Esser et al. 10.1073/pnas.0601149103. |
Supporting Table 3
Supporting Table 4
Supporting Figure 4
Supporting Figure 5
Supporting Table 5
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Materials and Methods
Supporting Figure 4
Fig. 4.
Sequence alignment and conservation of residues around the Qo pocket for the cyt b subunits of bc1 complexes. Seven sequences of cyt b are given as follows: BT, bovine; SC, S. cerevisiae; RR, R. rubrum; RV, R. viridis; RC, R. capsulatus; PD, P. denitrificans; and RS, R. sphaeroides. Residues with sequence similarity of >99.5% from the alignment of >22,000 sequences are in red boldface. Residues that are identical in this alignment are in magenta. Helices as determined crystallographically were mapped to the primary sequences as green rectangles. The orange box is for the ef1 helix in the bacterial insertion. Residues of importance to this work are indicated with red stars; those in direct contact with the ISP-ED are emphasized with a black dot. The top numbering is for the bovine sequence, and the bottom one is for R. sphaeroides.Fig. 5.
Stereo pair showing the binding environment of JG144 in the Qo pocket of the cyt b subunit. Bound inhibitor is shown in a ball-and-stick model with carbon atoms in black, oxygen in red, and nitrogen in blue. The inhibitor is enclosed in the cage of difference electron density calculated with refined phases with the inhibitor molecule omitted. Secondary structure elements around the Qo site of cyt b are given. Helices cd1, cd2, and ef as well as parts of the C, B, and E helices are shown and are labeled. The heme bL of cyt b and the [2Fe2S] cluster of the ISP are also shown in ball-and-stick form. Residues in the Qo pocket that are in direct contact with the inhibitor are rendered as stick models, in which carbon atoms are yellow, sulfur is green, oxygen is red, and nitrogen is blue.Fig. 6.
H-bonding and the Lys-287–Ser-151 (KS) dyad electron densities for the KS dyad in the presence of two different classes of Qo pocket inhibitors. The Lys-287 at the end of EF loop and the Ser-151 at the end of the cd1 helix are depicted in stick form. The residues near the [2Fe2S] cluster of the ISP subunit is also given in a stick model. The depicted residues are based on the structure of cyt bc1 with bound stigmatellin. Overlapping the atomic model in the green chicken-wire cage is the electron density for the stigmatellin-bound bc1 crystal, and that in red is obtained from the MOAS-bound bc1 crystal; both maps were calculated with Fourier coefficients of 2mFo – DFc and contoured at 1s level. H-bonds are shown as dashed lines, and distances are labeled in red. Parts of the cd1 helix and of EF loop near the F helix are shown.Fig. 7.
Structure of the Rsbc1 and its comparison with the bc1 of mitochondria. (A) Ribbon diagram of the dimeric bc1 complex of R. sphaeroides. The two symmetry-related cyt b subunits are in green, the cyt c1 subunits are in blue, and the ISP subunits are in yellow with the [2Fe2S] clusters drawn as spheres located at the tips. The heme groups for cyt b and c1 are drawn as ball-and-stick models with carbon atoms in black, oxygen in red, and nitrogen in blue. A few lipid molecules are also shown. The red helices and coils are insertions in cyt b, c1, and ISP when compared with the corresponding subunits in the bovine complex. (B) Superposition of cyt b subunits of bovine bc1 (mt-cyt b) and Rsbc1 (Rs-cyt b). The mt-cyt b is shown in a black Ca trace; the Rs-cyt b is in magenta. The ef1 insertion is in red. The ISP subunit is shown as a yellow Ca trace and is taken from the structure of Rsbc1. The heme bL and [2Fe2S] groups are given as ball-and-stick models with carbon atoms in black, nitrogen in blue, oxygen in red, iron in orange, and sulfur in yellow. (C) A more detailed view is given for the boxed area in B showing interactions between residues in the ef1 insertion and in ISP-ED.Fig. 8.
