text:
Structure and Functional Properties of Bacillus subtilis Endospore Biogenesis Factor StoA*S⃞

Bacillus subtilis StoA is an extracytoplasmic thiol-disulfide oxidoreductase (TDOR) important for the synthesis of the endospore peptidoglycan cortex protective layer. Here we demonstrate that StoA is membrane-associated in B. subtilis and report the crystal structure of the soluble protein lacking its membrane anchor. This showed that StoA adopts a thioredoxin-like fold with N-terminal and internal additions that are characteristic of extracytoplasmic TDORs. The CXXC active site of the crystallized protein was found to be in a mixture of oxidized and reduced states, illustrating that there is little conformational variation between redox states. The midpoint reduction potential was determined as -248 mV versus normal hydrogen electrode at pH 7 consistent with StoA fulfilling a reductive role in endospore biogenesis. pKa values of the active site cysteines, Cys-65 and Cys-68, were determined to be 5.5 and 7.8. Although Cys-68 is buried within the structure, both cysteines were found to be accessible to cysteine-specific alkylating reagents. In vivo studies of site-directed variants of StoA revealed that the active site cysteines are functionally important, as is Glu-71, which lies close to the active site and is conserved in many reducing extracytoplasmic TDORs. The structure and biophysical properties of StoA are very similar to those of ResA, a B. subtilis extracytoplasmic TDOR involved in cytochrome c maturation, raising important general questions about how these similar but non-redundant proteins achieve specificity. A detailed comparison of the two proteins demonstrates that relatively subtle differences, largely located around the active sites of the proteins, are sufficient to confer specificity.

Bacteria of the genera Bacillus and Clostridium can form endospores in response to nutrient starvation. The endospore, which is a dormant and very resistant state of the bacterium, can germinate back into a vegetative cell once nutrients become available again. Different layers help to protect the endospore: the dehydrated core, corresponding to the cytoplasm and containing the genome, is surrounded by a peptidoglycan layer, the cortex, which is required for extreme heat resistance. Outside the cortex, coat layers of mainly proteins protect the endospore against damaging chemicals and enzymes (1). The StoA protein (also known as SpoIVH and YkvV) of Bacillus subtilis is a predicted membrane-bound thiol-disulfide oxidoreductase (TDOR) 4 important for endospore cortex synthesis (2,3). Inactivation of the stoA gene results in spores deficient in the cortex layer that are much more sensitive than wild-type spores to heat, lysozyme, and chloroform treatment.
TDORs are proteins that catalyze the reduction of disulfide bonds and the oxidation of thiols. One pair of cysteine residues, often found in a -CXXC-motif, is present in the active site of TDORs, and although TDORs generally lack high overall sequence similarity, many of them share a common three-dimensional fold called the thioredoxin fold (4). Within the cell under normal circumstances, TDORs preferentially exhibit either a reducing or an oxidizing function as determined, at least in part, by the reduction potential of their disulfide/thiol active site. Their function is essential for the stabilization, folding, and activity of many proteins in bacterial cells, and they are involved in a wide range of processes, including cytochrome synthesis, cell motility, natural competence development, and toxin biosynthesis (5)(6)(7)(8). Known enzymes that function in bacterial cell wall peptidoglycan synthesis, e.g. transglycosidases and transpeptidases, do not depend on cysteine redox chemistry, and so an important role for StoA in cortex synthesis was unexpected. Studies of this protein can reveal hitherto unknown features of sporulation and peptidoglycan synthesis (9). From the primary sequence of StoA (see Fig. 1), it was predicted to have one transmembrane segment and a single membrane-extruded domain with a thioredoxin-like fold. It is therefore likely to function in the control of thiol disulfide chemistry of a substrate protein(s). In the absence of BdbD, which is an orthologue of Escherichia coli DsbA that catalyzes disulfide bond formation in proteins on the outside of the cytoplasmic membrane, StoA is no longer important for endospore cortex synthesis, indicating that it functions to specifically reduce disulfide bonds on the outside of the cytoplasmic membrane (2). It was also proposed that the protein most likely operates in the intermembrane space of the developing forespore where the cortex is synthesized. The substrate protein(s) of StoA with a function in cortex synthesis has not yet been identified, but the CcdA protein most likely functions in transmembrane transport of reducing equivalents from thioredoxin in the cytoplasm to StoA in the forespore intermembrane space (9,10).
StoA is similar in primary sequence to ResA (see Fig. 1), which is a ditopic membrane-bound TDOR that functions specifically as a reductase in cytochrome c maturation in B. subtilis and which has been well characterized (11). The soluble, membrane-extruded part of ResA has a typical thioredoxin fold augmented by an additional ␤-hairpin at the N-terminal end and a /␤ insertion between strand ␤2 and helix ␣2 of the classic thioredoxin fold (12). The cysteine residues of the ResA active site exhibit unusually high pK a values (both above 8) (13), yielding very low reactivity of the cysteine residues at neutral pH. In contrast to most other TDORs that have been characterized, both thiols of the ResA active site are reactive to thiol-modifying reagents. In addition, a glutamate (Glu-80) in the vicinity of the active site has been shown to play a key role in controlling the reactivity of the enzyme (13,14). StoA is not involved in cytochrome c synthesis and cannot functionally replace ResA. Likewise ResA cannot replace StoA in sporulation (2). Thus, StoA and ResA have distinctly different substrate specificities. Given their primary sequence similarity, it is of key importance to understand the basis of their specificity differences.
Here we report the isolation of the soluble domain of B. subtilis StoA (sStoA) and subsequent three-dimensional structure determination together with the biophysical characterization of the protein, including reduction potential and pK a values of the active site cysteines. Furthermore mutant variants of StoA with amino acid substitutions in the active site region have been studied in vitro and in vivo in B. subtilis. Common and discriminating features of StoA and ResA are discussed in the context of the distinctly different substrate specificities exhibited by these similar proteins.

