- Email: [email protected]
Projection structure of the bacterial oxalate transporter OxlT at 3.4 Å resolution
Journal of
Structural Biology Journal of Structural Biology 144 (2003) 320–326 www.elsevier.com/locate/yjsbi
Projection structure of the bacterial oxalate transporter resolution OxlT at 3.4 A J€ urgen A.W. Heymann, Teruhisa Hirai, Dan Shi, and Sriram Subramaniam* Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Received 25 July 2003, and in revised form 2 September 2003
Abstract OxlT is a bacterial transporter protein with 12 transmembrane segments that belongs to the Major Facilitator Superfamily of transporters. It facilitates the exchange of oxalate and formate across the membrane of the Gram-negative bacterium Oxalobacter formigenes. From an electron crystallographic analysis of two-dimensional, tube-like crystals of OxlT, we have previously deter resolution. Here, we report conditions to obtain crystalline, mined the three-dimensional structure of this transporter at 6.5 A two-dimensional sheets of OxlT with diameters exceeding 2 lm. Images of the crystalline sheets were recorded at liquid nitrogen temperatures on a transmission electron microscope equipped with a field-emission gun, operated at 300 kV. Computed optical , and electron diffraction patterns show spots diffraction patterns from the best images display measurable reflections to about 3.4 A resolution in the best cases. As in the case of the tube-like crystals, the new crystalline sheets also belong to the p221 21 to about 3.2 A 67.3 A are significantly smaller in one direction than those presymmetry group. However, the unit cell dimensions of 102.7 A 79.0 A . Different regions of OxlT are viously observed with the tube-like crystals that display unit cell dimensions of 100.3 A involved in intermolecular contacts in the two types of crystals, and the improved resolution of the sheet crystals appears to be mainly attributable to this tighter packing of the monomers within the unit cell. Published by Elsevier Inc. Keywords: Electron cryo-microscopy; Major facilitator superfamily; Membrane transporter; Two-dimensional crystallization
1. Introduction Analyses of several microbial genomes have indicated that as many as 30% of all membrane proteins may be involved in the transport of solutes and macromolecules across biological membranes (Paulsen and Sliwinski, 1998). Of these proteins, the Major Facilitator Superfamily (MFS) represents a large collection of evolutionarily related transporters whose members function in a range of activities such as uniport, symport, and antiport (Goswitz and Brooker, 1995; Marger and Saier, 1993; Pao and Paulsen, 1998; Saier et al., 1999), referring to the transport of a single species (uniport), the coupled transport of two solutes (symport), or the exchange of two different substrates in opposite directions (antiport) across the membrane, respectively. The
* Corresponding author. Fax: 1-301-480-3834. E-mail address: [email protected] (S. Subramaniam).
1047-8477/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.jsb.2003.09.002
bacterial oxalate transporter OxlT, a representative member of the MFS, is an antiporter that catalyzes the exchange of oxalate and formate across the inner membrane of the Gram-negative bacterium Oxalobacter formigenes (Allison et al., 1985; Maloney et al., 1992). OxlT facilitates the passage of divalent oxalate from the periplasmic space into the cytoplasm where it undergoes decarboxylation. It then exports monovalent formate, the product of this reaction, in the opposite direction. The net charge difference that is generated by this process and the consumption of protons in the decarboxylation step creates an inward directed proton gradient that is utilized for the synthesis of ATP (Anantharam et al., 1989; Kuhner et al., 1996). In this obligate anaerobic bacterium, oxalate serves as the sole energy source. We have recently reported a three-dimensional map resolution that we determined by the of OxlT at 6.5 A electron crystallographic analysis of two-dimensional crystals of OxlT (Heymann et al., 2001; Hirai et al., 2002), prepared in the presence of oxalate. Our analysis
J.A.W. Heymann et al. / Journal of Structural Biology 144 (2003) 320–326
of the structure of OxlT has established that OxlT has 12 membrane-spanning a-helices surrounding a central cavity that presumably represents the pathway of substrate transport. We find that the 12 a-helices (Fig. 1A) can be grouped into three classes (Hirai et al., 2002; Hirai et al., 2003): helices that are almost perpendicular with respect to the membrane plane (shaded in green), helices that include bends (shaded in yellow), and helices that include both a bend and curve in the transmembrane region (shaded in purple). Eight of the 12 helices are in direct contact with the central cavity, while the remaining four are located in the periphery. The arrangement of the helices (Fig. 1B) strongly suggests the presence of a pseudo twofold axis in the molecule that relates the first set of six helices to the second set, despite the lack of significant homology between the first and second halves of the protein sequence. Within each half, there is also an indication that each set of helices consists of a pair of three helices present in structurally similar environments (Fig. 1C), and has allowed us to propose an assignment of the 12 densities in the map to specific transmembrane regions in the primary sequence (Fig. 1B; (Hirai et al., 2003)). Computed diffraction patterns from the tube-like crystals that were used to construct our published 3D map of OxlT rarely displayed reflections with measurable . Further, the sizes of intensity at resolutions beyond 6 A the crystalline areas were too small to permit the collection of electron diffraction patterns that could be used to provide amplitude information to improve the quality of the map. As part of our efforts to increase the resolution of the map, we have explored conditions to test whether systematic variations in crystallization conditions could produce two-dimensional crystals of higher quality. Here, we report on the generation of a second crystal form of OxlT exhibiting near-atomic order and on the electron crystallographic analysis of these crystals.
