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1 Structure of an ABC transporter-binding protein complex Kaspar Hollenstein, Dominik C. Frei, and Kaspar P. Locher Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland Supplementary Information Methods Expression and Purification of A. fulgidus ModBC The genes encoding the binding protein ModA and the ABC transporter ModBC were cloned from Archaeoglobus fulgidus DSM 4304 genomic DNA, where they form an operon with the organization modabc, similar to those of E. coli or A. vinelandii. ModA and ModBC were separately overexpressed in E. coli. A modbc co-expression vector was constructed by amplification of the genes AF009 and AF0093 from Archaeoglobus fulgidus genomic DNA (ATCC 49558D) by polymerase chain reaction and insertion into a modified pet-19b (Novagen) expression vector, attaching a N-terminal decahistidine affinity tag to ModB. ModBC was over-expressed in Escherichia coli BL1-CodonPlus(DE3)-RIPL (Stratagene) grown in Terrific Broth supplemented with 1% (w/v) D-(+)-glucose in a 10-liter fermentor at 37 C. Protein production was induced by addition of 0.4 mm isopropyl-β-d-thiogalactopyranoside (IPTG) at an optical density (A 600 ) of for 1 hour. All subsequent steps were carried out at 4 C unless indicated differently. Cells were harvested by centrifugation for 8 min at x g, resuspended in 50 mm Tris-HCl ph 7.5, 0.5 M NaCl and ruptured using a M-110L microfluidizer (Microfluidics) at psi external pressure. Membranes were pelleted by ultracentrifugation at x g for 40 min, washed and resuspended in 50 mm Tris-HCl ph 7.5, 0.5 M NaCl. Membrane proteins were solubilized by addition of 1% (w/v) n-dodecyl-β-d-maltopyranoside (DDM, Anatrace) and 1% (w/v) octaethylene glycol monododecylether (C 1 E 8, Anatrace) and incubation 1 1

2 for 1 hour, whereas all subsequent buffers contained only C 1 E 8 as detergent. Solubilized membrane proteins were loaded onto a NiNTA superflow (Qiagen) affinity column, washed with 60 mm imidazole, and ModBC was eluted with 160 mm imidazole. The buffer was exchanged to mm Tris-HCl ph 7.5, 10 mm Na SO 4, 0.5 mm EDTA and the protein was concentrated to 0 mg / ml using an Amicon Ultra-15 concentrator unit (Millipore) with a molecular cutoff of 100 kda. Expression and Purification of A. fulgidus ModA The fragment of the moda (AF0094) gene encoding the predicted mature part of the periplasmic binding-protein (amino acids 3-34) was amplified from A. fulgidus genomic DNA by polymerase chain reaction and inserted into a modified pet-19b (Novagen) expression vector, attaching an N-terminal decahistidine affinity tag followed by a TEV protease cleavage site. The protein was over-expressed in the cytoplasm of E. coli BL1-CodonPlus(DE3)-RIPL (Stratagene). Cells were grown in Terrific Broth media supplemented with 1% (w/v) glycerol at 37 C. At an optical density (A 600 ) of the temperature was lowered to 5 C and cells were grown into stationary phase where expression of ModA was auto-induced. All subsequent purification steps were carried out at room temperature unless stated differently. Cells were harvested by centrifugation and disrupted using a M-110L microfluidizer (Microfluidics) at psi external pressure. Insoluble material was removed by centrifugation ( x g for 45 min at 4 C) and the supernatant was loaded onto a NiNTA superflow affinity column (Qiagen). The column was washed with 5 mm and 80 mm imidazole, and ModA was eluted with 300 mm imidazole. The buffer was exchanged to 50 mm Tris-HCl ph 8.0, 100 mm NaCl, 0.5 mm EDTA, and mm 1,4-dithio-DL-threitol (DTT) by desalting and the protein was incubated with TEV protease for hours. The buffer was exchanged to 5 mm Tris-HCl ph 8.0, 50 mm NaCl, and 5 mm imidazole prior to removal of cleavage fragments and uncleaved material by a re-run on a NiNTA superflow column. The protein was desalted into 5 mm Tris-HCl ph 7.5, 50 mm NaCl and was concentrated to between 0 and 30 mg / ml using an Amicon Ultra-15 concentrator unit (Millipore) with a molecular cutoff of 10 kda.