EXPERIMENTAL PROCEDURES
Bacterial Strains and Growth of Bacteria-Strains used in this work are presented in Table 1. E. coli strains were grown in lysogeny broth or on lysogeny broth plates, and B. subtilis strains were grown in nutrient sporulation medium with phosphate (15) with appropriate antibiotics added as follows: ampicillin, 100 g/ml; kanamycin, 10 g/ml (for B. subtilis) or 50 g/ml (for E. coli); and chloramphenicol, 3 or 4 g/ml (for B. subtilis) and 15 g/ml (for E. coli). Liquid cultures were grown in baffled E-flasks on a rotary shaker (200 rpm) at 37°C.
Construction of Plasmids Encoding sStoA-For production of sStoA plasmid pLMC19 was constructed by amplifying part of the stoA gene using oligonucleotides LE051 and LE052 (supplemental Table S1), Phusion polymerase (Finnzymes), and B. subtilis 1A1 chromosomal DNA as template. The PCR product was cloned into pCR-Blunt-II-TOPO (Invitrogen). The insert was cut out from the plasmid using PstI and HindIII and ligated into pBADmyc-HisC cut with the same enzymes resulting in plasmid pLMC19. The cloned stoA fragment was verified by DNA sequence analysis.
For production of a thrombin-cleavable GST-sStoA fusion protein, a fragment of the stoA gene encoding residues 21-165 of StoA was first amplified by PCR as above using oligonucleo- For the construction of plasmids encoding mutant StoA variants, pLLE83 was digested by HindIII and BamHI, and the 2-kb fragment containing stoA was cloned in pBluescript SK(ϩ), resulting in pLYM001. Site-directed mutagenesis was carried out with the QuikChange II kit and protocol (Stratagene) using pLYM001 and primers LY003-LY008 to generate plasmids pLYM009 (C65A), pLYM004 (C68A), and pLYM006 (E71Q), respectively, which were verified by sequencing. The HindIII/ BamHI fragment of each of these three plasmids was subsequently cloned into pDG148, generating, respectively, pLYM015, pLYM013, and pLYM012, which were used for expression of full-length mutant stoA genes in B. subtilis. Plasmids encoding GST-sStoA fusion protein with C65A or C68A in sStoA were obtained by first amplifying stoA as above using primers LY001 and LY002 and pLYM009 or pLYM004 plasmid DNA, respectively, as template. PCR products were then cloned into pGEX4T1 as described above for the wild-type variant, generating plasmids pLYM031 and pLYM032, which were verified by sequencing.
Purification of sStoA-Non-tagged sStoA, which was utilized in initial crystallization trials and to generate a StoA antiserum, was purified from E. coli TOP10/pLMC19 as described in the supplemental data. For the production of GST-sStoA fusion protein, E. coli BL21/pLYM028 was grown in 1-liter portions in 5-liter E-flasks. At A 600 ϭ 0.6 -0.8 expression was induced by addition of 1 mM isopropyl ␤-D-thiogalactoside (final concentration). After incubation for 5 h, cells were collected by centrifugation, washed in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.3), and stored as pellets at Ϫ20°C until required. The cell pellet from 1 liter of culture was suspended in 20 ml of ice-cold PBS containing 1 mM DTT and lysed by passage (three times) through a French pressure cell at 18,000 p.s.i. The lysate was centrifuged at 48,000 ϫ g for 40 min at 4°C, and the supernatant was centrifuged at 100,000 ϫ g for 1 h at 4°C. The final supernatant was mixed with 2 ml of 50% slurry of glutathione-Sepharose 4B (GE Healthcare), and the GST-sStoA fusion protein was purified according to the resin manufacturer's instructions. Affinity-purified GST-sStoA fusion protein was cleaved by 50 units of thrombin (GE Healthcare) at room temperature for 5 h and then loaded onto a Sephacryl S-100 HR gel filtration column. Protein was eluted using 20 mM Tris-HCl, pH 8.0, containing 100 mM NaCl and 1 mM DTT. Fractions containing sStoA were identified using SDS-PAGE and Western blot with StoA antiserum, pooled, and concentrated. The N-terminal amino acid residue sequence of the purified protein was verified by Edman degradation (see Fig.  1). Cysteine variants of sStoA were produced in E. coli BL21 containing pLYM031 or pLYM032 and purified as described for wild-type sStoA.
For the production of selenomethionine-labeled sStoA, E. coli B834(DE3)/pLYM028 was grown overnight in 10 ml of SelenoMet TM medium (AthenaES) supplemented with 50 g/ml methionine. The overnight culture was used to inoculate 1 liter of SelenoMet medium containing 50 g/l methionine to an A 600 of 0.1, and the culture was grown until A 600 was ϳ0.8. Cells were harvested by centrifugation for 10 min at 10,000 ϫ g at 4°C, and the pellet was resuspended in 1 liter of non-supplemented SelenoMet medium and incubated for 2 h. Selenomethionine was then added to a final concentration of 50 g/ml, and the culture was incubated for a further 30 min when production of GST-sStoA was induced by the addition of 1 mM isopropyl ␤-D-thiogalactoside (final concentration). Four hours after induction the culture was harvested by centrifugation for 10 min at 10,000 ϫ g at 4°C. The cells were washed in cold PBS and stored as a pellet at Ϫ20°C. Selenomethionine-labeled sStoA was purified as described above. MALDI mass spectrometry confirmed that selenomethionine incorporation was close to 100%.
Crystallization and Structure Determination of sStoA-sStoA was crystallized using the sitting drop vapor diffusion method. A 2-l sitting drop was formed by mixing equal volumes of protein solution (12 mg/ml sStoA in 25 mM Mops, pH 7.0) and crystallization reagent (27% (w/v) PEG 2000, 0.2 M ammonium acetate, 100 mM sodium acetate, pH 4.8) over an 800-l reservoir of the reagent alone. Crystals grew over a period of 1-2 days and were cryoprotected in a solution of 30% (w/v) PEG 2000, 100 mM sodium acetate, pH 4.8, 20% (v/v) ethylene glycol before flash freezing in liquid nitrogen. X-ray data sets for native and selenomethionine-labeled sStoA were collected on beam line ID23-1 of the European Synchrotron Radiation Facility (Grenoble, France). Structure determination utilized programs of the CCP4 (16) and PHENIX (17) software suites. Diffraction patterns were indexed and integrated with MOSFLM (18) and scaled with SCALA (19). Selenium sites were identified using a combination of automated methods implemented in PHENIX.HYSS and manual inspection of anomalous difference maps produced with FFT. An initial electron density map was obtained using SAD phases calculated with MLPHARE and subsequent density modification with DM. A key factor in producing an interpretable electron density map was the identification of the correct non-crystallographic symmetry relating each sStoA molecule in the asymmetric unit. Non-crystallographic symmetry averaging in the phase improvement procedure benefited from the use of a predefined protein mask derived from a monomer of ResA (12). Further phase improvement was obtained by cross-crystal averaging with a second SAD-phased selenomethionine data set composed of merged data from two individual sStoA crystals. Manual model building was conducted in COOT, and initial phased refinement of the model was conducted with REFMAC (20). Further refinement of the model (against a single selenomethionine data set with a "low" twin fraction (␣ ϭ 0.36)) utilized PHENIX.REFINE, which was essential for proper refinement of the twinned data. The final model of sStoA is composed of seven ordered protein chains and 99 water molecules. A small amount of residual density located at a coordinate of (42.34, 64.52, 19.53) may indicate the presence of an additional StoA monomer of low occupancy and high mobility that is insufficiently well resolved to enable further model building. The coordinates and structure factors have been deposited at the Protein Data Bank with accession code 3ERW.
Reduction Potential Determination-sStoA (0.2 M) in 50 mM potassium phosphate, pH 7, was added to 5 mM oxidized DTT in the same buffer to obtain the fully oxidized protein. The protein was subsequently titrated with reduced DTT in the same buffer, allowing 10 min for the protein to equilibrate to each new potential. The transition from oxidized to reduced protein was monitored by the increase in tryptophan fluorescence emission at 344 nm (excitation at 280 nm) measured at 25°C using a PerkinElmer Life Sciences LS-55 fluorescence spectrometer with 10-nm excitation and emission slits. Intensity was corrected for dilution effects. From the data at 344 nm, midpoint reduction potentials were determined as described previously (11,21); further details are given in the supplemental data.
pH Stability and Cysteine pK a Measurements-Reduced wild-type and variant sStoA protein stocks were prepared in 10 mM Mops, pH 7, 2 mM TCEP (Pierce) and subsequently diluted (30-fold to a final protein concentration of 0.15 M) with PCTC (potassium phosphate, sodium citrate, Tris, and Ches, all at 50 mM) buffer (pre-prepared at the appropriate pH) and equilibrated in a sealed cuvette for 1 h before measurement of tryptophan fluorescence spectra as above. For pK a measurements, reduced protein solutions (wild-type sStoA and single cysteine variants) were prepared in 10 mM Mops, pH 7, with 2 mM TCEP as reductant. Reaction with 6-bromoacetyl-2-dimethylaminonaphthalene was carried out under pseudo-first order conditions, and pK a values were determined as described previously (13). Further details are also given in the supplemental data.
Modification of sStoA with MAL-PEG-Wild-type sStoA and C65A and C68A variant proteins in 20 mM Tris-HCl, 100 mM NaCl, pH 8.0 were treated with 1 mM TCEP at room temperature for 30 min. Excess TCEP was removed using a YM10 column (Millipore), and each reduced sStoA sample (10 g) was incubated with 0.1 mM or 1 mM monomethyl polyethylene glycol 5000 2-maleimidoethyl ether (MAL-PEG) (Ն90%; Fluka) at room temperature for 30 min. The samples were then applied directly onto an SDS-polyacrylamide gel.
Antisera and Western Blot Analysis-Non-tagged sStoA was used to immunize rabbits (custom polyclonal antibody production service; MedProbe, Oslo, Norway). For Western blot analysis, proteins were separated by SDS-PAGE using the Schägger and von Jagow (22) system and subsequently electroblotted to a polyvinylidene difluoride membrane (Millipore) by wet blot using 20 mM Tris, 150 mM glycine, 20% (v/v) methanol. Transfer conditions were 30 V, 0.1 A overnight at 4°C. The membrane was blocked using 5% (w/v) nonfat dry milk in 0.1% (v/v) Tween 20 in PBS. StoA antiserum was used at 1500-fold dilution. Bound primary antibodies were detected using horseradish peroxidase-linked anti-rabbit antiserum from donkey (GE Healthcare) diluted 3000-fold. Immunodetection was carried out by chemiluminescence using SuperSignal West Pico substrate (Pierce) and an Eastman Kodak Co. image station.
Preparation of Cell-free Extracts from B. subtilis Strains-Samples of 200 ml were taken from a 1.5-liter culture at time points spanning from 1 h before entry into postexponential (T ϭ Ϫ1) to 5 h into stationary growth phase (T ϭ 5). Cells were harvested by centrifugation; immediately washed in 50 mM potassium phosphate, pH 8.0; and frozen as a pellet at Ϫ20°C. When required, cell pellets were thawed and suspended in 0.8 ml of phosphate buffer containing 0.7 mg/ml lysozyme, 25 g/ml DNase, 25 g/ml RNase, 4 mM MgSO 4 , and Complete protease inhibitor (Roche Applied Science; one tablet/50 ml of buffer)). After incubation at 37°C for 45 min, an aliquot was frozen for subsequent analysis of total cell lysate. The remaining main part of the lysate was centrifuged at 48,000 ϫ g for 60 min at 4°C: the supernatant was used for the analysis of the soluble cell fraction, whereas the pellet, after washing in phosphate buffer, was suspended in 0.3 ml of the buffer and used for the analysis of the membrane fraction.
Other Methods-Chromosomal DNA from B. subtilis was isolated according to Marmur (23). E. coli was transformed by electroporation (24). Plasmid DNA was isolated using Quantum miniprep (Bio-Rad) or by CsCl density gradient centrifugation. SDS-PAGE was carried out using the NuPAGE system (Invitrogen) or Schägger and von Jagow (22) system. Protein concentrations were determined by measuring the absorbance at 280 nm using an extinction coefficient of 15,460 Ϯ 100 M Ϫ1 cm Ϫ1 determined as described previously (25) or using the BCA reagent (Bio-Rad) with bovine albumin as reference.
N-terminal sequencing was carried out by Edman degradation (Protein Analysis Center, Karolinska Institutet, Sweden) on proteins separated by SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane as described for Western blotting. The membrane was stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 in 2% (v/v) acetic acid, 45% (v/v) methanol. Mass spectrometry analysis was performed using an UltraFlex-MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Coventry, UK) on samples prepared by mixing sStoA in a 1:1 ratio with a saturated solution of sinapinic acid matrix in 30% acetonitrile, 0.05% trifluoroacetic acid. 0.5 l of this combined mixture was spotted onto a polished stainless steel target and allowed to crystallize prior to analysis. The spectrometer was externally calibrated using a two-point linear calibration through the singly and doubly charged ions of trypsinogen.
The molecular mass of sStoA was determined using high performance liquid chromatography with an Ultraspherogel SEC 3000 column (Beckman). 1.2 nmol of sStoA in 10 l was applied to the column equilibrated with 20 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM DTT and eluted in the same buffer at a flow rate of 1 ml min Ϫ1 . Mass was calculated based on a calibration curve obtained using catalase (230 kDa), bovine serum albumin (67 kDa), ovalbumin (46 kDa), carboanhydrase (26 kDa), myoglobin (17.2 kDa), and horse heart cytochrome c (12.3 kDa).

