by them that are involved in the interaction of RsbT with RsbU (except for the T₁₃ mutant that is likely affected in ATP binding), and we tested the ability of these RsbT mutants to bind to the RsbR-RsbS complex and RsbU.

Three of the constructs that were made, RsbT^D14A (derived from T₃), RsbT^D35A (derivative of T₇), and RsbT^{V107A/K108A/R109A} (T₁₆), expressed at very low levels in E. coli and thus were not studied further. Woodbury et al. (33) reported previously that the rsbT₃ mutant, from which D14A is derived, failed to activate ^B but that it also accumulated poorly in B. subtilis. Perhaps the D14A mutation in RsbT reduces protein stability. For the purpose of this study we focused on the remaining RsbT mutants, RsbT^E12A (T₃ derivative), RsbT^R19A (T₄ derivative), RsbT^Q20A (T₄ derivative), RsbT^R23A/N24A (T₅ derivative), and RsbT^Q36A (T₇ derivative), which were purified to electrophoretic homogeneity. Mutant RsbT proteins were mixed with the purified RsbR-RsbS complex before unbound RsbT was separated by gel filtration chromatography. Wild type RsbT binds to RsbR-RsbS and all three proteins co-elute in the void volume of the gel filtration column (Fig. 4a). Of the RsbT mutants, RsbT^E12A, RsbT^Q20A, and RsbT^Q36A each retained the ability to interact with the RsbR-RsbS binding partner and co-eluted with RsbR-RsbS similarly to wild type RsbT (Fig. 4a). On the other hand, RsbT^R19A and RsbT^R23A/N24A each eluted as monomeric proteins of 14 kDa and were well separated from the much larger stressosome. These proteins, which we presume are folded correctly, are severely impaired in RsbRRsbS binding and may therefore identify key amino acids in this interaction.

A native PAGE assay was also used to study the interaction of N-RsbU with the RsbT mutants. Unlike wild type RsbT which electrophoreses poorly into native gels, RsbT^R19A, RsbT^R23A/N24A, and RsbT^Q36A each migrate into the gel (Fig. 4b), and RsbT^E12A does not enter the gel at all. Perhaps these differences in electrophoretic behavior reflect a change in the overall charge of the protein. The native gel analysis indicated that no RsbT-NRsbU complexes could be detected on mutation to alanine of RsbT residues Glu¹², Arg¹⁹, and Arg²³-Asn²⁴. Only RsbT^Q20A and RsbT^Q36A retained the ability to form a complex with N-RsbU. These results indicate that the mutations in RsbT that appear to disrupt the interaction with RsbU are also sufficient to disrupt the interaction with the RsbR-RsbS complex. The sole exception is RsbT^E12A, which does not appear to bind to N-RsbU in the native gel assay but can still bind to RsbR-RsbS.

To confirm the roles of RsbT residues Glu¹², Arg¹⁹, and Arg²³-Asn²⁴ in the interaction with RsbU, we performed SPR analyses with N-RsbU^WT immobilized on the SPR chip and used the mutant RsbT proteins as analytes (Table 3). For RsbT^Q20A and RsbT^Q36A, the measured K_d values were 2.1 and 4.0 µM in comparison with the wild type value of 2.14 µM. No binding to N-RsbU could be detected for RsbT^R19A, RsbT^R23A/N24A, observations that are consistent with the results from the native gel assay. However, we could detect N-RsbU binding to RsbT^E12A, with a K_d of 3.8 µM, indicating that a complex is formed between RsbU and RsbT^E12A, albeit one that is 75% weaker than that of wild type.

