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Supplementary information. Response Regulator YycF Essential for Bacterial Growth: X-ray Crystal Structure of the DNA-binding Domain and its PhoB-like DNA Recognition Motif Toshihide Okajima, Akihiro Doi, Ario Okada, Yasuhiro Gotoh, Katsuyuki Tanizawa, and Ryutaro Utsumi 1. Supplements to the material and methods 1.1. Crystallography The His-tagged YycF protein was purified to near homogeneity (purity, >95% by SDS–PAGE) as described previously [1s] and after dialysis against 5 mM sodium phosphate buffer, pH 7.0, containing 0.1 mM EDTA and 0.1 mM dithiothreitol, it was concentrated to 10 mg/ml by ultrafiltration. Diffraction-quality YycF crystals were grown in a dialysis button at 16 ºC for 2–3 months against 90 mM HEPES, pH 6.6, containing 9 mM MgCl2 and 3% dioxane. After 1-day soaking of the crystals in the same solution containing 30% glycerol as a cryoprotectant, diffraction data were collected at 100 K with a synchrotron X-radiation at SPring8 (Hyogo, Japan) using an imaging plate DIP6040 (Bruker AXS) in the beam-line station BL44XU ( = 0.9 Å). The collected data were processed, merged, and scaled using MOSFLM [2s] and SCALA [3s]. The structure was solved by molecular replacement with MOLREP [4s] using the coordinates of a carboxyl terminal domain (residues 127–213) of an OmpR homologue DrrB (PDB entry: 1P2F) as a search model because the crystallized YycF –1– contained only the carboxyl terminal half (see “Results and discussion”). Refinements and manual model building were performed with CNS [5s] and XtalView [6s], respectively. The details of X-ray crystallography are provided in Supplementary TABLE 1. The atomic coordinates and structure factors for YycF-C have been deposited in the Protein Data Bank with the accession codes 2D1V. 1.2. Gel mobility shift assay DNA fragments corresponding to the promoter regions of yocH, rpsJ, and yycF genes (abbreviated as PyocH, PrpsJ, and PyycF, respectively) of B. subtilis 168, and that of phoA gene (PphoA) of E. coli W3110 were amplified by PCR using Ex Taq DNA polymerase (Takara Bio, Japan) and genomic DNA of each bacterium as a template. The primers are summarized in Supplementary TABLE 2. For preparing the radio-labeled PyocH and PphoA, corresponding primers were labeled with [-32P]ATP prior to PCR and the PCR products were gel-purified. The binding reactions were performed by incubating 32P-labeled PyocH or PphoA (2 fmol) with the purified His-tagged YycF (70 pmol) in the presence or absence of unlabeled competitor DNA (6 fmol) at 37 ºC for 10 min in 10 l of 50 mM Tris-HCl, pH 7.8, containing 50 mM NaCl, 3 mM magnesium acetate, 0.1 mM EDTA, and 0.1 mM dithiothreitol. The reaction mixtures were immediately subjected to PAGE using a 6% non-denaturing gel. Radioactive bands were detected with a Bio-imaging analyzer BAS100MAC system (Fuji Photo Film, Japan). 1.3. Alkaline phosphatase (AP) assay –2– E. coli M15 (pREP4) cells carrying pYycF [1s] were cultured at 37 °C in a 60-ml phosphate-limited or -rich minimum medium [7s] containing 100 g/ml ampicillin and 25 g/ml kanamycin. At an early logarithmic phase of the bacterial growth (OD600 = 0.2), the culture was divided into 10-ml portions, each supplemented with 0, 0.25, 0.5, 1.0, or 1.25 mM IPTG to induce YycF expression. After further incubation for 1 h, the cells were harvested and used for measurements of the AP activity [8s]. 1.4. DNase I footprint assay 32 P-labeled PphoA or PyycF (5x104 cpm) was pre-incubated with the purified His-tagged YycF for 10 min at 37 ºC in 25 l of 50 mM Tris-HCl (pH 7.8 at 37 ºC), containing 50 mM NaCl, 3 mM magnesium acetate, 0.1 mM EDTA, and 0.1 mM dithiothreitol. DNA digestion was initiated by the addition of 6.25 ng of DNase I (Takara Bio, Japan). After incubation for 30 s at 25 ºC, the reaction was terminated by adding 45 l of the stop solution [20 mM EDTA (pH 8.0 at 4 ºC), 1% SDS, 0.2 M NaCl, and 250 g/ml tRNA]. DNA was precipitated with ethanol, dissolved in the formamide-dye solution, and analyzed by PAGE with a 6% gel in the presence of 8 M urea. Radioactive signals were detected as described above. 