RESULTS AND DISCUSSION
StoA Is a Membrane-bound Protein-From its amino acid sequence (Fig. 1), StoA was predicted to be a ditopic membrane-bound protein with an N-terminal ϳ30-residue segment that anchors the ϳ135-residue TDOR domain to the membrane. As shown previously by the use of a StoA-PhoA (alkaline phosphatase) fusion protein in E. coli cells, the transmembrane segment of StoA functions as a signal sequence to direct translocation and to membrane anchor the C-terminal TDOR domain (2). Experiments in B. subtilis indicated that the function of StoA is not dependent on the membrane anchor (3), and prediction programs suggested that the N-terminal segment might be cleaved off after the TDOR domain has been translocated across the membrane. To establish whether StoA is a membrane-bound protein, B. subtilis strain 1A1 was grown in nutrient sporulation medium with phosphate for sporulation, and samples taken at different time points during growth were analyzed for StoA by using Western blot with polyclonal antiserum directed against the TDOR domain of StoA. No StoA antigen could be detected in cell-free extracts, although BdbD, a protein very similar to StoA, was readily detected in all sam-ples (not shown). To facilitate detection of StoA by increasing the level of the protein in cells, plasmid pLLE83 containing stoA under control of the pSpac promoter was used. StoA was found in the membrane fraction of 1A1/pLLE83 cells from early stationary phase cultures but not in late stationary phase cultures (Fig. 2). The results indicate that StoA is membrane-associated but is present in very low amounts and is degraded or trapped in maturing endospores so that it is not detectable by the Western blot procedure used.
In Vivo Functional Analysis of Active Site Variants of StoA-To establish that the function of StoA in endospore biogenesis is dependent on the cysteine residues of the protein, B. subtilis strain LUL20 in which the stoA gene is inactivated and strain LUL30 in which stoA is deleted from the chromosome were used (2). These two strains containing a plasmid encoding wild-type StoA (pLLE83), C65A StoA (pLYM015), C68A StoA (pLYM013), or empty vector (pDG148) were grown for sporulation and tested for production of heat-resistant cells. Western blot analysis showed that StoA proteins were present in membranes of strains containing pLYM015 and pLYM013 (see supplemental Fig. S1). Compared with wild type, the presence of either StoA variant resulted in a ϳ100-fold reduction in sporulation efficiency (Table 2). Cells completely lacking StoA, however, showed more than a 1000-fold reduction in sporulation efficiency indicating some residual activity of StoA even when one of the two cysteine residues is missing.
The High Resolution Structure of the Soluble Domain of StoA in a Mixture of Oxidized and Reduced States-sStoA (residues 22-165) was produced in E. coli and purified. Gel filtration analysis indicated a molecular mass of 17 Ϯ 3 kDa (data not shown) consistent with the protein being monomeric in solution. The crystal structure of sStoA was solved using the selenomethionine SAD method of phase determination. The crystals used in structure determination belong to space group P3 1 and contained seven molecules of sStoA per asymmetric unit (see supplemental Fig.  S2). Although structure determination was hampered by the fact that sStoA crystals were merohedrally twinned, it was nonetheless possible to refine the structure to acceptable R work and R free values using twin refinement against x-ray data obtained from a single sStoA crystal with a twin fraction (␣) of 0.36. Data collection and refinement statistics for the sStoA structure are given in Table 3.
Overall the structure of each sStoA monomer (Fig. 3A) can be described as a modified thioredoxin-like fold that is highly reminiscent of B. subtilis ResA (12), CcmG (from E. coli and Bradyrhizobium japonicum) (26,27), and Mycobacterium tuberculosis DsbE (28), which are all extracytoplasmic TDORs (see Fig. 3B). Like these proteins, the classical thioredoxin-like motif of StoA is embellished by a central  ␣␤ insertion and an N-terminal ␤-hairpin (in addition to the transmembrane helix predicted from primary sequence analysis). Unlike other extracytoplasmic TDORs, StoA also possesses a short insertion of residues between strand ␤4 and helix ␣2 that forms an ordered loop at the surface of the protein.