View larger version (30K): [in this window] [in a new window]   FIGURE 5. Schematic of the environmental stress response. Pre-stress, the RsbT kinase is sequestered by the stressosome, represented here as a round disk with peripheral turrets. On the imposition of stress, the kinase activity of RsbT results in the phosphorylation of the stressosome, and RsbT is released to interact with RsbU and stimulate its phosphatase function toward RsbV-P. Dephosphorylated RsbV attacks the RsbW-B complex and liberates B to bind to the crab claw-shaped core RNA polymerase (cRNAP) to form the holoenzyme (hRNAP) that then transcribes the B regulon. To reset the switch, the RsbX phosphatase must dephosphorylate the phosphorylated stressosome, which can then re-capture RsbT, down-regulating RsbU phosphatase activity. The kinase function of RsbW then predominates, and RsbV is maintained in a phosphorylated form, and B is retained by RsbW. In the scheme, enzymatic activities are italicized, and partner switching modules linked by dashed arrows.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reversible phosphorylation of proteins is one of the most prevalent biological control mechanisms (42). The general stress response of B. subtilis and close relatives is underpinned by phosphoryl transfer and by transient protein-protein interactions. The interplay between the RNA polymerase sigma factor subunit, ^B, and its anti-sigma factor, RsbW, and anti-antisigma factor, RsbV, a phenomenon termed "partner switching," is regulated by phosphorylation and protein complex remodeling (Fig. 5). Here we report an analysis of the partner switching of the RsbW homologue, RsbT, with its alternative binding partners RsbU and a simplified model of the stressosome, the RsbR-RsbS complex. The experimental design utilized reflects the absence of any high resolution structural data on the complexes that RsbT forms with its binding partners.

Previously, we have determined that RsbT binding to RsbU is mediated by the N-terminal domain of RsbU (19). In this study we have identified and characterized key residues in RsbU that are required for the interaction with RsbT. These data complement those of Murray et al. (32) who identified three surface-exposed residues in N-RsbR that, in the context of the RsbRRsbS complex, also cannot detectably bind RsbT. The determination of the structure of N-RsbR revealed, quite unexpectedly, that N-RsbR and N-RsbU are structurally homologous (32). N-RsbU can be superimposed on (the core of) N-RsbR with an r.m.s.d. on equivalent C atoms of 3 Å. This structural homology conserves the functional ability for these protein domains to interact with RsbT. The three N-RsbR residues affected in RsbT binding, Glu⁶⁰, Lys⁸², and Glu¹²⁶ have spatial counterparts in N-RsbU; Leu²⁷, Glu⁴⁵, and Glu⁷⁵, respectively. Leu²⁷ and Glu⁷⁵ are each just a single amino acid away from residues Tyr²⁸ and Ile⁷⁴ that this study has revealed are central to the interaction of RsbU with RsbT (Fig. 3, e and f). Although the region that RsbT binds to in RsbR is structurally equivalent to that in RsbU, the physicochemical properties of the amino acids involved are quite different. The glutamic acids at positions 60 and 126 in RsbR have near-spatial equivalents in RsbU at 28 and 74, but the chemistries of tyrosine and isoleucine at these positions are not equivalent to glutamate. Therefore, only the global site and not the specific mode of interaction of RsbT to RsbU and RsbR are conserved. Perhaps RsbR^Glu60 and RsbU^Glu24 are functionally equivalent, each positioned so as to interact with either RsbT^Arg19 or RsbT^Arg23. These two arginines are extremely well conserved across RsbT orthologues in those organisms coding for the rsbRST gene cluster (43), underlining their functional significance.

From the data we present it is clear that mutation of residues in the RsbT binding region of RsbU diminishes or renders undetectable the RsbU binding to RsbT. For RsbU, it appears that the loss of RsbT binding ability is because of a combination of the "filling" of the RsbT binding pocket by amino acid side chains and the removal of possible charge-charge interactions between residues of RsbU and RsbT. The crystallographic studies reveal, in the three structures solved, that the overall fold of the RsbT binding domain in N-RsbU is barely changed by the mutagenesis. The structural studies are compatible with our biochemical data and with our in vivo data, where ^B activity cannot be induced in these mutants. Therefore, all our data, from quite disparate experimental procedures, are self-consistent and point to crucial roles for RsbU residues Glu²⁴, Tyr²⁸, Ile⁷⁴, and Ile⁷⁸ in the RsbU-RsbT interaction.