Stereo pair: electron density in the vicinity of Lys-329 of the Rsbc1. Rsbc1 was crystallized in the presence of the Pf inhibitor stigmatellin, making the ISP-ED in a fixed conformation. Residues Lys-329 and Gly-167 of the cyt b subunit in the Rsbc1 are the equivalents of Lys-287 and Ser-151, respectively, of the bovine cyt b. In the absence of the KS dyad, the electron density for Lys-329 is well defined, and Lys-329 still forms two H-bonds with backbone carbonyl oxygen atoms of ISP. Coefficients of 2mFo – DFc were used for calculating the Fourier map, which was contoured at 1s level.Table 3. Crystallographic data collection and model refinement statistics
| Myxothiazol* | Azoxystrobin | MOAS | Native | Stigmatellin | UHDBT1 | Famoxadone | JG144† | Rsbc 1 |
Data collection |
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Resolution, Å (outer shell) | 50-2.7 (2.80-2.70) | 40-2.64 (2.73-2.64) | 50-3.0 (3.11-3.00) | 50-2.4 (2.49-2.40) | 50-2.5 (2.59-2.50) | 50-2.85 (2.95-2.85) | 50-2.20 (2.25-2.20) | 40-2.26 (2.34-2.26) | 50-3.2 (3.11-3.20) |
R merge‡ | 0.066 (0.433)§ | 0.073 (0.434) | 0.058 (0.384) | 0.058 (0.392) | 0.068 (0.449) | 0.072 (0.436) | 0.064 (0.431) | 0.064 (0.477) | 0.137 (0.480) |
<I>/<sI> | 15.8 (1.33) | 14.1 (2.15) | 21.9 (2.38) | 17.7 (1.76) | 24.1 (1.77) | 17.1 (1.14) | 17.9 (5.25) | 23.4 (1.11) | 10.2 (2.2) |
Completeness, % | 91.8 (90.7) | 95.1 (85.9) | 95.5 (94.7) | 85.7 (77.8) | 94.7 (89.4) | 85.4 (20.6) | 86.7 (70.5) | 96.9 (87.3) | 99.6 (99.4) |
Ano. Rmerge | 0.060 (0.382) | 0.068 (0.334) | 0.049 (0.338) | 0.055 (0.366) | 0.062 (0.389) | 0.065 (0.397) | 0.060 (0.396) | 0.062 (0.419) | - |
Ano. Completeness, % | 86.3 (79.2) | 89.5 (72.7) | 91.8 (85.3) | 77.4 (61.5) | 89.3 (78.5) | 81.4 (13.0) | 78.8 (56.9) | 93.8 (75.7) | - |
No. unique reflections | 90,291 | 99,945 | 69,377 | 119,745 | 117,666 | 70,581 | 155,226 | 160,625 | 135,428 |
No. free reflections | 2,776 | 2,866 | 2,112 | 3,584 | 3,133 | 2,124 | 2,461 | 3,230 | 2,102 |
Wavelength, Å | 1.0 | 0.92 | 1.736 | 0.97 | 1.0 | 1.2 | 1.0 | 0.97 | 1.0 |
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Model refinement |
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R work/Rfree | 0.245/0.297 | 0.223/0.270 | 0.259/0.307 | 0.232/0.282 | 0.238/0.281 | 0.216/0.279 | 0.239/0.290 | 0.244/0.282 | 0.224/0.254 |
R work/Rfree (outer shell) | 0.32/0.38 | 0.35/0.36 | 0.28/0.37 | 0.48/0.49 | 0.28/0.34 | 0.29/0.38 | 0.38/0.37 | 0.31/0.31 | 0.325/0.347 |
No. atoms | 17,125 | 7,131 | 17,233 | 17,454 | 17,064 | 17,371 | 17,497 | 17,304 | 41,688 |
No. res. (% cmpl.) ¶ | 2,081 (96.0) | 2,078 (95.9) | 2,104 (97.1) | 2,099 (96.9) | 2,101 (97.0) | 2,110 (97.4) | 2,111 (97.4) | 2,110 (97.4) | 5,178 (90.2) |
No. cofactors | 5 | 5 | 5 | 4 | 5 | 5 | 5 | 5 | 30 |
No. sol. mol.|| | 237 | 243 | 189 | 454 | 298 | 322 | 567 | 271 | 0 |
rmsd bond length, Å | 0.021 | 0.018 | 0.029 | 0.015 | 0.021 | 0.015 | 0.018 | 0.022 | 0.013 |
rmsd bond angle, ° | 2.0 | 1.7 | 2.2 | 1.9 | 2.0 | 2.0 | 1.8 | 2.1 | 1.7 |
0.361 | 0.322 | 0.454 | 0.279 | 0.304 | 0.424 | 0.254 | 0.215 | N/A | |