TABLE 2 Efficiency of B. subtilis strains in producing heat-resistant cells after growth for 2 days at 37°C in nutrient sporulation medium with phosphate supplemented with 1 mM isopropyl ␤-D-thiogalactoside
Presented are typical results obtained from at least two independent experiments with each strain, including analysis of two sister clones. B. subtilis LUL20 and LUL30 are StoA-deficient, and 1A1 is the parental strain (Table 1).   In the structure determined here, the active site cysteines of sStoA appear as a mixture of oxidized and reduced redox states (in each monomer). Crystallization of sStoA utilized solely the oxidized form of the protein, and thus it is likely that partial reduction of the disulfide bond was induced by photoreduction in the x-ray beam. The electron density associated with the partially broken disulfide is shown in Fig. 4A along with separated models of the oxidized and reduced conformations shown in Fig. 4, B and C, respectively.

Strain
The oxidized (disulfide-bonded) conformation of the active site dominates the electron density and is best described as adopting a classical right-handed hook conformation with a 3 angle of 73.5 Ϯ 1.8°formed between the two cysteine residues. The sulfur-to-sulfur bond distance for the oxidized conformation is 2.06 Å, whereas in the reduced conformation an average sulfur-to-sulfur distance of 3.4 Å separates the cysteine residues. This distance is considerably shorter than that observed for the structure of ResA that has an exceptionally long sulfurto-sulfur distance of 4.5 Å in the reduced state (12). Partial reduction of the disulfide does not seem to cause significant rearrangement of the local protein structure, and thus, unlike ResA, there is no evidence for any redox-linked conformational changes due to reduction of the cysteines in StoA.
In almost all known natural thioredoxin-like proteins, at least one of the two residues that intervene between the active site cysteines residues (i.e. within the CXXC motif) is a proline. In StoA, both of these intervening residues are proline. Pro-66 and Pro-67 both adopt the trans conformations and have backbone angles that are consistent with an ␣-helical conformation. Like all other thioredoxin-like TDORs, the CXXC motif of StoA is found at the N terminus of a reasonably long ␣-helix (␣1 in StoA), and the macrodipole arising from this helix is often invoked as a primary cause of the lowered pK a values associated with the more N-terminal cysteine residue of the CXXC motif in most TDORs (29). The presence of proline residues at the cap of this active site helix in StoA is therefore likely to have important consequences for the distribution of the electrostatic field near the cysteines as proline does not possess a standard peptide group. Furthermore the limited conformational freedom of proline (in comparison with other residues) may be an important factor in maintaining rigidity of the CPPC motif and may be one of the reasons that the reduced form of sStoA is so similar to its oxidized form.
A further proline residue (Pro-135), which is in the cis conformation and is conserved in all thioredoxin-like proteins, is found in van der Waals contact with two buried polar residues, a histidine and a glutamate, which are located immediately behind the second cysteine of the active site motif. His-59 is located on strand ␤3, whereas Glu-71 arises from helix ␣1 directly opposite. A buried water molecule is observed in the space between these two residues within hydrogen-bonding distance of the Cys-68 sulfur (see Fig. 3C).
The arrangement of these two buried polar residues and the intervening water molecule (in StoA) is very similar to that observed in ResA where the glutamate is conserved (Glu-80) and an asparagine (Asn-68) residue takes an equivalent position to that of His-59. Substitution of Glu-80 in ResA has been shown previously to have a significant effect on the active site properties of the enzyme; for example, the pK a values of both active site cysteines were significantly lowered in an E80Q variant (13), and a B. subtilis strain containing E80Q ResA was also severely impaired in its ability to mature c-type cytochromes (14). Thus, it may be that these buried polar residues are also important in StoA function. To analyze the functional role of residue Glu-71 in StoA, the StoA-deficient B. subtilis strains LUL20 and LUL30 containing plasmid pLYM012 encoding E71Q StoA were studied. The presence of the variant StoA protein in membranes was confirmed by Western blot (supplemental Fig. S1), and the efficiency in production of heat-resistant endospores was found to be 10 -150-fold (depending on strain) lower in these strains compared with wild-type controls ( Table 2). This indicates that Glu-71 is functionally important in StoA.
StoA Is a Low Potential TDOR-The reduction potential of the active site cysteines of sStoA was measured using the difference in tryptophan fluorescence intensity of oxidized and reduced sStoA to follow oxidation state as a function of reduc-  tion potential (see Fig. 5). The data fitted well to the Nernst equation, giving a midpoint reduction potential of Ϫ248 Ϯ 2 mV versus normal hydrogen electrode at pH 7 with n ϭ 2.18 Ϯ 0.16, as expected for a two-electron reduction process. This value is similar to that measured for B. subtilis ResA (Ϫ256 mV at pH 7) (21) and E. coli thioredoxin (Ϫ270 mV at pH 7) (30,31) and is entirely consistent with the structural similarity between these proteins and a role for StoA in the reduction of (as yet unidentified) proteins involved in endospore cortex synthesis (2,9).
Both Active Site Cysteines Can Be Modified by Alkylating Agents-The solvent accessibility of the active site cysteines of wild-type sStoA and two variants, C65A and C68A sStoA, was investigated using MAL-PEG, a high molecular mass, cysteinespecific alkylating agent. sStoA proteins were incubated with either 0, 0.1, or 1 mM MAL-PEG as described under "Experimental Procedures" and analyzed by SDS-PAGE. Samples with exposed thiols are able to react with the MAL-PEG to form covalent complexes of significantly greater molecular mass relative to non-alkylated samples and thus are retarded during subsequent migration in the electrophoretic gel (see Fig. 6). Unmodified wild-type, C65A, and C68A sStoA variants migrated with an apparent molecular mass of ϳ18 kDa, which is in reasonable agreement with the actual mass (16.4 kDa). In the presence of MAL-PEG, both single cysteine variants reacted to give a single species with a significantly lower mobility. Protein molecular mass standards cannot be used to judge the mass of MAL-PEG-modified proteins, but the significant retardation of sStoA variants is consistent with the alkylation of a single cysteine residue in each. In contrast, wild-type sStoA gave rise to two bands when incubated with 0.1 mM Mal-PEG. The lower (faster running) band corresponded to the singly modified single cysteine variants, and we conclude that under these conditions sStoA is present as a mixture of singly and doubly modified molecules. This was confirmed by incubating sStoA with a higher (1 mM) concentration of MAL-PEG that resulted in the observation of only the larger, slower running band corresponding to the doubly modified protein. In addition to these major bands, other much fainter bands were observed on the gel; these most likely arise from a small degree of heterogeneity in the size of the PEG adducts in the MAL-PEG reagent and therefore do not represent additional (non-cysteine) alkylation events or protein heterogeneity. Certainly no protein heterogeneity was observed in any of the untreated sStoA samples.
From the structure it is apparent that the more N-terminal of the two active site cysteines is exposed to the solvent, whereas the other is not. Solvent-accessible surface area calculations (using a solvent probe of 1.2 Å) on the structure of reduced sStoA showed that the Cys-65 sulfur has an accessible surface area of 5.43 Å 2 , whereas the sulfur atom of Cys-68 is inaccessible from the bulk solvent. A similar arrangement exists in all structurally characterized thioredoxin-like proteins, and in virtually all of them, the second, buried cysteine thiol does not react with modifying reagents in solution (32,33). This is not the case for B. subtilis ResA in which we showed previously that both cysteines are readily modified by alkylating reagents (13). To our knowledge, ResA and now StoA are the only examples where this behavior has been demonstrated. For StoA, one possibility is that alkylation of the solvent-exposed Cys-65 might cause a structural rearrangement that allows subsequent modification of Cys-68. Alternatively the reduced protein in solution may undergo dynamic motion that would allow occasional access to the sulfur of Cys-68.
pK a Values of StoA Active Site Cysteines-The pH stability profiles of the wild-type and single cysteine variant sStoA proteins were first determined to verify the range of values over which the acid-base properties of each protein could be investigated. The intrinsic tryptophan fluorescence was measured as a function of pH for each protein under reducing conditions. Significant changes in the character of the tryptophan fluorescence emission spectrum, resulting from unfolding of the proteins, were observed at extremes of pH. Both the emission wavelength maxima and fluorescence intensity maxima were affected by pH-induced unfolding. The former has the advantage of being independent of protein concentration and was thus used preferentially in monitoring pH stability (see Fig. 7). The data showed that wild-type sStoA is stable between pH 3.5 and 9.3, whereas C65A and C68A sStoA variants are stable between 4.4 and 9.6 and between 3.7 and 9.3, respectively.