2. Materials and methods 2.1. Overexpression and purification of OxlT The OxlT transporter was expressed as a C-terminally His9 -tagged polypeptide in the strain XL-1 Blue (Stratagene, La Jolla, CA) of Escherichia coli under lac-promoter control (Ruan et al., 1992). Cultures were grown in LB medium (QBiogene, Carlsbad, CA) at 36 °C and expression was induced at 34 °C and an OD600 0.1 by addition of isopropyl-b-D -thiogalactoside (RPI, Mt. Prospect, IL) to a final concentration of 1 mM. Cells were harvested 2 h after induction at OD600 values of 0.4–0.6. Membranes were prepared by osmotic lysis of cells by resuspending them in lysis buffer (10 mM Tris– HCl, 5 mM MgSO4 , and 1 mM phenylmethylsulfonylfluoride, pH 7.4), in the presence of lysozyme (2 mg/ml),
321
and DnaseI (4 mg/ml) for 30 min at RT and then diluted with ice-cold water 20-fold. Water-washed membranes were then extracted with 1.5% b-D -octylglucopyranoside (OG) (Anatrace, Maumee, OH) in the presence of 0.5% E. coli polar lipids (Avanti Polar Lipids, Alabaster, AL) for 30 min at 4 °C. Cleared extracts were batch-absorbed to Ni2þ –NTA (Qiagen, Valencia, CA) for 4 h at 4 °C. The column packed Ni2þ –NTA-beads were then washed at RT with 100 volumes of a buffer (pH 7) containing 200 mM NaF, 40 mM imidazole, 20 mM 3-(N-morpholino)propanesulfonic acid potassium salt (MOPS/K), 10 mM potassium oxalate, 6 mM b-mercaptoethanol, 20% glycerol, and 1% 1,2-diheptanoyl-sn-glycero-3phosphocholine (DHPC) (Avanti Polar Lipids, Alabaster, AL). Purified OxlT (>95%) was eluted at room temperature on addition of a solution (pH 4.5) containing 100 mM potassium oxalate, 50 mM potassium acetate, 6 mM b-mercaptoethanol, 20% glycerol, and 0.25% DHPC. Typical preparations yielded OxlT at a concentration between 2 and 3 mg/ml based on quantitation using commercially available protein detection kits (Micro-BCA assay, Pierce, Rockford, IL). 2.2. Two-dimensional crystallization of OxlT OxlT was crystallized by dialysis from solutions containing purified protein, either 0.3% (w/v) (tube-like crystals) or 0.4% (sheet-like crystals) n-cyclohexyl-heptylb-D -maltoside (CYMAL-7) (Anatrace, Maumee, OH) and either E. coli polar lipids or 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) (Avanti Polar Lipids), at lipid-to-protein ratios of either 0.2 to 0.4 (w/w) (tubelike crystals) or 0.15 to 0.25 (sheet-like crystals). Prior to dialysis, OxlT samples were combined with lipid solubilized in CYMAL-7 and incubated for 1hr at RT. Dialysis was performed in ÔSlide-a-lyzersÕ (Pierce) in a temperature controlled environment: 100 ll aliquots of OxlT-lipiddetergent mixtures were loaded and dialyzed for 3 days with daily buffer changes. Dialysis was carried out at either fixed temperature (27 °C; for tube-like crystals) or using a temperature profile (37 °C for 12 h followed by lowering temperature to RT by 1 °C every 3.5 h for sheetlike crystals) against a solution (pH 4.5) containing either 150 mM potassium acetate (tube-like crystals) or 50 mM potassium citrate (sheet-like crystals), 100 mM potassium oxalate, 6 mM b-mercaptoethanol, and 20% glycerol. The quality of crystals obtained with POPC was generally superior to those obtained with E. coli polar lipids as judged by visual inspection of computed diffraction patterns calculated from images of negatively stained specimen captured on CCD. 2.3. Microscopy and image processing Typically, 2 ll of a suspension of OxlT crystals in dialysis buffer was injected into 20 ll of 2–4% trehalose
322
J.A.W. Heymann et al. / Journal of Structural Biology 144 (2003) 320–326
Fig. 1. (A) Model for secondary structure of the oxalate–formate antiporter OxlT from O. formigenes. OxlT is an integral membrane protein with 12 membrane-spanning helices arranged with a topology such that both N and C termini face the cytoplasm. An extended cytoplasmic loop region connects two sets of six transmembrane helices. The transmembrane helices are color coded according to our previously published model (Hirai et al., 2003). Green indicates helices that are almost perpendicular to the membrane plane, yellow indicates helices that include bends, and purple indicates helices that include both a bend and a curve in the transmembrane region. The single lysine residue in transmembrane helix 11 that is believed to be involved in substrate transport (Fu and Maloney, 1998) is shown enlarged. A 9-histidine tag was added to the C terminus to facilitate purification resolution, superimposed with models using Ni2þ –NTA affinity chromatography. (B) Top view of the three-dimensional density map of OxlT at 6 A for the 12 helices, indicating the symmetrical arrangement of the helices and the central transport channel pathway. The color code is as described in (A). (C) Schematic depicting the topological relationship of the four sets of three-helix units, viewed from the cytoplasmic side. The units are related by three quasi-twofold symmetries and occupy structurally similar environments.
