Structure of a bacterial multi-drug ABC transporter

Transcription

1 1 Structure of a bacterial multi-drug ABC transporter Roger J. P. Dawson and Kaspar P. Locher Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland Supplementary Information Methods Expression and purification of S. aureus Sav1866. The Sav1866 gene was amplified from Staphyloccus aureus genomic DNA (ATCC no D-S) by polymerase chain reaction and inserted via NdeI/BamHI restriction sites into a modified pet19b (Novagen) expression vector, attaching a N-terminal decahistidine affinity tag to the protein. The resulting plasmid was verified by DNA sequencing. The protein was overexpressed in E.coli BL21-codon plus (DE3)-RIPL (Stratagene) grown in an 10 liter fermentor at 37 C in terrific broth medium supplemented with 1 % (w/v) glucose. Expression was induced by the addition of 0.4 mm isopropyl-β-dthiogalactopyranoside (IPTG) at an optical cell density (A 600 ) of for 1.5 hours. All subsequent procedures were performed at 4 C unless specified differently. Cells were harvested by centrifugation, resuspended in 50 mm Tris ph 8.2, 500 mm NaCl and disrupted using a M-110L microfluidizer (Microfluidics) at psi external pressure. Cell membranes were produced by ultracentrifugation at 100,000xg. Membrane proteins were solubilized in 100 mm sodium phosphate ph 8.0, 200 mm NaCl, 15 % (w/v) glycerol, 20 mm imidazole, 0.1 % (w/v) n-dodecyl-β-dmaltopyranoside (DDM, Anatrace) and 1 % (w/v) polyoxyethylene-8-dodecylether (anapoe-c 12 E 8, Anatrace) for 1.5 hours, while all subsequent buffers only contained C 12 E 8 as detergent. The solubilized membrane proteins were loaded onto a NiNTA superflow affinity column (Qiagen), washed with 50 mm imidazole, and Sav1866 was eluted with 200 mm imidazole. The buffer was exchanged to 10 mm Tris ph 8.2, 100 mm NaCl by desalting and the protein was concentrated to 15 mg/ml using an Amicon Ultra-15 concentrator unit (Millipore) with a molecular cutoff of 100 kda.

2 2 ATPase activity assay. ATPase activity of purified S.aureus Sav1866 in C 12 E 8 detergent was measured at room temperature as described earlier for the E. coli vitamin B 12 transporter BtuCD 1. For inhibition, 1 mm freshly boiled sodium ortho-vanadate was added to the solution. Inorganic phosphate was assayed by a modified molybdate protocol 2. Reconstitution in proteoliposomes. Purified Sav1866 protein was reconstituted in proteoliposomes as described earlier for the E. coli vitamin B 12 transporter BtuCD 1. Drug-stimulated ATPase activity was monitored by adding various drugs to the proteoliposomes. Crystallization and structure determination. Sav1866 was crystallized by vapor diffusion in sitting drops at 10 C against a reservoir containing 20 % (w/v) polyethylene glycol 6000, 50 mm Li 3 -citrate, 150 mm K 3 -citrate, 100 mm Na 2 HPO 4 and 3 mm MgCl 2. ADP was added to the concentrated protein to 1 mm, and the protein to reservoir ratio in the sitting drop was 2:1. Box-shaped crystals appeared after 5 days and matured to full size within 2-3 weeks. They belonged to the space group C2 with one full transporter (Sav1866) 2 in the asymmetric unit. The crystals were cryo-protected in 15% (w/v) glycerol before flash-freezing in liquid nitrogen. Diffraction data were collected at the protein crystallography beamline S06 PX at the Swiss Light Sourse (SLS) and processed with Denzo and Scalepack 3. The structure was solved by multiple isomorphous replacement with anomalous scattering using data from xenon (collected at a wavelength of Å), Ta 6 Br 14 (collected at a wavelength of Å), selenomethionine (collected at a wavelength of Å), 2 -iodo-adp (collected at a wavelength of Å), and ethyl mercury phosphate (EMP, collected at a wavelength of Å) derivative crystals (Table S1). Native data was collected at a wavelength of Å. Initial phases were obtained using SOLVE 4 and were used to calculate anomalous cross-fourier maps using programs from the CCP4 suite 5. Additional heavy atom positions were refined using SHARP 6 and the FOM of centric/acentric reflections was 0.32/0.26 overall for phasing from 30 to 3.0 Å resolution. Solvent flattening and non-crystallographic averaging was performed using Solomon 7 and DM 8. This yielded electron density of excellent quality (Fig. S2), and a

3 3 model of the entire protein (residues 1 to 578) was built using O 9. Chain tracing was aided by the known positions of methionines from the selenomethioinine data, and the location of ADP was indicated by the presence of the iodine signal from the 2'-iodo- ADP data (Fig. S3). Refinement was carried out using CNS 10, and except for crystal contact regions in the extracellular loops and a few residues with evidently different density in the two subunit, strict two-fold non-crystallographic symmetry was imposed. Ramachandran analysis revealed 86.0% in the most favorable, 14.0% in the additional allowed, and no residues in the generously allowed or disallowed regions.