The acid-base properties of the active site cysteines of wildtype sStoA and the C65A and C68A variants were investigated by measuring rates of reaction with the fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene as described under "Experimental Procedures" (see Fig. 8A). The fluorescence is sensitive to the environment of the modified cysteine with emission occurring in the range of ϳ440 -550 nm, depending on the solvent exposure of the modified cysteine (34). Here the emission maxima for Cys-65 and Cys-68 were 510 nm, indicating that the fluorescent group of both modified residues is located in a relatively solvent-exposed position. For the single cysteine variants of sStoA, data fitted well to an equation describing a single protonation/deprotonation event, giving pK a values of 7.0 Ϯ 0.1 and 7.1 Ϯ 0.1 for Cys-65 and Cys-68, respectively (see Fig. 8, C and D). For the wild-type protein in which both cysteine residues are intact, the data fitted well to two independent protonation/deprotonation events, giving pK a values of 5.5 Ϯ 0.4 and 7.8 Ϯ 0.2 (Fig. 8B). We tentatively ascribe the first transition to Cys-65 and the latter to Cys-68. Both of these values are lower than the typical value of ϳ8.5-9.0 observed for cysteine, and the data are consistent with the reactivity of both residues toward alkylating reagents (which react with the deprotonated form only). A low pK a value is normally observed for the N-terminal active site cysteine of thioredoxinlike proteins, which in some cases exhibit pK a values as low as 3.5 (35). However, the pK a of the second cysteine is normally estimated to be Ͼ9 (32,36,37). In this respect, StoA is similar to ResA in that the C-terminal cysteinyl has a pK a value low enough to be measurable in the stable pH range of the protein.
This large separation of pK a values is consistent with the close proximity of the two thiol groups, indicating that the ionization of one significantly influences that of the other. The wide separation of active site thiol pK a values appears to be a general feature of TDORs that act with low specificity (33,38). For sStoA, we also observed an interdependence of the cysteine acid-base properties: the sStoA single cysteine variants have pK a values that are very similar, but in the wild-type protein they differ by more than 2 pH units. This suggests that in the wild-type protein Cys-65 and Cys-68 have a significant effect on one another such that the presence of both cysteines causes the pK a of the N-terminal cysteine to drop, whereas that of C-ter-minal cysteine rises (relative to the respective single cysteine variants). Such interdependence of pK a values was not observed for ResA for which respective cysteines in single cysteine variants showed acid-base properties similar to those for the wildtype protein. The stronger interdependence of the cysteine pK a values in StoA may well be linked to the significantly shorter sulfur-to-sulfur distance observed in reduced StoA (ϳ3.4 Å) compared with that of ResA (ϳ4.5 Å) (12).
Specificity Determinants of StoA and ResA-Here we have demonstrated that StoA and ResA have many features in common. The three-dimensional structure, redox properties, and acid-base properties of active site cysteine residues are similar in these proteins. Furthermore in vivo, the two proteins are believed to interact with the same integral membrane protein, CcdA, which functions to supply electrons from thioredoxin (TrxA) in the cytoplasm to the extracytoplasmic compartment (10, 39 -41); thus, structural/physical features important for this interaction are expected to be shared by StoA and ResA. Despite the similarities, the two proteins do not exhibit any functional redundancy (2). So how do StoA and ResA achieve specificity for their particular substrates? To try to answer this question, it is important to identify regions of the proteins that do show differences.
First, the two proteins differ in the active site sequence motif: CPPC in StoA and CEPC in ResA. Recently we reported the effects of altering the dipeptide intervening sequence on the properties of ResA, and this included a ResA CPPC variant (21). Significant effects were observed: the redox potential increased by ϳ25 mV, and the pK a values of the two cysteines decreased by 1.8 and 1.6 pH units, respectively. These findings are consistent with the midpoint reduction potential and pK a values measured here for StoA and those previously reported for ResA (13). Alteration of the dipeptide sequence was also shown to impair the in vivo activity of ResA (21). Beyond their effect on the biophysical properties of the active site cysteines (i.e. redox potential and pK a values), the intervening two residues may also be important for interaction with potential substrates. The close proximity of these residues to the active site cysteines and the fact that both are exposed on the surface of the protein make it highly likely that they contact partner proteins, at least transiently, and thus affect specificity of interaction.
Second, the protein surfaces close to the active site are subtly different in StoA and ResA. The structures of oxidized and reduced ResA previously revealed redox-linked conformational changes, the most significant of which was the opening up of a hydrophobic cavity close to the active site upon reduction (12). Despite the conservation or conservative substitution of several of the residues that line the ResA cavity, reduction of StoA does not lead to the opening up of an equivalent cavity (see Fig. 9). The lack of a cavity in StoA may be the result of the substitution of Thr-159 (in ResA) with Pro-153 in StoA; Thr-159 undergoes one of the biggest conformational movements upon formation of the hydrophobic cavity in ResA, and thus its replacement by a proline (Pro-153) in StoA might well restrict conformational change in this region of the protein. Alternatively the lack of a cavity in StoA might be linked to the much smaller conformational change in the CXXC motif itself, which is likely to be the driving force for the larger conformational changes around the active site motif in ResA.
It has been demonstrated that Glu-80 of ResA plays a key role in vivo (14), and in vitro studies showed that it is important for the elevated pK a values of the active site cysteines and that it is capable of hydrogen bonding to amino acid side chain residues bound in the cavity (13,21). This led us to propose that Glu-80 is important for the binding of apocytochrome substrates to ResA (12)(13)(14)42). This residue is conserved in StoA (Glu-71), and we have shown here that it is also functionally important in StoA (Table 2). Sequence alignments showed that it is conserved in many extracytoplasmic TDORs that are proposed to have a reductive function (13). The data presented here suggest that it fulfils a similar role in ResA and StoA and also in other TDORs. This could be in controlling the acid-base properties of the active site cysteines or through direct participation in the reduction mechanism. Therefore, it is highly unlikely that this glutamate is itself an important determinant for specificity. It remains a possibility that, in ResA, it interacts directly with substrates, but this would be an additional role facilitated by the formation of the hydrophobic cavity upon reduction of the protein. Interaction of Glu-71 with substrate is not favored in StoA because the residue remains buried in both oxidation states.
Third, with the exception of the aforementioned hydrophobic cavity in (reduced) ResA, the only major difference in the electrostatic surfaces of each protein is in helix ␣3 (StoA numbering), which is positively charged in StoA and negatively charged in ResA (Fig. 9). However, this helix is quite distant from the active site, and it seems unlikely that this feature is responsible for differences in substrate recognition by ResA and StoA.
The final major difference is the presence in StoA of an extended loop between strand ␤4 and helix ␣2. In the primary sequence alignment (Fig. 1), this can be clearly seen as an apparent insertion/deletion of several residues that are present in the StoA but not in ResA. The sequence alignment also shows that there is little similarity between the two proteins in this region. The structure shows that the extended ␤4-␣2 loop (composed of Ser-97, Glu-98, Gln-99, and Asn-100) lies close to the active site of StoA (Fig. 9) and represents the most significant difference in the surface shape of StoA compared with ResA and is, therefore, likely to be important for the differential substrate selectivity/specificity of these proteins. For example, this loop could be involved in specific binding interactions with StoA  substrate(s) or could serve to sterically hinder interactions with non-substrate molecules.
Concluding Remarks-The structural, biochemical, and in vivo characterization of B. subtilis StoA reported here provides new knowledge about this unprecedented endospore biogenesis factor whose physiological function is not completely understood (9). Furthermore the data reveal that this low potential extracytoplasmic TDOR is remarkably similar to ResA, another well characterized extracytoplasmic TDOR from the same organism that is required for cytochrome c maturation. It is thought that both proteins interact with the same integral membrane protein, CcdA, which supplies them with electrons from the cytoplasm. The high structural similarity of ResA and StoA is no doubt connected with their shared need to interact with this protein. Despite the large extent of their similarity (in both sequence and structure), the proteins cannot functionally substitute for one another in vivo. Bacteria usually contain several thioredoxin-like proteins, soluble in the cytoplasm as well as membrane-bound. B. subtilis contains at least 10 such proteins, and none of these are essential for growth, indicating a narrow substrate specificity for each protein (43). The results of this work raise important general questions about how TDORs achieve substrate specificity: ResA can recognize at least four different apo-c-type cytochrome polypeptides as substrates, whereas StoA recognizes a different but as yet unknown substrate(s) critical for endospore cortex biosynthesis. From the structures of StoA and ResA, we have identified four principal structural differences between the two proteins that we believe provide the basis of substrate specificity/selectivity. The work demonstrates that protein-substrate specificity/selectivity can apparently be achieved through remarkably subtle variations in amino acid sequence and three-dimensional structure.