Fig. 2. Morphologies (A and B) and IQ plots (C and D) of tube-like or sheet-like two-dimensional crystals of OxlT visualized by negative staining with uranyl acetate. Scale bar is equivalent to 1 lm. The tube-like crystals are flattened into two bilayers upon deposition on the carbon surface (A), while the sheet-like crystals are deposited occasionally as single bilayer crystals (B). Insets: Idealized schematic cross-sections of the tube-like and sheet-like crystals are shown as insets in (A) and (B), respectively. Image-quality (IQ) plots of images recorded from tube-like (C) or sheet-like (D) plunge-frozen OxlT crystals recorded at liquid nitrogen temperatures on a Tecnai 30 (FEI) equipped with a field-emission gun, operated at 300 kV. ) at the indicated distances. Tube-like crystals show reflections up to about 6 A whereas The concentric circles indicate resolutions (3, 4, 6, 8, and 20 A . Periodic absence of reflections is due to the planar p221 21 symmetry present in both crystal types. sheet-like crystals display reflections up to 3.4 A
J.A.W. Heymann et al. / Journal of Structural Biology 144 (2003) 320–326
(in 50 mM citrate and 50 mM oxalate, pH 4.5) on continuous carbon. Excess liquid was removed by sideblotting using Whatman #1 filter paper upon which grids were flash-frozen in liquid nitrogen. Images were recorded using a Tecnai 30 electron microscope (FEI, OR) operating at 300 kV, equipped with a field-emission gun and a 626 single-tilt cryo-transfer system (Gatan, Pleasanton, CA), at a magnification of 59 000 and with measured specimen temperatures between )178 and )183 °C. Specimens were imaged under low-dose con2 ) either in the flood-beam ditions (10 electrons per A or in the spot-scan mode of illumination using SO-163 electron image film (Eastman Kodak, Rochester, NY). The exposed films were developed for 12 min in fullstrength D19 developer. Micrographs were screened using an optical diffractometer (Fujiyoshi Ironworks), to select for the best images. Useful images were obtained under both types of illumination conditions. Electron diffraction patterns from the sheet-like crystals were collected on a GATAN 2k by 2k CCD camera. The three best electron diffraction patterns used for analysis were obtained from a single specimen grid. Well-ordered
Table 1 Crystallographic data for sheet-like OxlT crystals Plane group Lattice constants Resolution limit for merging
p221 21 , b ¼ 67:3 A , c ¼ 90° a ¼ 102:7 A 3.4 A
No. of images Total No. of observations No. of unique reflections Completeness Overall phase residual
12 4224 (IQ 6 6) 463 99.4% 26.6°
No. of diffraction patterns Total No. of observations No. of unique reflections Completeness Friedel R-factor Merging R-factor
3 2011 390 82.8% 25.5% 29.2%
323
areas (0.7 lm 0.7 lm) were scanned and digitized using a Zeiss SCAI scanner (Z/I Imaging, Huntsville, AL). Images and diffraction patterns were processed using established methods (Henderson et al., 1986). Plane group symmetry of p221 21 was established by analysis of the images using ALLSPACE (Valpuesta et al., 1994). The phase residual values shown in Tables 1 and 2 reflect deviations determined before imposing p221 21 symmetry.