We have also investigated whether mutations in RsbT that disrupt the interaction with RsbU also disrupt the interaction with the RsbR-RsbS complex, a simplified model of the stressosome. In the original analysis by Woodbury et al. (33), the RsbT mutants Q20A and Q36A were both constructed as "double mutants," as R19A/Q20A and D35A/Q36A (called T₄ and T₇, respectively). From our results, the effect on ^B activation caused by T₄ is due solely to R19A, which we have shown here is defective in RsbR-RsbS and RsbU binding and RsbU activation (as is R23A/N24A, T₅), whereas Q20A behaves like wild type. For T₇, the effect on ^B activation is not because of Q36A, which also behaves as though a wild type, but more likely to D35A, which we have not been able to study. Taken together, our data indicate that RsbT utilizes overlapping surfaces to bind to its two alternative binding partners, perhaps explaining why no constitutively active mutant was obtained in the genetic analysis by Woodbury et al. (33). The behavior of RsbT^E12A is a little unusual; a complex with N-RsbU is not (easily) detectable by the qualitative native gel electrophoresis assay, yet the K_d value for this interaction by SPR is about double that of wild type. Furthermore, RsbT^E12A cannot stimulate RsbU phosphatase activity. Perhaps RsbT^Glu12 also contacts the C-terminal domain of RsbU (and/or the substrate, RsbV-P), which of course could not be measured with the isolated N-terminal domain of RsbU as the ligand.

The first description of partner switching was between the B. subtilis sporulation sigma factor, ^F and SpoIIAB, the antisigma, and SpoIIAA, the anti-anti-sigma factor (44, 45). This system is extremely well characterized (46). Structural analysis of the ^F-SpoIIAB and SpoIIAA-SpoIIAB complexes has revealed some features of this system that are distinct from the N-RsbU-RsbT and N-RsbR-RsbT interactions. For instance, ^F and SpoIIAA have no sequence homology, and this is mirrored by their three-dimensional structures, which are also discrete (47, 48). Although the sequences of the N-terminal domains of RsbU and RsbR are unalike, their three-dimensional structures are highly similar (Fig. 3, e and f). Moreover, our analysis indicates that RsbT utilizes a highly overlapped set of amino acids in the N-terminal domains of both RsbR and RsbU, albeit with different chemical characteristics, to form its alternative complexes.

By contrast, only one residue, Arg²⁰ in SpoIIAB, equivalent to Arg¹⁹ in RsbT, can contact either ^F or SpoIIAA (48). Other than for Arg²⁰, the ^F- and SpoIIAA-binding surfaces on SpoIIAB do not overlap (48). The binding footprint on SpoIIAB created by ^F permits the simultaneous binding of SpoIIAA to cause release of ^F because of the asymmetry of the SpoIIAB-^F complex, a dimer of SpoIIAB binds a monomer of ^F (49, 50). Therefore, one copy of SpoIIAB Arg²⁰ is always available for SpoIIAA to gain a "toehold" on SpoIIAB inducing release of ^F. The highly overlapped surfaces on RsbT for its binding partners raise the possibility that a different, and arguably simpler, mechanism is utilized by the monomeric RsbT to effect partner switching between stressosomes and RsbU. RsbT binds exclusively to one or the other of its partners, the identity of which would appear to depend purely on the phosphorylation state of the stressosome.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 2J6Y, 2J6Z, and 2J70) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the Biotechnology and Biological Sciences Research Council, the Wellcome Trust, and the University of Newcastle-upon-Tyne. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1a and 1b.

¹ Present address: Wolfson Laboratories, Dept. of Biological Sciences, Imperial College, London, London SW7 2AZ, UK.

² To whom correspondence should be addressed. Tel.: 44-191-222-5482; Fax: 44-191-222-7424; E-mail: R.Lewis{at}ncl.ac.uk.

³ The abbreviations used are: IPTG, isopropyl 1-thio--D-galactopyranoside; DTT, dithiothreitol; MES, 4-morpholineethanesulfonic acid; r.m.s.d., root mean square deviation.

	ACKNOWLEDGMENTS

 
We are grateful for access to synchrotron beamlines at the European Synchrotron Radiation Facility and the Synchrotron Radiation Source. We thank W. G. Haldenwang for generously providing strains and plasmids and W. G. Haldenwang and U. Völker for providing the anti-RsbU antibodies. We also thank R. Lightowlers for help and guidance with SPR data collection and analysis and to M. Quin and L. Graham for technical assistance.

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