extfieldsofstudy: Biology Medicine

text:
HTS-Compatible β-Lactamase Transcriptional Reporter Gene Assay for Interrogating the Heat Shock Response Pathway

Moderate environmental and physiological stressors are known to initiate protective heat shock response (HSR) leading to cell survival. HSR is largely mediated by the activation of heat shock factor (HSF), resulting in increased heat shock protein expression. Dysregulation of the HSR signaling has been associated with various diseases including cancer, inflammation and neurodegenerative disorders. Compounds that can modulate HSR have been pursued for the treatment of these diseases. To facilitate the discovery of HSR modulators, we developed a high-throughput amenable betalactamase transcriptional reporter gene assay for monitoring the function of HSF. HeLa cells were engineered to express the beta-lactamase reporter under the control of HSF response elements (HSE) present in the HSP70 gene promoter. The HSE-beta lactamase (HSE-bla) reporter gene assay was validated by using HSF-specific siRNAs and known small molecule modulators. Taking the advantage of fluorescence resonance energy transfer (FRET)-based cell permeable betalactamase substrate, this assay can be miniaturized into 1536-well format. Our results demonstrate that the assay is robust and can be applied to high-throughput screening (HTS) for modulators of HSR.

INTRODUCTION
Environmental and physiological stressors such as elevated temperature, chemical toxicants and oxidative stress are known to induce heat shock response (HSR) leading to the increased expression of molecular chaperones such as heat shock proteins (HSPs) and other cytoprotective proteins [1]. HSR and the induction of HSPs are mediated by a family of transcription factors called heat shock factors (HSFs), which bind to the heat shock elements (HSEs) present in the promoter regions of HSP genes [1]. Among the three human HSFs, HSF-1, -2, and -4, HSF1 is the best characterized and essential for HSR [2]. Under normal conditions HSF1 exists as an inert monomer held in a repressed state, presumably by interaction with HSP90 and HSP70 [1,3]. Upon exposure to stress, the rising level of unfolded proteins results in the dissociation and derepression of HSF1. HSF1 then trimerizes, undergoes post-translational modifications, and accumulates in the nucleus where it activates HSP gene expression ( Fig.  1) [3].
Aberrations in HSR have been associated with various human diseases, such as neurodegenerative disorder, inflammation and cancer [4]. Small molecule modulators of HSR including those directly targeting HSP chaperones have been pursued for the treatment of these diseases [4]. For example, HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) is currently in phase II clinical trial in patients with various cancers including melanoma and breast cancer [4]. Importantly, inhibition of HSP90 by 17-AAG also derepressses HSF1 and induces HSR [5]. Besides inhibitors of HSP90, proteasome inhibitors, amino acid ana-logues, and molecules such as arachidonic acid and prostaglandins that induce inflammatory responses have been reported to induce HSR [1,4]. Such inducers of HSR may prove beneficial to counteracting the protein misfolding and aggregation associated with various neurodegenerative disorders.
On the other hand, since tumor cells often express high levels of HSPs to survive the stressful environment in solid tumors, inhibitors of the HSR could be beneficial for cancer patients. Several inhibitors of the HSR have been reported. Quercetin (3,3',4',5,7-pentahydroxyflavone) is a natural compound that inhibits HSR in many cell types by downregulating HSF1 activity [6]. Benzylidene lactam KNK437 is reported to be a more selective compound that inhibits the expression of certain HSP family members [7]. Both quercetin and KNK437 inhibit cancer proliferation and render the cells more sensitive to hyperthermia and chemotherapy [6,7]. Other reported inhibitors of HSR include triptolide, NZ28 and emunin, each with different mechanisms of action [4]. In order to move forward for clinical development a significant effort needs to be invested to improve the potency and selectivity of these compounds.
The lack of specific HSR modulators is partially due to a lack of robust cell-based assay tools for high-throughput screening. Here, we describe the development of a reporter gene assay for HSR applicable to high-throughput screening. HeLa cells were engineered to express the beta-lactamase reporter gene [8] under the control of HSF response elements present in the HSP70 gene promoter. The HSE-bla reporter cell line was validated using HSF1-specific siRNAs and known small molecule modulators, and the assay was miniaturized into 1536-well format. Our results demonstrate that the assay is robust and can be applied to high-throughput screening for modulators of HSR.

PRINCIPLE OF ASSAY DESIGN
HeLa cells were transduced with lentivirus to express a beta-lactamase reporter gene under the control of three copies of HSE sequence (CTGGAATATTCCCGACCT GGCAG) present in the HSP70 gene promoter (Fig. 1). Cells expressing beta-lactamase after 42°C heat shock were isolated using fluorescence-activated cell sorting (FACS). In its un-induced state HSF1 exists as an inert monomer that is presumably repressed by interactions with HSP90 and HSP70 ( Fig. 1) [1,3]. Heat shock or a known HSR inducer such as 17-AAG treatment leads to trimerization and phosphorylation of HSF1, which in turn binds to DNA response elements driving the expression of the downstream betalactamase reporter gene (Fig. 1). Beta-lactamase activity can be detected by adding its substrate CCF4-AM directly into the cells [8,9]. In the absence of HSR inducer the pathway is inactive, therefore there is little beta-lactamase expression. When the cells are loaded with CCF4-AM and are excited at 406 nm, the substrate emits at around 530 nm (green). However, when cells are stimulated with 17-AAG the pathway is activated, leading to beta-lactamase reporter gene expression. The substrate is then cleaved by beta-lactamase, thereby disrupting the fluorescence resonance energy transfer (FRET) and resulting in emission at 460 nm (blue) following excitation at 406 nm.