3. Results and discussion 3.1. Optimization of crystallization conditions We obtained two-dimensional crystals of the transporter OxlT from protein-detergent-lipid micelles by removal of detergent using dialysis. Transmission electron microscopy of negative stained crystals showed vesicular crystals of an elongated tube-like shape (Fig. 2A). Typically, such crystals were 0.4–0.7 lm wide and up to several micrometers long. Upon deposition on continuous carbon, crystalline OxlT vesicles flatten out with the crystallinity often being preserved in both bilayers. These tube-like crystals displayed p221 21 planar symmetry and had lattice constants of 100.3 A (sd ¼ 1.0 A) by 79.0 A (sd ¼ 0.4 A), but generally did not display order that extended much beyond about 6 A (Fig. 2C). To increase the size, as well as to improve the quality of the crystals, we carried out a systematic analysis of the effect of lipid-to-protein ratios, detergent concentration, and temperature on crystal quality. Lowering the lipid-to-protein ratio (LPR) in the crystallization reaction to a range of 0.15–0.25 (weight lipid to weight protein), and using higher initial temperatures (37 °C) led to the formation of a different crystal morphology, that we refer to as Ôsheet-likeÕ based on its appearance in negatively stained specimens (Fig. 2B). Both parameters appear to be important:
Table 2 Analysis of images and diffraction pattern Resolution range ) (A
Image analysis
Diffraction pattern analysis
No. of unique reflections
Completeness (%)
Phase residual
No. of unique reflections
Completeness (%)
110–11.2 11.2–7.9 7.9–6.5 6.5–5.6 5.6–5.0 5.0–4.6 4.6–4.2 4.2–4.0 4.0–3.7 3.7–3.5 3.5–3.4
42 45 43 43 45 43 41 41 44 41 35
100.0 100.0 100.0 100.0 100.0 100.0 100.0 95.3 100.0 100.0 97.2
9.3 8.6 11.4 21.6 25.8 26.8 34.7 33.8 37.3 43.8 47.3
0 8 43 43 44 43 41 43 44 41 36
0 17.8 100.0 100.0 97.8 100.0 100.0 100.0 100.0 100.0 100.0
R-Friedel
R-merge
—
—
0.2281 0.2224 0.3237 0.2591 0.1980 0.1874 0.2208 0.3354 0.3686 0.3561
0.2858 0.2661 0.3216 0.2920 0.2775 0.2790 0.2780 0.3013 0.3527 0.3297
324
J.A.W. Heymann et al. / Journal of Structural Biology 144 (2003) 320–326
lowering the LPR without the use of higher temperatures led to precipitation of most of the protein, while increasing the temperature without lowering the LPR led to the formation of non-crystalline vesicles and occasionally tube-like crystals. Sheet-like crystals formed preferentially when a slow ÔcoolingÕ process followed the initial high temperature treatment as described in Section 2. Increasing the concentration of the detergent CYMAL-7 from 0.3% (w/v) to about 0.4% (w/v) in the crystallization reaction improved the yield of crystals, possibly by keeping the protein more soluble at a later stage of the dialysis. Low salt concentrations (<150 mM) promoted the formation of sheet-like crystals whereas higher salt concentrations (>200 mM) were beneficial for the formation of tube-like crystals. Replacement of acetate buffer with citrate improved the overall appearance of OxlT crystals, possibly by chelating Niþ -ions that co-eluted from the Ni–NTA matrix used in the purification step. Acidic buffer conditions (pH 4.5–5.5) favored the formation of both tube- and sheet-like crystals. At neutral or slightly alkaline conditions, vesicles were obtained, but these were rarely found to be crystalline. 3.2. Analysis of sheet-like crystals Under the most favorable conditions for sheet formation, about 10% of the protein was found in crystalline sheets, with the remainder in non-crystalline vesicles, tube-like crystals, and protein aggregates. The reconstitution of OxlT into two-dimensional crystals was about 2.5 times less efficient for the formation of sheet-like crystals than for the formation of tube-like crystals, i.e., there was much more material present in aggregates under conditions that were optimal for the formation of sheet-like crystals. This is likely due to the decreased stability of OxlT at lower lipid concentrations. The largest sheets ranged in size from about 0.5 lm to about 2 lm, and in some instances, sheets with sizes of up to 2 lm 5 lm could be observed. Inspection of the larger crystals by TEM revealed that they contained several smaller crystalline areas of varying sizes, while smaller crystals tended to be single crystals with welldefined order. Sheet-like crystals were sometimes present as single bilayers on the carbon film, but also as double bilayers, as seen in the case of the flattened tube-like crystals. Analysis of images from sheet-like crystals in (Fig. 2D), in dicated a resolution limit at about 3.4 A observed with the best contrast to the value of about 6 A tube-like crystals (Fig. 2C). These sheet-like crystals were of the same planar symmetry (p221 21 ) as the tubelike crystals previously described (Heymann et al., 2001). Lattice constants observed here (102.7 A ) by 67.3 A (sd ¼ 0.4 A )) were, however, sig(sd ¼ 0.9 A nificantly shorter in one direction than in the tube-like