3 ATPase activity assays The ATP hydrolysis activity of purified ModBC in detergent solution was assayed at room temperature essentially as described earlier for BtuCD 1. Reactions contained 0.3 mg / ml ( μm) ModBC, 0 mm Tris-HCl ph 8.0, 10 mm MgCl, 100 mm NaCl, 1 mm Na SO 4, and mm ATP. Inhibition was achieved by addition of mm freshly boiled sodium ortho-vanadate. Vanadate trapping of ModB C A complex ModBC and ModA were incubated in detergent solution at a molar ratio of :1 (ModA to ModBC) in the presence of mm ATP, 0.5 mm freshly boiled sodium orthovanadate, and 1.0 mm CoCl. Cobalt chloride was used because the complex was more stable than in magnesium salts. After 30 minutes incubation at room temperature, the mixture was moved to 4 C and applied to a desalting column to remove ATP, vanadate, and cobalt chloride. The desalted sample was concentrated using a Amicon Ultra-15 concentrator unit (Millipore) with a molecular cutoff of 50 kda and applied to a Superdex 00 10/300 GL gel filtration column (GE Healthcare). Samples were collected and analyzed by SDS PAGE. Crystallization and structure determination of the ModB C A complex Purified ModBC and ModA were mixed at an equimolar ratio resulting in a final protein concentration of 19.6 mg/ml, and Na WO 4 was added to 1 mm. The complex was crystallized by vapor diffusion in sitting drops at 0 C against a reservoir containing 17% (w/v) polyethylene glycol (PEG) 4000, 30 mm Tris-HCl ph 8., 100 mm MgCl, 0.0% (w/v) C 1 E 8, and 0.1% (w/v) n-decyl-β-d-maltopyranoside (DM, Anatrace). The protein to reservoir volume ratio was 1:1. Crystals appeared after 4 days and reached their full size within 10 to 15 days. They belonged to the space group P 1 with two full transporter-binding protein complexes (ModB C A) in the asymmetric unit. Crystals were flash-frozen by immersion in liquid nitrogen, and diffraction data were collected at the protein crystallography beamline X06SA at the Swiss Light Source (SLS) and processed using programs from the HKL000 software package. Phases were obtained by collecting three-wavelength anomalous diffraction (MAD) data (Table S1) using WO 4 bound to ModA in the complex. Initial phases were obtained using SHELX 3 and 3

4 the heavy atom positions were refined and phases calculated using SHARP 4. The obtained FOM of centric / acentric reflections was 0.66 / 0.45, respectively, for phasing from 30 to 3.15 Å resolution. Solvent flattening was performed using Solomon 5. This yielded electron density of excellent quality (Fig. Sa), and the backbone and side chains of 195 out of 1313 amino acid residues were built into the map using the program O 6. A selenium peak data set of selenomethionine derivatized ModB C A crystals provided direct visualization of the methionine positions via anomalous cross Fourier maps calculated with CCP4 programs 7 and thus valuable help in tracing the chains (Fig. Sb). Structure refinement was carried out using CNS 8. Except for crystal contact regions in ModA and ModC and some residues involved in the ModA-ModB interface with evidently different electron density, strict -fold noncrystallographic symmetry was imposed. All residues are in the most favored or additional allowed regions of the Ramachandran plot. Crystallization of ModA and Structure Determination ModA was crystallized by vapor diffusion in sitting drops at 0 C against a reservoir containing 40% (w/v) PEG 400, 50 mm bicine-naoh ph 9.0, 100 mm Mg(NO 3 ), and either 1.5 mm Na WO 4 or 1.5 mm Na MoO 4. The protein to reservoir volume ratio was 1:1. Bipyramidal crystals matured to their final size within 3-7 days. The crystals belonged to space group P4 1 1 with one ModA per asymmetric unit. Crystals were flash-frozen in liquid nitrogen and diffraction data were collected at the protein crystallography beamlines X06SA and X10SA at the Swiss Light Source (SLS) and processed using programs from the HKL000 software package. The structure was solved by single-wavelength anomalous diffraction (SAD) of a selenomethionine ModA crystal with bound WO 4 at the peak wavelength of the selenium absorption edge (Table S). Initial phases were obtained using the programs SHELX 3, and the structure was automatically built using ARP/WARP 9. The structure was checked and missing segments built using the program O 6 and refinement was carried out using CNS 8 (Table S). Individual B-factors were refined throughout and all residues were in the most favored or additional allowed regions of the Ramachandran plot. 4

5 Table S1: Data collection and refinement statistics of the ModB C A complex with bound WO 4 Crystal 1 Crystal Crystal 3 SelenoMet high remote high remote peak inflection peak Data Collection Space group P 1 P 1 P 1 Wavelength (Å) Cell dimensions (Å) a b c β (º) Resolution (Å)* ( ) ( ) ( ) ( ) ( ) R sym or R merge (%)* 7. (57.9) 6.1 (53.1) 8.0 (4.3) 6.4 (51.3) 10.5 (39.8) I/σI* 17.6 (.4) 0. (.6) 18.1 (4.).9 (3.1) 15.4 (4.3) Completeness (%)* 100 (100) 100 (100) 100 (100) 100 (100) 100 (100) Redundancy* 4.7 (4.8) 4. (4.) 6.3 (6.3) 6. (6.0) 6.5 (6.5) Refinement Resolution (Å)* ( ) No. reflections 8435/4387 R work /R free * 0.56/0.83 (0.393/0.407) No. atoms Protein 0187 WO 4 10 Mg 6 PO 4 0 B-factors Protein WO Mg 77.5 PO R.m.s deviations Bond lengths (Å) Bond angles (º) 1.58 *Highest resolution shell is shown in parenthesis. 5