Generation of the Stable HSE-bla Reporter Line
Three contiguous copies of the HSE sequence (CTGGAATATTCCCGACCTGGCAG) present in the HSP70 gene promoter was cloned into pLenti-bsd/MCS-bla vector (Invitrogen, Carlsbad, CA) via ClaI and NheI sites. Lentivirus was prepared according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). HeLa cells were transduced with the lentivirus expressing HSE-bla and selected for blasticidin resistance. Blasticidin resistant cells were left untreated or treated with heat shock at 42°C for 30 minutes, and then returned to 37°C. Cells were allowed to recover for 4.5 hours and then loaded with CCF4-AM substrate for 2 hours at room temperature prior to collecting a pool of heat shock induced beta-lactamase expressing cells by FACS. The cells were expanded and further tested in 384-well plate format for dose response using HSR inducer 17-AAG and HSF-specific RNAi knock-down.

HSE-bla Assay in 384-well Plate Format
Cells in a sub-confluent state (or cryopreserved cells) were resuspended in Assay Medium (DMEM with Glu-taMAX TM (Invitrogen, Carlsbad, CA, catalog number 10569) supplemented with 0.1% dialyzed FBS, 0.1 mM NEAA, 25 mM HEPES, 100 U/mL Penicillin and 100 g/mL Streptomycin) and plated in a 384-well assay plate (Corning, Lowell, MA, catalog number 3712) at 8,000 cells (36 μL) per well ( Table 1). 36 μL of Assay Medium without cells were plated in cell-free control wells on the same plate. Following overnight incubation at 37ºC, the cells were then stimulated with 4 L/well of 10x 17-AAG (LC Laboratories, MA, catalog number A-6880) over the indicated concentration range for 5 hours before adding 8 L/well of 6x Live-BLAzer TM -FRET B/G Substrate mixture (Invitrogen, catalog number K1096) for 2 hours. Fluorescence intensity at excitation 406 nm and emission 460 nm and 530 nm were obtained using a Tecan Safire 2 fluorescence plate reader (Tecan, Durham, NC). After subtracting the average fluorescence intensity from the cell-free control wells, the 460 nm/530 nm emission ratio was calculated. Response Ratio is a measurement of the assay window and is calculated as the 460 nm/530 nm emission ratio of the stimulated wells divided by the 460 nm/530 nm emission ratio of the unstimulated wells. Response Ratio provides a consistent way for data comparison, especially when data generated from different plates are compared and when different plate reader settings are used which usually result in large variables in the 460 nm/530 nm emission ratio. Response Ratios were plotted against test ligand concentrations in log scale and then analyzed using Prism software (GraphPad Software, Inc. San Diego, CA). A sigmoidal dose-response equation with varying slope was used to fit the data and generate EC 50 values. Z'-factor values were calculated as: Z'-factor = 1 -[(3 x stdev unstim + 3 x stdev maxstim ) / (avg maxstim -avg unstim )].
For the compound inhibition experiment, cells were seeded into a 384-well assay plate at 8,000 cells (32 μL) per well. Following overnight incubation at 37°C, cells were at a final concentration of 65 nM (EC 80 ) for 5 hours before the beta-lactamase assay was performed as described above.

HSE-bla Assay in 1536-well Plate Format
Assay protocol is described in Table 2. Briefly, HSE-bla HeLa cells were resuspended in OPTI-MEM ® I medium (Invitrogen, CA, catalog number 51895) containing 0.1% dialyzed FBS, and dispensed at 1500 cells/5 μL/well in 1536well black wall/clear bottom plates (Kalypsys, San Diego, CA) using a Flying Reagent Dispenser (Aurora Discovery, Carlsbad, CA). After the cells were incubated at 37ºC overnight, 23 nL of DMSO or 17-AAG at concentration range of 0.1 nM to 3.83 M was transferred to the assay plate by a pin tool (Kalypsys, San Diego, CA) resulting in a 217-fold dilution. The plates were incubated at 37°C for 6 hours. After 1 L of LiveBLAzer ™ B/G FRET substrate (Invitrogen, Carlsbad, CA) was added, the plates were incubated at room temperature for an additional 2 hours and then fluorescence intensity at 405 nm excitation and 460 nm and 530 nm emission was measured using an Envision plate reader (Perkin Elmer, Shelton, CT). Data was expressed as the ratio of 460 nm/530 nm emissions without background subtraction.

RNAi Experiment
HSE-bla HeLa cells were plated in 384-well format in Growth Medium (DMEM containing 10% dialyzed FBS, 0.1 mM NEAA and 25 mM HEPES) at 2,000 cells/well and reverse transfected using Lipofectamine TM RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA) and 20 nM of Stealth™ RNAi duplexes against HSF1, HSF2, and HSF4. RNAi oligo targeting beta-lactamase was used as the positive control and a random-sequence oligo with 48% GC content (MedGC) as the negative control. A set of 2 oligos for each HSF was used: HSF1-1(GGAUGCUAUGGACUCCAACCUGGAU) HSF1-2(UGCGGCAGCUCAACAUGUAUGGCUU) HSF2-1(GCAUAGACCCAGAUCUCCUGGUUGA) HSF2-2 (CCCUUUGGAAGGAGGUGUCAGAAUU) HSF4-1 (GGUCAUUGGCAAGCUGAUCCAGUGU) HSF4-2 (CCCUACUUCAUCCAGUCGCCUUCUA) Cells were incubated with RNAi oligos at 37°C for 40 hours, followed by a medium change to Assay Medium and then stimulation with 17-AAG for 6 hours before the beta-lactamase assay was performed as described above. For heat shock treatment, 40 hours post-transfection the plate was subjected to heat shock at 42°C for 1 hour followed by a 5hour incubation at 37°C prior to performing the betalactamase assay.

Assay Development
To generate a cell-based assay suitable for highthroughput screening for modulators of HSR, we stably integrated an HSF-responsive beta-lactamase reporter element, HSE-bla, into the HeLa human cervical cancer cell background as detailed in Methods. Previous studies have established that HeLa cells respond to heat shock leading to the activation of HSF1 and therefore serve as a suitable cell background for interrogating this pathway [10]. Following antibiotic selection, a highly responsive pool of stable integrants was isolated that provides an effective HSR readout, with low beta-lactamase activity detected from unstimulated cells (i.e. mostly green cells) and efficient induction (i.e. mostly blue cells) observed following incubation at 42°C or by using HSP90 inhibitor 17-AAG ( Fig. 1 and Fig. 2A). Fig. (2). HSE-bla HeLa cells respond to HSR inducers and heat shock. (A) HSE-bla HeLa cells were left untreated (unstim), or treated with 17-AAG (0.45 μM), Celastrol (1 μM) and Bortezomib (10 μM) for 5 hour, or incubated at 42°C for 1 hour and then recovered for 5 hours in a 37°C/5% CO2 incubator before the betalactamase assay was performed as described in Methods. Response Ratios were plotted for each condition (n=4 for each data point). (B) Dividing or cryopreserved HSE-bla HeLa cells were resuspended in Assay Medium and plated out in 384-well format as described in Methods. Following an overnight incubation at 37°C, the cells were treated with the indicated concentrations of 17-AAG for 5 hours before the beta-lactamase assay was performed as described in Methods. The calculated EC 50 value for 17-AAG was 49 nM for dividing cells, and 36 nM for cryopreserved cells (n=4 for each data point).