79.0 A ). The tighter packing, likely crystals (100.3 A due to reduction in the number of lipid molecules in the lattice, appears to be responsible for the higher resolution achieved. Because the sizes of the crystalline areas were frequently larger than 1 lm, electron diffraction patterns could be recorded, and in the best cases, they displayed detectable reflections to a resolution of about (Figs. 3A and B). Quantitative aspects of the im3.2 A age and diffraction pattern analysis are summarized in Tables 1 and 2. Using the images collected from the sheet-like crystals we have calculated new projection maps of OxlT at resolution in which the amplitudes are derived 3.4 A either from images (Fig. 3D) or from electron diffraction patterns (Fig. 3E). The higher resolution projection map is more featured as expected, but its overall appearance is very similar to the map obtained previously at 6 A resolution (Fig. 3C). In both cases, the projections of the monomers occupy an elliptical area in which the 12 transmembrane helices are tightly packed, although subtle differences in the position of each of the densities corresponding to the transmembrane helices can be found. The origins of these differences are likely to be due to the differences in order between tube and sheetlike crystals, and the different intermolecular interactions of monomers in the two crystal lattices. It is also plausible that the different lipid packing in the two crystal morphologies contributes to these differences. Closer inspection of the crystal packing in the tube and sheet-like crystals (Fig. 4) provides useful insights into specific differences in the molecular arrangements. In tube-like crystals, monomers align such that alternating OxlT molecules are Ôout-of-registerÕ by half the width of the molecule along both axes leading to a ÔzigzagÕ arrangement (Fig. 4A). In contrast, in sheet-like crystals, monomers arrange in almost parallel rows (Fig. 4B). Thus, they are in register with each other along the long axis, and Ôout-of-registerÕ by half the width of the molecule along the short axis. As a result, the distances and therefore the contacts between the monomers are different in the two crystal forms. The superposition of the 3D model of OxlT on the projection structure (Figs. 4A and B) further clarifies the nature of the interaction between monomers in the lattice: different regions of the protein are involved in the intermolecular contacts in the two crystal forms, arguing against the presence of a preferred interface for dimer formation. The better order in the sheet-like crystals may simply arise form the fact that a larger surface area of the protein is involved in protein–protein contacts. Our observations are consistent with findings for several transporters of the MFS that identified monomers and not dimers as the functional unit (Auer et al., 2001; Burant and Bell, 1992; Sahin-Toth et al., 1994). However, there are also examples such as the lactose transport protein LacS, that can undergo reversible
J.A.W. Heymann et al. / Journal of Structural Biology 144 (2003) 320–326
325
Fig. 3. Electron diffraction experiment on OxlT sheet-like crystals and comparison of monomers from OxlT projection density maps. (A), diffraction ) at the indicated distances with the highest resolution pattern, (B) indexed diffraction pattern. The concentric circles indicate resolutions (3, 6, and 8 A being at the outer circle. Diffraction patterns of trehalose-embedded OxlT crystals were recorded at liquid nitrogen temperatures with a 2k 2k cooled CCD camera (Gatan) on a Tecnai 30 electron microscope (FEI) equipped with a field-emission gun, operated at 300 kV. Lattice dimensions derived from the electron diffraction patterns were identical to the values calculated from the computed diffraction patterns of images with ; (0, 20), 3.37 A ; and (30, 0), 3.24 A . (C) Contour a =b ¼ 0:655 in both cases. Spots at the highest resolutions were determined to be: ()11, 20), 3.17 A resolution using merged data from from tube-like crystals as reported previously (Heymann et al., 2001). (D) map of OxlT monomer calculated at 6 A resolution using merged data from 10 sheet-like crystals. (E) Diffraction amplitude-corrected Contour map of OxlT monomer calculated at 3.4 A resolution. Amplitudes derived from diffraction patterns were used to contour map of OxlT monomer (from sheet-like crystals) calculated at 3.4 A correct the contour map obtained from sheet-like crystals leading to a noticeable sharpening of helix contours (E) when compared with the map calculated using OxlT image amplitudes (D).
Fig. 4. Comparison of intermolecular helix–helix interactions (proximities) in the two OxlT crystal morphologies. (A) Projection density map from resolution overlaid with model from three-dimensional structure derived from tube-like OxlT crystals. The tube-like OxlT crystals calculated at 6 A transmembrane helices of an OxlT monomer are depicted as idealized cylinders as in Fig. 1(B). (B) Amplitude corrected projection density map from resolution. The OxlT monomers are arranged in a ‘‘zig-zag’’ fashion in the map derived from tube-like crystals sheet-like crystals calculated at 3.4 A (A), and arranged in rows parallel to each other along the long axis in the map derived from sheet-like crystals (B). The 3D-model of OxlT obtained from tube-like crystals was overlayed on the projection map from sheet-like crystals for the sole purpose of indicating the approximate positions of the peripheral (green) transmembrane segments and their proximity to the neighboring monomers (pale blue shaded area).
self-association, and may function in the membrane as a cooperative dimer with two sugar translocation pathways (Veenhoff et al., 2001).