2. Supplements to results and discussion 2.1. Gel mobility shift with the purified YycF As shown in Fig. 1S, A, the mobility of radio-labeled PphoA DNA in the gel was –3– markedly retarded in the presence of YycF (lane 2). Addition of a non-competitive DNA, PrpsJ (the promoter of the gene for ribosomal protein S10), did not affect the retarded mobility of PphoA (lane 3). However, addition of excess unlabeled PphoA released most of the radio-labeled PphoA into the free DNA mobility (lane 4), most likely by absorbing YycF. A promoter in the well-known YycF regulon, PyocH, containing the YycF boxes [9s], allowed the free mobility of PphoA nearly completely (lane 5). Interestingly, the promoter-like DNA (PyycF) of the yycF gene itself (Fig. 2B) also suppressed the retarded shift of PphoA (lane 6). Similar results were also obtained using radio-labeled PyocH DNA and the same set of competitors (Fig. 1S, B). Although a large amount of YycF was applied in these experiments, competitive effects of PyycF, PyocH, and PphoB support the specific interactions between YycF and these promoters. References [1s] Furuta, E., Yamamoto, K., Tatebe, D., Watabe, K., Kitayama, T. and Utsumi R. (2005) Targeting protein homodimerization: A novel drug discovery system. FEBS Lett. 579, 2065–2070. [2s] Leslie, A.G.W. (1992) Joint CCP4 and EESF-EACMB newsletter on protein crystallography. (Warrington, UK: SERC Daresbury Laboratory, 1992). [3s] Collaborative Computational Project Number 4 (1994) The CCP 4 suite: programs for protein crystallography. Acta Crystallogr. D50, 760–763. [4s] Vagin, A.A. (1997) MOLREP: An automated program for molecular replacement. J. Appl. Crystallogr. 10, 1022–1025. –4– [5s] Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T. and Warren, G.L. (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921. [6s] McRee, D.E. (1999) XtalView/Xfit - A versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165. [7s] Brickman, E. and Beckwith, J. (1975) Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and 80 transducing phages. J. Mol. Biol. 96, 307–316. [8s] Amemura, M., Shinagawa, H., Makino, K., Otsuji, N. and Nakata, A. (1982) Cloning of and complementation tests with alkaline phosphatase regulatory genes (phoS and phoT) of Escherichia coli. J. Bacteriol. 152, 692–701. [9s] Howell, A., Dubrac, S., Andersen, K.K., Noone, D., Fert, J., Msadek, T. and Devine, K. (2003) Genes controlled by the essential YycG/YycF two-component system of Bacillus subtilis revealed through a novel hybrid regulator approach. Mol. Microbiol. 49, 1639–1655. –5– Supplementary TABLE 1. Crystal parameters and statistics of data collection and refinement Crystal Size (mm) 0.3 0.3 0.2 (Thick rhombic) Unit cell dimensions (Å) a = 45.8, b = 65.6, c = 79.1 Space group C2221 Data collection Resolution limits (Å) a 33.9 – 2.40 (2.53 – 2.40) Number of observations 23625 Number of unique reflections 4659 Completeness (%) a 96.6 (98.2) I/ (I) a 4.2 (1.9) Redundancy a 5.1 (5.2) Rmerge (%) a, b 10.3 (37.7) Refinement statistics Resolution limits (Å) a 33.9 – 2.40 (2.51 – 2.40) Number of non-hydrogen atoms 901 Number of solvent atoms 37 Average temperature factors 70.9 RMS deviation from ideal values Bond lengths (Å) 0.007 Bond angles (deg) 1.3 Rwork (%) a, c 22.8 (35.5) Rfree (%) a, d 23.2 (40.1) a Values in parentheses refer to data in the highest resolution shells b Rmerge = h i Ih,i <Ih> / h i Ih,i, where Ih,i is the intensity value of the ith measurement of h and <Ih> is the corresponding mean value of Ih for all i measurements. c Rwork = Fo Fc / Fo. d Rfree is an R factor of the refinement evaluated for 5% of reflections that were excluded from the refinement. –6– Supplementary TABLE 2. Oligonucleotide primers used and amplified region. Primer Sequence Amplified regionb name yocH-Fa 5’-AATACAGGCTTATGCAAGGATGACAGACTA-3’ yocH-Ra 5’-ATGCAGTTGTTGAAAGTGCAGCAACTGCCA-3’ rpsJ-F 5’-CGCGAACACATTAAGTCGGCATGCACGCAA-3’ rpsJ-R 5’-CGGAATCGGACCAGATACGCTGGCACCAGA-3’ yycF-F 5’-AAGACGTCATTGATAAAGACGCACTCCGGT-3’ yycF-R 5’-AGCCGTCAGCATAATGATTGGCATATCGTA-3’ phoA-Fa 5’-TTCCAGACACTATGAAGTTGTGAAACATAA-3’ phoA-Ra 5’-TCTGGTGCCGGGCTTTTGTCACAGGGGTA-3’ a 32 b –268 to +55 –230 to +110 –320 to +130 –429 to +74 P-labeled PyocH and PphoA were prepared by using 5’-32P -labeled primers. The nucleotide positions are shown with respect to the first ‘A’ of the translation initiation codon. –7– (Figure legend) Fig. 1S. Interactions of YycF with phoA and yocH promoters. The gel mobility shifts of 32 P-labeled PphoA (A) and PyocH (B) were assayed with no addition (only labeled probes) (lane 1), His-tagged-YycF (lane 2), His-tagged-YycF and PrpsJ (lane 3), His-tagged-YycF and PphoA (lane 4), His-tagged-YycF and PyocH (lane 5), and His-tagged-YycF and PyycF (lane 6). –8– –9–