A Response to Known HSR inducers B 17-AAG Dose Response
A. HSE-bla HeLa cells responded to 17-AAG in a dose dependent manner with an EC 50 value of 49 nM (Fig. 2B), consistent with literature results [5]. At the highest two concentrations tested, a slight drop of response ratio was observed, presumably due to the negative feedback signal since no significant cytotoxicity was observed at these concentrations of 17-AAG. It is possible that high concentration of 17-AAG leads to high expression of HSP70 which then sequesters HSF1 from activating downstream reporter gene expression.
The HSE-bla HeLa cells were also shown to be responsive, to a lesser extent, to additional HSR inducers such as the triterpenoid antioxidant celastrol and proteasome inhibitor bortezomib ( Fig. 2A), consistent with previously reported activities for these inducers [1,3].
Assay-ready cryopreserved cells have recently become an important option for HTS, which demands a large quantity of cells that can provide consistent assay results [11]. To further test the suitability of the HSE-bla HeLa cells for HTS applications, cryopreserved cells were thawed and immediately used for setting up the assay. A nearly super-imposable 17-AAG dose response curve (EC 50 of 36 nM) to that generated with dividing cells was obtained (Fig. 2B). The Z' values at the maximum assay window for both dividing and cryopreserved cells were 0.83 and 0.88, respectively, indicating that cryopreserved assay-ready HSE-bla HeLa cells can be generated and applied for HTS.

RNAi Validation
HSF1 is known to be essential for HSR induced by various stressors [1]. To validate the assay and to determine which HSF is required for HSR in the HSE-bla HeLa cell line, RNAi experiments were performed where the effect of HSF-specific RNAi oligos on 17-AAG or heat shockinduced beta-lactamase reporter activity was studied (Fig. 3). A beta-lactamase specific RNAi oligo (b-lac) was used as a positive control, which resulted in complete knock down of the reporter activity induced by both 17-AAG (Fig. 3A) and 42°C heat shock (Fig. 3B). In contrast, the negative control oligo (MedGC) with random sequence and medium GC content showed minimal effects. Two RNAi oligos directed at HSF1 effectively blocked reporter activity to a similar extent as the beta-lactamase positive control under both of these HSR conditions. RNAi oligos against HSF2 and HSF4 did not show significant effect on the HSR reporter activity. These results suggest that HSF1 is essential for HSR reporter activity in HSE-bla HeLa cells, consistent with previous findings [2]. Moreover, these results also demonstrate the suitability of this reporter line for RNAi-mediated screening for HSR modulators.

Inhibitor Activity
To demonstrate screening for inhibitors of HSR signaling, dose response inhibition was performed using the flavonoid quercetin, which has been reported to inhibit HSR by down-regulating HSF1 activity [6]. As shown in Fig. (4), quercetin effectively inhibited 17-AAG treated HSE-bla HeLa cells with an IC 50 of 5.8 μM, suggesting that this assay can be used to screen for HSR inhibitors. Fig. (4). Inhibition of 17-AAG-induced beta-lactamase reporter activity in the HSE-bla HeLa cells. Cells were pretreated with the indicated concentrations of quercetin and then stimulated with 65 nM 17-AAG for 5 hours before the beta-lactamase assay was performed as described in Methods. Response Ratios were plotted for the indicated concentrations of quercetin (n=4 for each data point). The calculated IC 50 for quercetin was 5.8 μM.

Assay Optimization
To optimize the assay for 384-well format, several parameters were evaluated, including cell plating density, Fig. (3). Effects of various HSF-specific RNAi oligos on 17-AAG and heat shock-induced beta-lactamase reporter activity. (A) Cells were incubated with the indicated RNAi oligos for 40 hrs, followed by a medium change to Assay Medium. Cells were then stimulated with 400 nM 17-AAG for 6 hours before the beta-lactamase assay was performed as described in Methods. (B) Cells were incubated with the indicated RNAi oligos for 40 hrs, followed by heat shock at 42°C for 1 hour and a 5-hour recovery at 37°C before the betalactamase assay was performed as described in Methods (n=3 for each data point). Controls were as follows: nontransfected and unstimulated cells (NT unstim), nontransfected and stimulated cells (NT stim), mock transfected (mock), Stealth™ RNAi Negative Control Med GC (MedGC), and beta-lactamase positive control RNAi duplex (b-lac).

Response Ratio
DMSO tolerance, 17-AAG stimulation time, and betalactamase substrate loading time. First, the effect of cell density on assay performance was tested using a range of plating densities, from 2,000 cells/well to 16,000 cells/well (Fig.  5A). A robust assay window was observed with a wide range of cell plating densities, from 4,000 cells/well to 12,000 cells/well with EC 50 values of 17-AAG ranging from 33 nM to 47 nM. The 8,000 cells/well plating density was selected as the basis for further assay optimization because it provided both a good assay window (7-fold) and minimal variability as exemplified by a CV (coefficient of variation) of 5% at the maximal response.
To satisfy the HTS requirement for compounds to be dissolved in DMSO, a cell-based assay needs to be tolerant to certain amounts of DMSO. DMSO tolerance testing results (Fig. 5B) indicated that the HSE-bla HeLa reporter assay can tolerate up to 0.5% final DMSO concentration with only a modest drop in assay window (from ~7.2-fold in the presence of 0.1 % DMSO to ~5.2-fold in the presence of 0.5% DMSO in this experiment). The highest concentration of DMSO (1%) tested adversely affected the reporter assay by reducing the assay window by nearly two-fold and producing a substantial right-shift in the 17-AAG EC 50 value. Thus, 0.1% DMSO was consistently used in all other assay optimization experiments described here.
It usually takes a minimum of 4 to 5 hours of ligand stimulation to activate downstream gene transcription. Depending on the pathway, longer stimulation may be required for generating a robust assay window. To test the effect of 17-AAG stimulation time on assay performance, HSE-bla HeLa cells were stimulated with 17-AAG for 4, 6, or 16 hours prior to assaying beta-lactamase activity (Fig. 5C).
Among the three time points tested, a 6-hour incubation with 17-AAG resulted in the highest response with a maximal response ratio of ~7.9, nearly two-fold higher as compared to the shorter 4-hour incubation (maximal response ratio of ~4.0). Overnight incubation with 17-AAG resulted in a substantially diminished assay window (maximal response ratio of ~2.0).
The last parameter evaluated was the beta-lactamase substrate loading time. It is known that when the amount of beta-lactamase present in cells is low, longer substrate loading can result in more substrate conversion [8]. To evaluate the effect of substrate loading time on the HSR assay outcome, an assay plate was read at various time points following addition of the substrate to the wells (Fig. 5D). Increasing substrate loading time clearly resulted in marked increases in assay window up to a maximum of 3 hours of loading tested. Both 1.5-hour and 2-hour loading conditions generated sufficient assay windows and good Z' values (0.80). Together a 5-hour 17-AAG stimulation and a loading  Response Ratio time of 2 hours or shorter allows for the assay to be conveniently performed within the context of an 8-hour workday.

Assay Miniaturization to 1536-well Plate Format
The HSE-bla assay was further miniaturized into 1536well plate format with a final 5 μl assay volume. To check the assay quality the assay was tested with a DMSO plate containing a final DMSO concentration of 0.46%. In the DMSO/CV plate testing, the signal to background ratio (S/B) was 3.3 fold, CV was 6.4% and Z' factor was 0.66 (Fig. 6A). Fig. (6B) shows the dose response curve of 17-AAG that was generated in 1536-well format, yielding an EC 50 of 54 nM that correlates well with the EC 50 (49 nM) obtained in 384-well format. These results demonstrate that the HSE-bla assay can be run in 1536-well format and is robust and suitable for high-throughput screening.

CONCLUSIONS
In conclusion, we have developed a beta-lactamase reporter gene assay that can be used for high-throughput screening for modulators of the HSR pathway, which can be then deconvoluted with lower-throughput follow-up assays, such as HSP90 binding assays and/or assays monitoring the phosphorylation of HSF1. Taking together the advantages of the beta-lactamase reporter technology, which allows for a ratiometric and sensitive read-out, this reporter assay can be miniaturized to 384-well and 1536-well formats. We have demonstrated the suitability of this assay for detecting both inducers and inhibitors of HSR signaling using known small molecule modulators and HSF-specific RNAi oligos. It is anticipated that compound library screening using the HSEbla HeLa assay will provide new leads in the search for novel modulators of HSR.