The transporter OxlT belongs to the MFS, the largest family of transporters in the class of porters (Pao and Paulsen, 1998; Saier et al., 1999). Although there are no
326
J.A.W. Heymann et al. / Journal of Structural Biology 144 (2003) 320–326
atomic structures that have been published yet of proteins in this family, rapid progress is being made, and progress towards three-dimensional crystallization of GlpT, a bacterial sn-glycerol-3-phosphate transporter has been recently reported (Auer et al., 2001). Interestingly, the architecture of OxlT does not bear a resem blance to the three-dimensional structure at 7 A resolution (Williams, 2000) of the sodium-proton exchanger NhaA, also a 12-helix transporter. The progress in obtaining larger, and well-ordered, sheet-like crystals may provide an opportunity to extend our current analysis of the three-dimensional structure of OxlT in a lipid bilayer membrane towards atomic resolution, and to obtaining new biological insights into this family of membrane proteins under physiologically relevant conditions. After submission of this manuscript, atomic models for the structures of two other MFS proteins have been reported (lactose permease (Abramson et al., 2003) and glycerol-3-phosphate transporter (Huang et al., 2003)). Inspection of these structures reveals that the relative arrangement of transmembrane helices in both transporters is identical with the model that we previously reported for OxlT (Hirai et al., 2002; Hirai et al., 2003), although the proteins appear to have been crystallized in a different conformation. Acknowledgments We thank Drs. Rafiquel Sarker, Liwen Ye, and Peter Maloney for providing purified OxlT during the initial stages of this project, and Drs. Jacqueline Milne and Mario Borgnia for valuable advice and helpful discussions. References Abramson, J., Smirnova, I., Kasho, V., Verner, G., Kaback, H.R., Iwata, S., 2003. Structure and mechanism of the lactose permease of Escherichia coli. Science 301, 610–615. Allison, M.J., Dawson, K.A., Mayberry, W.R., Foss, J.G., 1985. Oxalobacter formigenes gen. nov., sp. nov.: oxalate-degrading anaerobes that inhabit the gastrointestinal tract. Arch. Microbiol. 141, 1–7. Anantharam, V., Allison, M.J., Maloney, P.C., 1989. Oxalate: formate exchange. The basis for energy coupling in Oxalobacter. J. Biol. Chem. 264, 7244–7250. Auer, M., Kim, M.J., Lemieux, M.J., Villa, A., Song, J., Li, X.D., Wang, D.N., 2001. High-yield expression and functional analysis of Escherichia coli glycerol-3-phosphate transporter. Biochemistry 40, 6628–6635. Burant, C.F., Bell, G.I., 1992. Mammalian facilitative glucose transporters: evidence for similar substrate recognition sites in functionally monomeric proteins. Biochemistry 31, 10414–10420.
Fu, D., Maloney, P.C., 1998. Structure–function relationships in OxlT, the oxalate/formate transporter of Oxalobacter formigenes. Topological features of transmembrane helix 11 as visualized by sitedirected fluorescent labeling. J. Biol. Chem. 273, 17962–17967. Goswitz, V.C., Brooker, R.J., 1995. Structural features of the uniporter/symporter/antiporter superfamily. Protein Sci. 4, 534– 537. Henderson, R.H., Baldwin, J.H., Downing, K.H., Lepault, J., Zemlin, F., 1986. Structure of purple membrane from Halobacterium halobium: recording, measurement and evaluation of electron resolution. Ultramicroscopy 19, 174–178. micrographs at 3.5 A Heymann, J.A., Sarker, R., Hirai, T., Shi, D., Milne, J.L., Maloney, P.C., Subramaniam, S., 2001. Projection structure and molecular architecture of OxlT, a bacterial membrane transporter. EMBO J. 20, 4408–4413. Hirai, T., Heymann, J.A., Shi, D., Sarker, R., Maloney, P.C., Subramaniam, S., 2002. Three-dimensional structure of a bacterial oxalate transporter. Nat. Struct. Biol. 9, 597–600. Hirai, T., Heymann, J.A., Maloney, P.C., Subramaniam, S., 2003. Structural model for 12-helix transporters belonging to the major facilitator superfamily. J. Bacteriol. 185, 1712–1718. Huang, Y., Lemieux, M.J., Song, J., Auer, M., Wang, D.N., 2003. Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli. Science 301, 616–620. Kuhner, C.H., Hartman, P.A., Allison, M.J., 1996. Generation of a proton motive force by the anaerobic oxalate-degrading bacterium Oxalobacter formigenes. Appl. Environ. Microbiol. 