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*Statistics for all data sets were computed at –1 s cutoff for reflection intensity; space group symmetry for all crystals is I4122. † The chemical structure of JG144 is S-3-anilino-5-methyl-5-(4,6-difluorophenyl)-1,3-oxazolidine-2,4-dione.‡ Rmerge is defined as S|Ih,i - <Ih>| / SIh,i, where Ih,i is the intensity for ith observation of a reflection with Miller index h, and <Ih> is the mean intensity for all measured Ihs and Friedel pairs.§ Values in parentheses are for the highest-resolution shells.¶ No. of residues in the model and percentage of completeness.|| No. of solvent of molecules in the model.**Diffraction-component precision index [Blow, D. M. (2002) Acta Crystallogr. D 58, 792–797]. | |||||||||
Table 5. Sequence identities for various structural elements in the cyt b subunit
Secondary structural elements | Database: 31,568*, % | Data base id 0.95: 5355,† % | Residue range | Number of residues |
a | 67.5 | 63.5 | 10–17 | 8 |
A | 84.1 | 79.0 | 29–53 | 25 |
ab | 87.2 | 81.8 | 62–71 | 10 |
B | 83.7 | 79.8 | 78–101 | 24 |
C | 82.4 | 78.1 | 113–133 | 21 |
cd1 | 96.0 | 93.1 | 136–151 | 16 |
cd2 | 73.5 | 68.2 | 156–165 | 10 |
D | 75.8 | 71.1 | 177–203 | 27 |
E | 62.0 | 57.1 | 221–245 | 25 |
ef | 97.2 | 93.9 | 272–282 | 11 |
F1 | 82.0 | 77.5 | 288–296 | 9 |
F2 | 61.3 | 59.1 | 299–306 | 8 |
G | 72.0 | 66.1 | 319–341 | 23 |
H1 | 75.7 | 68.9 | 344–361 | 18 |
H2 | 61.2 | 58.9 | 365–376 | 12 |
Overall | 79.3 | 74.6 | 1–379 | 379 |
*Alignments were done with BLAST-P on the National Institutes of Health Supercomputer (biowulf.nih.gov) using the nr database: All nonredundant GenBank CDS translations, PDB, SwissProt, PIR, PRF excluding environmental samples. Number of sequences included: 31,568.
†The sequences obtained were filtered so that no pair of any given sequences has >95% identity. Number of sequences included: 5,355.
Supporting Materials and Methods
Purification of Rsbc1.
The purification was published in ref. 1; it was slightly modified for the purpose of crystallization. Chromatophore membranes were prepared from cells harboring plasmids (pRKDfbcFmBCHQ) coding for either wild-type or mutant Rsbc1 by disrupting cells with a French Press followed by differential centrifugations. To purify the His-6-tagged Rsbc1, the chromatophore suspensions were adjusted to a cyt b concentration of 25 mM with 50 mM Tris•HCl (pH 8.0 at 4°C), containing 20% glycerol, 1 mM MgSO4, and 1 mM phenylmethylsulfonyl fluoride. Dodecyl-D-maltopyranoside solution (10% wt/vol) was added dropwise to a final detergent to protein ratio of 0.57 (mg/mg). After centrifugation, the supernatant was loaded on a Ni-NTA column, which was washed thoroughly with 6 column volumes (CV) of buffer A (50 mM Tris•HCl, pH 8.0 at 4°C, 200 mM NaCl, and 0.01% DDM), 6 CV of buffer A in the presence of 5 mM histidine and 4 CV of buffer B [50 mM Tris•HCl, pH 8.0 at 4°C, 200 mM NaCl, and 0.5% b-octyl-D-glucopyranoside (b-OG)] in the presence of 5 mM histidine. The protein fractions were eluted with buffer B in the presence of 200 mM histidine and concentrated with a Centriprep-50 concentrator to a final concentration of 300 mM.Crystallization and Structure Determination of the Rsbc1.