4 4 Table S1 Data collection and refinement statistics Native Xenon Ta 3 Br 14 SelenoMet 2 -I-ADP EMP Data Collection Space group C2 C2 C2 C2 C2 C2 Cell dimensions a, b, c (Å) α, β, γ (º) Resolution (Å) R sym or R merge * 8.1 (56.9) 10.9 (45.7) 10.8 (27.8) 8.1 (35.1) 7.9 (45.9) 9.9 (53.3) I/σI 20.9 (2.18) 14.5 (2.00) 13.3 (3.96) 16.7 (2.83) 19.5 (1.99) 15.0 (2.40) Completeness (%) 99.1 (87.9) 98.0 (85.9) 95.2 (73.9) 97.6 (79.2) 98.0 (79.9) 100 (100) Redundancy 7.4 (5.3) 7.1 (5.1) 6.6 (5.5) 6.0 (4.4) 6.2 (5.1) 6.4 (5.9) Refinement Resolution (Å) No. reflections 54627/4176 R work/ R free 0.255/0.272 No. atoms Protein 9170 ADP 54 Na 2 Water 16 B-factors Protein Ligand 83.2 Ion 27.5 Water 30.9 R.m.s deviations Bond lengths (Å) Bond angles (º) 1.4 *Highest resolution shell is shown in parenthesis.

5 5 Table S2 Comparison of TM helix arrangements of Sav1866 and inverted S. typhimurium MsbA. TM helix of inverted S.typhimurium MsbA TM helix of S. aureus TM helix direction Sav same yes 2 4' same no 3 5' same no 4 2 same yes 5 6 opposite yes 6 3 opposite yes TM helix in same subunit

6 6 Supplementary References 1. Borths, E. L., Poolman, B., Hvorup, R. N., Locher, K. P. & Rees, D. C. In vitro functional characterization of BtuCD-F, the Escherichia coli ABC transporter for vitamin B-12 uptake. Biochemistry 44, (2005). 2. Chifflet, S., Torriglia, A., Chiesa, R. & Tolosa, S. A method for the determination of inorganic phosphate in the presence of labile organic phosphate and high concentrations of protein: Application to lens ATPases. Anal. Biochem. 168, 1-4 (1988). 3. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, (1997). 4. Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D55, (1999). 5. CCP4. The CCP4 (Collaborative Computational Project Number 4) suite: programs for protein crystallography. Acta Crystallogr. D50, (1994). 6. de la Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, (1997). 7. Abrahams, J. P. & Leslie, A. G. W. Methods used in the structure determination of bovine mitochondrial F-1 ATPase. Acta Crystallogr. D52, (1996). 8. Cowtan, K. D. & Main, P. Phase combination and cross validation in iterated density-modification calculations. Acta Crystallogr. D52, (1996). 9. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved Methods for Building Protein Models in Electron- Density Maps and the Location of Errors in These Models. Acta Crystallogr. A47, (1991). 10. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D54, (1998).

7 7 Supplementary Figure Legends Figure S1 ATPase activity of purified Sav1866. a, Sav1866 protein in C 12 E 8 detergent solution. Inorganic phosphate generated by ATP hydrolysis is shown as a function of time (closed symbols). A control reaction (open symbols) was inhibited by addition of 1 mm ortho-vanadate at 10 minutes. Error bars indicate standard deviations, all reactions were performed in triplicates. b, Similar to a, but using Sav1866 reconstituted in liposomes (closed symbols). A moderate stimulation of the ATPase activity upon addition of 1 mm doxorubicin is evident (open symbols). c, Drugstimulated ATPase activity. Initial ATPase rates were determined in triplicates by linear regression of time points similar to experiments shown in panel b. Closed squares indicate relative ATPase rates upon addition of Hoechst33342, whereas open symbols indicate those with vinblastine. Figure S2 Experimental electron density. Stereo view of the backbone of the Sav1866 transmembrane domains in red, with the experimental electron density map contoured at 1σ (blue mesh). Figure S3 Anomalous electron density maps. The backbone of Sav1866 is in white. Anomalous cross-fourier maps were calculated using experimental phases and the anomalous differences of the following data sets: Selenomethiononine (yellow, contoured at 7σ), Ta 6 Br 14 (green, contoured at 5σ), 2'-iodo-ADP (purple, countoured at 8σ), and xenon (red, contoured at 15σ). The selenomethiononine peaks were helpful in chain tracing. Figure S4 Superposition of Sav1866 and MsbA. a, Stereo views of protein backbones of the transmembrane domains of a single subunit of Salmonella typhimurium MsbA (pdb entry 1Z2R, purple) and Sav1866 (green) after manual superpositions of the entire transporters. No agreement in the helix arrangement is apparent. b, The structure of MsbA was inverted using the coordinate manipulation program pdbset 5, and the resulting molecule was superimposed onto Sav1866. The transmembrane domain of a single monomer of inverted MsbA is shown in purple, while that of subunit A of Sav1866 is shown in green and transmembrane helices TM1 and TM2 of subunit B are shown in yellow. The approximate superposition of TM helices is evident. The differences in helix directionality and subunit assignment are detailed in Table S2.

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