62, 2494– 2500. Maloney, P.C., Anantharam, V., Allison, M.J., 1992. Measurement of the substrate dissociation constant of a solubilized membrane carrier. Substrate stabilization of OxlT, the anion exchange protein of Oxalobacter formigenes. J. Biol. Chem. 267, 10531–10536. Marger, M.D., Saier Jr., M.H., 1993. A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport. Trends Biochem. Sci. 18, 13–20. Pao, S.S., Paulsen, I.T., Saier Jr., M.H., 1998. Major facilitator superfamily. Microbiol. Mol. Biol. Rev. 62, 1–34. Paulsen, I.T., Sliwinski, M.K., Saier Jr., M.H., 1998. Microbial genome analyses: global comparisons of transport capabilities based on phylogenies, bioenergetics and substrate specificities. J. Mol. Biol. 277, 573–592. Ruan, Z.S., Anantharam, V., Crawford, I.T., Ambudkar, S.V., Rhee, S.Y., Allison, M.J., Maloney, P.C., 1992. Identification, purification, and reconstitution of OxlT, the oxalate: formate antiport protein of Oxalobacter formigenes. J. Biol. Chem. 267, 10537– 10543. Sahin-Toth, M., Lawrence, M.C., Kaback, H.R., 1994. Properties of permease dimer a fusion protein containing two lactose permease molecules from Escherichia coli. Proc. Natl. Acad. Sci. USA 91, 5421–5425. Saier Jr., M.H., Beatty, J.T., Goffeau, A., Harley, K.T., Heijne, W.H., Huang, S.C., Jack, D.L., Jahn, P.S., Lew, K., Liu, J., Pao, S.S., Paulsen, I.T., Tseng, T.T., Virk, P.S., 1999. The major facilitator superfamily. J. Mol. Microbiol. Biotechnol. 1, 257–279. Valpuesta, J.M., Carrascosa, J.L., Henderson, R., 1994. Analysis of electron microscope images and electron diffraction patterns of thin crystals of phi 29 connectors in ice. J. Mol. Biol. 240, 281– 287. Veenhoff, L.M., Heuberger, E.H., Poolman, B., 2001. The lactose transport protein is a cooperative dimer with two sugar translocation pathways. EMBO J. 20, 3056–3062. Williams, K.A., 2000. Three-dimensional structure of the ion-coupled transport protein NhaA. Nature 403, 112–115.
Structural Biology Journal of Structural Biology 144 (2003) 320–326 www.elsevier.com/locate/yjsbi
Projection structure of the bacterial oxalate transporter resolution OxlT at 3.4 A J€ urgen A.W. Heymann, Teruhisa Hirai, Dan Shi, and Sriram Subramaniam* Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Received 25 July 2003, and in revised form 2 September 2003
Abstract OxlT is a bacterial transporter protein with 12 transmembrane segments that belongs to the Major Facilitator Superfamily of transporters. It facilitates the exchange of oxalate and formate across the membrane of the Gram-negative bacterium Oxalobacter formigenes. From an electron crystallographic analysis of two-dimensional, tube-like crystals of OxlT, we have previously deter resolution. Here, we report conditions to obtain crystalline, mined the three-dimensional structure of this transporter at 6.5 A two-dimensional sheets of OxlT with diameters exceeding 2 lm. Images of the crystalline sheets were recorded at liquid nitrogen temperatures on a transmission electron microscope equipped with a field-emission gun, operated at 300 kV. Computed optical , and electron diffraction patterns show spots diffraction patterns from the best images display measurable reflections to about 3.4 A resolution in the best cases. As in the case of the tube-like crystals, the new crystalline sheets also belong to the p221 21 to about 3.2 A 67.3 A are significantly smaller in one direction than those presymmetry group. However, the unit cell dimensions of 102.7 A 79.0 A . Different regions of OxlT are viously observed with the tube-like crystals that display unit cell dimensions of 100.3 A involved in intermolecular contacts in the two types of crystals, and the improved resolution of the sheet crystals appears to be mainly attributable to this tighter packing of the monomers within the unit cell. Published by Elsevier Inc. Keywords: Electron cryo-microscopy; Major facilitator superfamily; Membrane transporter; Two-dimensional crystallization
1. Introduction Analyses of several microbial genomes have indicated that as many as 30% of all membrane proteins may be involved in the transport of solutes and macromolecules across biological membranes (Paulsen and Sliwinski, 1998). Of these proteins, the Major Facilitator Superfamily (MFS) represents a large collection of evolutionarily related transporters whose members function in a range of activities such as uniport, symport, and antiport (Goswitz and Brooker, 1995; Marger and Saier, 1993; Pao and Paulsen, 1998; Saier et al., 1999), referring to the transport of a single species (uniport), the coupled transport of two solutes (symport), or the exchange of two different substrates in opposite directions (antiport) across the membrane, respectively. The
* Corresponding author. Fax: 1-301-480-3834. E-mail address: [email protected] (S. Subramaniam).
