SUPPLEMENTAL MATERIAL FOR Structure of Bacterial Transcription Factor SpoIIID and Evidence for a Novel Mode of DNA Binding Bin Chen, Paul Himes, Yu Liu, Yang Zhang, Zhenwei Lu, Aizhuo Liu, Honggao Yan, and Lee Kroos Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 USA 1
Table S1. Intermolecular NOEs observed in the SpoIIID DNA complex SpoIIID resonances Possible DNA resonances a DNA resonance chemical shifts b (ppm) Ser35 HN CYT H5 or any ribose H1 6.26 Thr36 HN ADE H6 or CYT H4 6.73 Glu43 HN Ribose H2, H2, H4, H5 or H5 3.25 Arg44 HN Ribose H2, H2, H4, H5 or H5 3.52, 3.26 Lys64 HN Ribose H2, H2, H4, H5 or H5 4.33 Arg67 HN Ribose H2, H2, H4, H5 or H5 3.52 ADE H2, H8 or CYT H6 or THY H6 or GUA 7.56 H8 or ADE H6 or CYT H4 or GUA H2 Gly71 HN Ribose H2, H2, H4, H5 or H5 3.62, 3.96 CYT H5 or any ribose H1 5.91, 6.14 Gly72 HN CYT H5 or any ribose H1 6.04 ADE H2, H8 or CYT H6 or THY H6 or GUA 7.79 H8 or ADE H6 or CYT H4 or GUA H2 Ala74 HN Ribose H2, H2, H4, H5 or H5 3.54, 3.96 ADE H2, H8 or CYT H6 or THY H6 or GUA 7.79 H8 or ADE H6 or CYT H4 or GUA H2 Thr75 HN Ribose H2, H2, H4, H5 or H5 3.96, 4.37 CYT H5 or any ribose H1 6.08, 6.40 Lys76 HN Ribose H2, H2, H4, H5 or H5 3.96 CYT H5 or any ribose H1 6.08 a The ambiguous chemical shift assignments were based on the DNA chemical shift data in the Biological Magnetic Resonance Data Bank (http://www.bmrb.wisc.edu/ref_info/statsel_dna.htm). b All intermolecular NOEs were obtained from the 3D 13 C, 15 N-filtered (F1), 15 N-edited (F3) NOESY spectrum in comparison with the 3D 15 N-edited NOESY spectrum. 2
Table S2. Substitutions in SpoIIID and corresponding plasmid and E. coli strain designations SpoIIID substitution pph4 derivative a E. coli strain b K34A pph251 PH251B H38A pph252 PH252B K39A pph253 PH253B E43A pph254 PH254B R44A pph255 PH255B K76A pph258 PH258B K78A pph259 PH259B K80A pph260 PH260B K81A pph261 PH261B a pph4 is a pet-21b (Novagen) derivative in which spoiiid is expressed from a T7 RNA polymerase promoter (1). b Plasmids were transformed into E. coli BL21 (DE3) (Novagen). 3
Table 3S. Oligonucleotides used in this study Oligonucleotide a Sequence b LK2331 (K34A) c 5 -ggaatttggtgtttccgcaagtacagtacacaagg-3 LK2332 5 -ccttgtgtactgtacttgcggaaacaccaaattcc-3 LK2333 (H38A) 5 -ggtgtttccaaaagtacagtagccaaggatttaacagagcgtc-3 LK2334 5 -gacgctctgttaaatccttggctactgtacttttggaaacacc-3 LK2335 (K39A) 5 -caaaagtacagtacacgcggatttaacagagcgtc-3 LK2336 5 -gacgctctgttaaatccgcgtgtactgtacttttg-3 LK2337 (E43A) 5 -gtacacaaggatttaacagcgcgtctgcctgaaattaac-3 LK2338 5 -gttaatttcaggcagacgcgctgttaaatccttgtgtac-3 LK2339 (R44A) 5 -cacaaggatttaacagaggcgctgcctgaaattaaccccg-3 LK2340 5 -cggggttaatttcaggcagcgcctctgttaaatccttgtg-3 LK2345 (K76A) 5 -ggaggagaagcgacagcgctcaaatataaaaaag-3 LK2346 5 -cttttttatatttgagcgctgtcgcttctcctcc-3 LK2347 (K78A) 5 -gaggagaagcgacaaagctcgcgtataaaaaagatg-3 LK2348 5 -catcttttttatacgcgagctttgtcgcttctcctc-3 LK2349 (K80A) 5 -gcgacaaagctcaaatatgcgaaagatgaaattctcgaag-3 LK2350 5 -cttcgagaatttcatctttcgcatatttgagctttgtcgc-3 LK2351 (K81A) 5 -gacaaagctcaaatataaagcggatgaaattctcgaagg-3 LK2352 5 -ccttcgagaatttcatccgctttatatttgagctttgtc-3 a Oligonucleotides are shown in pairs that were used for mutagenesis. b Boldface type indicates a mutation. c For each pair of oligonucleotides used for mutagenesis, the substitution is indicated in parentheses. 4
Table S4. Ambiguous interaction restraints for modeling the SpoIIID DNA complex Active residues for SpoIIID Passive residues for dsdna Method determined Lys34, His38, Lys39, Lys78, Any DNA nucleotide Mutational analysis Lys80, Lys81 HN of Ser35, Thr36, Glu43, See Supplementary Table S1 Intermolecular NOEs Arg44, Lys64, Arg67, Gly71, Gly72, Ala74, Thr75, Lys76 Active residues for dsdna a Passive residues for SpoIIID CYT4, THY5, THY6, GUA7, THY8, CYT9, CYT10, GUA19, GUA20, ADE21, CYT22, Any SpoIIID residue (1-81) Mutational analysis and/or sequence conservation ADE23, ADE24, GUA25 a See Figure S2 for base numbering. 5
