7
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. 700699D-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 0.4mM isopropyl-b-D-thiogalactopyranoside (IPTG) at an optical cell density (A600) of 12-15 for 1.5 hours. All subsequent procedures were performed at 4°C unless specified differently. Cells were harvested by centrifugation, resuspended in 50mM Tris pH 8.2, 500mMNaCl and disrupted using a M110L microfluidizer (Microfluidics) at 15000 psi external pressure. Cell membranes were produced by ultracentrifugation at 100,000xg. Membrane proteins were solubilized in 100mM sodium phosphate pH 8.0, 200mM NaCl, 15%(w/v) glycerol, 20mM imidazole, 0.1%(w/v) n-dodecyl-b-D-maltopyranoside (DDM, Anatrace) and 1%(w/v) polyoxyethylene-8-dodecylether (anapoe-C12E8, Anatrace) for 1.5 hours, while all subsequent buffers only contained C12E8 as detergent. The solubilized membrane proteins were loaded onto a NiNTA superflow affinity column (Qiagen), washed with 50mM imidazole, and Sav1866 was eluted with 200mM imidazole. The buffer was exchanged to 10mM Tris pH8.2, 100mM NaCl by desalting and the protein was concentrated to 15mg/ml using an Amicon Ultra-15 concentrator unit (Millipore) with a molecular cutoff of 100kDa.
ATPase activity assay. ATPase activity of purified S.aureus Sav1866 in C12E8 detergent was measured at room temperature as described earlier for the E. coli vitamin B12 transporter BtuCD1. For inhibition, 1 mM freshly boiled sodium ortho-vanadate was added to the solution. Inorganic phosphate was assayed by a modified molybdate protocol2.
Reconstitution in proteoliposomes. Purified Sav1866 protein was reconstituted in proteoliposomes as described earlier for the E. coli vitamin B12 transporter BtuCD1. 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, 50mM Li3citrate, 150mM K3citrate, 100mM Na2HPO4 and 3mM MgCl2. 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 flashfreezing 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 Scalepack3. The structure was solved by multiple isomorphous replacement with anomalous scattering using data from xenon (collected at a wavelength of 1.54004 Å), Ta6Br14 (collected at a wavelength of 1.25447 Å), selenomethionine (collected at a wavelength of 0.97894 Å), 2´-iodo-ADP (collected at a wavelength of 1.06994 Å), and ethyl mercury phosphate (EMP, collected at a wavelength of 1.00799 Å) derivative crystals (Table S1). Native data was collected at a wavelength of 1.07252 Å. Initial phases were obtained using SOLVE4 and were used to calculate anomalous cross-Fourier maps using programs from the CCP4 suite5. Additional heavy atom positions were refined using SHARP6 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 Solomon7 and DM8. This yielded electron density of excellent quality (Fig. S2), and a model of the entire protein (residues 1 to 578) was built using O9. 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 CNS10, and except for crystal contact regions in the extracellular loops and a few residues with evidently different density in the two subunit, strict twofold noncrystallographic 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.
Table S1 | Data collection and refinement statistics
Data Collection
Space group / C2 / C2 / C2 / C2 / C2 / C2
Cell dimensions
a, b, c (Å) / 161.279
103.955
181.013 / 161.391
103.839
181.948 / 160.895
104.163
181.937 / 161.399
103.886
181.644 / 160.621
105.045
181.245 / 161.510
104.074
181.546
a, b, g (º) / 90.000
97.987
90.000 / 90.000
97.471
90.000 / 90.000
97.849
90.000 / 90.000
97.501
90.000 / 90.000
98.021
90.000 / 90.000
97.672
90.000
Resolution (Å) / 30-3.0 / 30-3.3 / 30-3.8 / 30-3.3 / 30-3.1 / 30-3.3
Rsym or Rmerge* / 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/sI / 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) / 20-3.0
No. reflections / 54627/4176
Rwork/ Rfree / 0.255/0.272
No. atoms
Protein / 9170
ADP / 54
Na / 2
Water / 16
B-factors
Protein / 102.3
Ligand / 83.2
Ion / 27.5
Water / 30.9
R.m.s deviations
Bond lengths (Å) / 0.0097
Bond angles (º) / 1.4
*Highest resolution shell is shown in parenthesis.
Table S2 | Comparison of TM helix arrangements of Sav1866 and inverted S. typhimurium MsbA.
1 / 1 / same / yes
2 / 4' / same / no
3 / 5' / same / no
4 / 2 / same / yes
5 / 6 / opposite / yes
6 / 3 / opposite / yes
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, 16301-16309 (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, 307-326 (1997).
4. Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D55, 849-861 (1999).
5. CCP4. The CCP4 (Collaborative Computational Project Number 4) suite: programs for protein crystallography. Acta Crystallogr. D50, 760-763 (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, 472-494 (1997).
7. Abrahams, J. P. & Leslie, A. G. W. Methods used in the structure determination of bovine mitochondrial F-1 ATPase. Acta Crystallogr. D52, 30-42 (1996).
8. Cowtan, K. D. & Main, P. Phase combination and cross validation in iterated density-modification calculations. Acta Crystallogr. D52, 43-48 (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, 110-119 (1991).
10. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D54, 905-921 (1998).
Supplementary Figure Legends
Figure S1 | ATPase activity of purified Sav1866. a, Sav1866 protein in C12E8 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, Drug-stimulated 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 1s (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 7s), Ta6Br14 (green, contoured at 5s'-iodo-ADP (purple, countoured at 8s), and xenon (red, contoured at 15s). 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 pdbset5, 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.
Fig. S1
Fig. S2
Fig. S3
Fig. S4