Crystals of Rsbc1 grew in sitting drops containing protein (15 mg/ml) in 50 mM Tris•HCl (pH 7.5), 0.5% b-OG, 0.12% sucrose monocaprate, 200 mM histidine, and 10% PEG 400 and were equilibrated against a well solution consisting of 50 mM Tris•HCl (pH 8.0), 20% glycerol, 26% PEG 400, and 500 mM NaCl. The Rsbc1 crystallized in three different crystal forms and typically diffracted x-rays anisotropically to 3-Å resolution. However, after optimizations in crystallization and cryoprotection conditions, a crystal form belonging to the space group C2 gave rise to a stable and relatively isotropic diffraction under cryogenic conditions.The crystal structure of Rsbc1 was determined by the molecular replacement method implemented in the program MOLREP (2) with an initial phasing model constructed from the bovine enzyme in a dimeric form (Protein Data Bank ID code 1NTZ) and subsequently refined.
Structure Refinement and Analysis.
Bovine structures in complex with various inhibitors were refined with the program Refmac (3). Atomic models of stigmatellin, UHDBT, MOAS, azoxystrobin, myxothiazol, and JG144 were constructed based on similar structures in the Cambridge Crystallographic Data Centre (4) using the program Conquest. For Rsbc1, the structure was refined with the program CNS (5), and the model was built with the molecular graphics program O (6).Site-Directed Mutagenesis, Enzyme Preparations, and Activity Assay.
Mutants in the cyt b subunit of Rsbc1 were constructed following a published procedure (7). Chromatophore and purified proteins were prepared as described (8) and stored at –80°C in the presence of 20% glycerol until use. To assay bc1 activity, the protein preparation was diluted with 50 mM Tris•Cl buffer (pH 8.0) containing 200 mM NaCl to a final concentration of cyt b of 5 mM. Five microliters of the diluted samples was added to 1 ml of assay mixture containing 100 mM Na+/K+ phosphate buffer (pH 7.4), 0.3 mM EDTA, 100 mM cyt c, and 25 mM 2,3-dimethoxy-5-methyl-6-geranyl-1,4-benzoquinol [Q2H2 (9)]. Activities (i.e., the reduction of cyt c) were determined by measuring the increase in absorbance at a wavelength of 550 nm in a Shimadzu UV 2101 PC spectrophotometer at 23°C. The specific activity of Rsbc1 was calculated using a millimolar extinction coefficient of 18.5 liters/mmol per cm from the observed absorbance after correcting it for the effect of nonenzymatic oxidation of Q2H2.1. Yu, L. & Yu, C. A. (1991) Biochemistry 30, 4934–4939.
2. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. (1997) Acta Crystallogr. D 53, 240–255.
3. Allen, F. H. (2002) Acta Crystallogr. B 58, 380–388.
4. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-
Kanstleve, R. W., Jiang, J., Kuszewski, J., Nilges, M., Pannu, N. S., et al. (1998) Acta Crystallogr. D 54, 905–921.
5. Jones, T. A., Zou, J. Y. & Cowan, S. W. (1991) Acta Crystallogr. A 47, 110–119.
6. Mather, M. W., Yu, L. & Yu, C. A. (1995) J. Biol. Chem. 270, 28668–28675.
7. Tian, H., Yu, L., Mather, M. W. & Yu, C. A. (1998) J. Biol. Chem. 273, 27953–27959.
8. Yu, C. A. & Yu, L. (1982) Biochemistry 21, 4096–4101.
9. Vagin, A. & Teplyakov, A. (2000) Acta Crystallogr. B 56, 1622–1624.