6 Table S: Data collection and refinement statistics of ModA Native MoO 4 Native WO 4 SelenoMet WO 4 peak Data Collection Space group P4 1 1 P4 1 1 P4 1 1 Wavelength (Å) Cell dimensions (Å) a = b c α = β = γ (º) Resolution (Å)* ( ) ( ) ( ) R sym or R merge (%)* 7.9 (36.) 5.1 (3.6) 11.4 (31.4) I/σI* 40.5 (8.0) 44.5 (6.7) 8.7 (6.6) Completeness (%)* 100 (100) 97.6 (94.9) 100 (99.4) Redundancy* 1.3 (17.0) 16.0 (14.7) 3.1 (18.1) Refinement Resolution (Å)* ( ) ( ) No. reflections 4497/ /474 R work /R free * 0.178/0.03 (0.0/0.49) No. atoms Protein WO 4 5 MoO 4 5 Mg 1 1 NO H O B-factors Protein WO MoO Mg NO H O R.m.s deviations Bond lengths (Å) Bond angles (º) *Highest resolution shell is shown in parenthesis /0.1 (0.08/0.09) 6

7 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-1 uptake. Biochemistry 44, (005).. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 76, (1997). 3. Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. D58, (00). 4. de la Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 76, (1997). 5. Abrahams, J. P. & Leslie, A. G. W. Methods used in the structure determination of bovine mitochondrial F-1 ATPase. Acta Crystallogr. D5, 30-4 (1996). 6. 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). 7. CCP4. The CCP4 (Collaborative Computational Project Number 4) suite: programs for protein crystallography. Acta Crystallogr. D50, (1994). 8. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D54, (1998). 9. Morris, R. J., Perrakis, A. & Lamzin, V. S. ARP/wARP and automatic interpretation of protein electron density maps. Methods Enzymol. 374, 9-44 (003). 10. Shannon, R. D. Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides. Acta Crystallogr. A3, (1976). 11. Marcus, Y. Ionic radii in aqueous solutions. Chem. Rev. 88, (1988). 7

8 Supplementary Figure Legends Figure S1 ATPase activity of purified ModBC. Inorganic phosphate release by ATP hydrolysis is shown as a function of time. At t=10 min (arrow), mm ortho-vanadate was added to the protein, and inhibition is shown (dashed line). (B) Size exclusion chromatography of vanadate-trapped ModBC-A. (C) SDS PAGE of peak 1 and peak from the gel filtration analysis shown in b. The gel was stained with Coomassie Blue. Molecular masses (in kda) and protein components are indicated to the left and the right of the gel, respectively. Lane 1: marker proteins; lanes and 3, pure ModA and ModBC for reference; lanes 4 and 5, peak 1 from gel filtration experiment (lane 5 half the amount of lane 4), corresponding to the vanadate-trapped ModBC-A complex; lane 6, peak from gel filtration experiment, corresponding to excess ModA. Note that the membrane-spanning ModB stains weaker than ModC, presumably due to its increased hydrophobicity. Figure S Experimental electron density maps. a, Stereo view of the backbone of the ModB C A complex, with a section of the experimental electron density map (blue mesh) contoured at 1σ. ModA and ModB backbones are colored in red and yellow, respectively. The ModC subunits are not visible in this view. b, Stereo view of the Cα trace (yellow) of the full ModB C A complex. The red mesh represents an anomalous cross-fourier map calculated from the selenomethionine peak data at 3.7 Å resolution and contoured at 4.5σ. The methionine positions were helpful in chain tracing. Figure S3 Architecture of ModB subunit. Stereo views of the transmembrane ModB subunits, with one subunit in yellow ribbon representation, the other as a blue backbone trace. TM helices are numbered, short helices following a TM helix additionally carry letters. Amino- and carboxy-termini are indicated with N and C, respectively. Figure S4 Sequence alignment of TMDs. Alignment of the A. fulgidus ModB sequence with those of other ABC importers carrying molybdate (Mod), sulfate (Cys), and phosphate (Pst). Amino acids of the gate regions 1 and are shaded in green and red, respectively. The conserved Phe00 is in white letters and shaded black, while 8

9 amino acids of the "EAA-motif" are shaded yellow. The transmembrane helices and conserved motifs are indicated above the sequences. Figure S5 ModA structure and binding site. a, Stereo view of superimposed backbone traces of A. fulgidus ModA (red) and E. coli ModA (black). Bound molybdate is visible at the center of the molecule. The additional 3-stranded β-sheet present in A. fulgidus is visible at the lower left side. b, Stereo view of bound tungstate in the ModA binding site. The W atom is shown as a green sphere, all other elements are shown in ball-and-stick representation, and the residues are labeled. The blue mesh represents a Fo-Fc omit map contoured at a level of σ and shown only around the 5 atoms of the tungstate ion. Note that the tungstate and molybdate binding sites are indistinguishable at the current resolution, which reflects the similar ionic radii of the two ions 10,11. c, Schematic representation of the binding site observed in the A. fulgidus ModA structure, with relevant aminoacid residues labeled. Note the octahedral coordination of the central molybdenum atom. 9

10 Supplementary Figures Figure S1 10

11 Figure S a b 11

12 Figure S3 1

13 Figure S4 13

14 Figure S5 14