mother cell forespore engulfment SpoIIID E polar septum coat K mother cell lysis Figure S1. Morphological changes and transcription factors during B. subtilis sporulation. Nutrient limitation initiates the process, causing a polar septum to form, which divides the cell into mother cell and forespore compartments. Differential transcription in the two cell types is directed by specific sigma subunits of RNA polymerase (2,3). After E RNA polymerase becomes active in the mother cell, it directs transcription of the gene for SpoIIID. The mother cell membrane of the septum engulfs the forespore (orange arrows), and channels (green) form between the two cell types (4-7). Fission of the mother cell membrane near the pole pinches the forespore off as a free protoplast inside the mother cell. In the absence of SpoIIID, some genes under E control are still expressed and engulfment is completed, but then the process of endospore formation stops. Although the blockage is not completely understood, one crucial function of SpoIIID is to activate transcription by E RNA polymerase of the gene for K (8,9). Genes under K control produce proteins that assemble a coat around the forespore, lyse the mother cell, and prepare the spore to germinate when nutrients are sensed (10). 6
5 1 GC 28 2 CG 27 3 GC 26 4 CG 25 5 TA 24 6 TA 23 7 GC 22 8 TA 21 9 CG 20 10 CG 19 11 TA 18 12 AT 17 13 AT 16 14 TA 15 5 Figure S2. Numbering of bases in the 14-bp DNA duplex bound by SpoIIID. The idealized binding site consensus sequence indicated by the arrow in Figure 5A and in Figure S5A is bases 17-26. 7
105 78 55 45 Wt Wt or Position of Ala substitution M 0.5 76 44 Wt 34 38 39 43 78 80 81 34 22 16 6 SpoIIID Figure S3. Purified SpoIIID proteins. SDS-PAGE followed by Coomassie blue staining of SeeBlue Plus 2 Prestained markers (M) (Invitrogen) with sizes (kda) indicated, a 2-fold dilution series of wild-type (Wt) SpoIIID starting at 0.5 g, unknown amounts of SpoIIID K76A and SpoIIID R44A, and 1 g of wild-type or single-ala-substituted SpoIIID as indicated. Based on the result, it was estimated that 0.5 g and 0.25 g of SpoIIID K76A and SpoIIID R44A had been loaded on the gel. 8
9
Figure S4. Secondary structural elements and NOEs of SpoIIID. (a) The secondary structural elements of SpoIIID and a summary of the sequential and medium-range NOEs, presence of HN- H 2 O cross-peaks, 13 C chemical shift indices for C and C, and cartoon representation of - helices. Residues that exhibited a cross-peak between HN and H 2 O resonances in the 3D 1 H- 15 N NOESY-HSQC spectrum are marked by *. Triangles indicate proline residues. Chemical shift indices (CSI) for C, C, and C characteristic for -helical regions are represented by ( ). (b) Distribution of NOEs along the sequence of SpoIIID. The number of intra-residue (white), sequential (light gray), medium-range (dark gray), and long-range (black) NOEs at each position is graphed. 10
a b Probe 10 CATTAGGACAAGCGCT 18 CATTAGGACAAACGCT 19 CATTAGGACAAGTGCT 20 CATTAGGACAAGCACT 21 CATTAGGACAAGCGTT 22 CATTAGGACGAGCGTT K d Position of Ala substitution SpoIIID: 44 39 (nm) 2.4 + 0.2 2.7 + 0.2 1.4 + 0.2 1.5 + 0.3 1.3 + 0.1 1.2 + 0.1 B Probe: 18 19 20 21 22 18 19 20 21 22 U Figure S5. Binding of SpoIIID to DNA. (a) Sequences of DNA probes and apparent K d for binding of wild-type SpoIIID. Only one strand of each probe is shown. The arrow denotes the idealized binding site consensus sequence in probe 10 and underlined bases in the other probes indicate differences from probe 10. Apparent K d s are the average of a least 3 determinations ± 1 standard deviation. (b) EMSAs of SpoIIID R44A and SpoIIID K39A binding to probes 18-22. Altered SpoIIID proteins (840 nm) were tested with different probes (indicated below the panel) (0.1 nm). Bound (B) and unbound (U) probe are indicated. 11
Figure S6. Schematic summary of hydrogen bonding interactions between SpoIIID and DNA in the top 10 models (a) and in the best model (b). Black lines in (a) indicate that at least one hydrogen bonding interaction is formed in all 10 models between a residue of SpoIIID and a phosphate oxygen, sugar, or base of DNA, while red lines indicate at least one interaction in greater than 8 models, blue lines greater than 5 models, and green lines less than 5 models. In the best model (b), some residues of SpoIIID form 2 or 3 hydrogen bonds with phosphate oxygens of DNA, as indicated by the numbers. 12
Helix 1 Helix 2 Helix 3 Helix 4 Helix 5 Figure S7. Alignment of SpoIIID orthologs. The highest scoring sequence from each species was identified using blastp (11, 12) with B. subtilis SpoIIID as the query, and the sequences were aligned using Clustal W (13). The results were visualized using ESPript (14). Numbers refer to residues in B. subtilis SpoIIID, which is marked with a star to the left. White, red, and black letters indicate identical, conserved, and less-conserved residues, respectively. Regions of B. subtilis SpoIIID that are -helical are indicated above the sequences. Adapted from (1).
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