1047-8477/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.jsb.2003.09.002
bacterial oxalate transporter OxlT, a representative member of the MFS, is an antiporter that catalyzes the exchange of oxalate and formate across the inner membrane of the Gram-negative bacterium Oxalobacter formigenes (Allison et al., 1985; Maloney et al., 1992). OxlT facilitates the passage of divalent oxalate from the periplasmic space into the cytoplasm where it undergoes decarboxylation. It then exports monovalent formate, the product of this reaction, in the opposite direction. The net charge difference that is generated by this process and the consumption of protons in the decarboxylation step creates an inward directed proton gradient that is utilized for the synthesis of ATP (Anantharam et al., 1989; Kuhner et al., 1996). In this obligate anaerobic bacterium, oxalate serves as the sole energy source. We have recently reported a three-dimensional map resolution that we determined by the of OxlT at 6.5 A electron crystallographic analysis of two-dimensional crystals of OxlT (Heymann et al., 2001; Hirai et al., 2002), prepared in the presence of oxalate. Our analysis
J.A.W. Heymann et al. / Journal of Structural Biology 144 (2003) 320–326
of the structure of OxlT has established that OxlT has 12 membrane-spanning a-helices surrounding a central cavity that presumably represents the pathway of substrate transport. We find that the 12 a-helices (Fig. 1A) can be grouped into three classes (Hirai et al., 2002; Hirai et al., 2003): helices that are almost perpendicular with respect to the membrane plane (shaded in green), helices that include bends (shaded in yellow), and helices that include both a bend and curve in the transmembrane region (shaded in purple). Eight of the 12 helices are in direct contact with the central cavity, while the remaining four are located in the periphery. The arrangement of the helices (Fig. 1B) strongly suggests the presence of a pseudo twofold axis in the molecule that relates the first set of six helices to the second set, despite the lack of significant homology between the first and second halves of the protein sequence. Within each half, there is also an indication that each set of helices consists of a pair of three helices present in structurally similar environments (Fig. 1C), and has allowed us to propose an assignment of the 12 densities in the map to specific transmembrane regions in the primary sequence (Fig. 1B; (Hirai et al., 2003)). Computed diffraction patterns from the tube-like crystals that were used to construct our published 3D map of OxlT rarely displayed reflections with measurable . Further, the sizes of intensity at resolutions beyond 6 A the crystalline areas were too small to permit the collection of electron diffraction patterns that could be used to provide amplitude information to improve the quality of the map. As part of our efforts to increase the resolution of the map, we have explored conditions to test whether systematic variations in crystallization conditions could produce two-dimensional crystals of higher quality. Here, we report on the generation of a second crystal form of OxlT exhibiting near-atomic order and on the electron crystallographic analysis of these crystals.
2. Materials and methods 2.1. Overexpression and purification of OxlT The OxlT transporter was expressed as a C-terminally His9 -tagged polypeptide in the strain XL-1 Blue (Stratagene, La Jolla, CA) of Escherichia coli under lac-promoter control (Ruan et al., 1992). Cultures were grown in LB medium (QBiogene, Carlsbad, CA) at 36 °C and expression was induced at 34 °C and an OD600 0.1 by addition of isopropyl-b-D -thiogalactoside (RPI, Mt. Prospect, IL) to a final concentration of 1 mM. Cells were harvested 2 h after induction at OD600 values of 0.4–0.6. Membranes were prepared by osmotic lysis of cells by resuspending them in lysis buffer (10 mM Tris– HCl, 5 mM MgSO4 , and 1 mM phenylmethylsulfonylfluoride, pH 7.4), in the presence of lysozyme (2 mg/ml),
321
and DnaseI (4 mg/ml) for 30 min at RT and then diluted with ice-cold water 20-fold. Water-washed membranes were then extracted with 1.5% b-D -octylglucopyranoside (OG) (Anatrace, Maumee, OH) in the presence of 0.5% E. coli polar lipids (Avanti Polar Lipids, Alabaster, AL) for 30 min at 4 °C. Cleared extracts were batch-absorbed to Ni2þ –NTA (Qiagen, Valencia, CA) for 4 h at 4 °C. The column packed Ni2þ –NTA-beads were then washed at RT with 100 volumes of a buffer (pH 7) containing 200 mM NaF, 40 mM imidazole, 20 mM 3-(N-morpholino)propanesulfonic acid potassium salt (MOPS/K), 10 mM potassium oxalate, 6 mM b-mercaptoethanol, 20% glycerol, and 1% 1,2-diheptanoyl-sn-glycero-3phosphocholine (DHPC) (Avanti Polar Lipids, Alabaster, AL). Purified OxlT (>95%) was eluted at room temperature on addition of a solution (pH 4.5) containing 100 mM potassium oxalate, 50 mM potassium acetate, 6 mM b-mercaptoethanol, 20% glycerol, and 0.25% DHPC. Typical preparations yielded OxlT at a concentration between 2 and 3 mg/ml based on quantitation using commercially available protein detection kits (Micro-BCA assay, Pierce, Rockford, IL). 2.2. Two-dimensional crystallization of OxlT OxlT was crystallized by dialysis from solutions containing purified protein, either 0.3% (w/v) (tube-like crystals) or 0.4% (sheet-like crystals) n-cyclohexyl-heptylb-D -maltoside (CYMAL-7) (Anatrace, Maumee, OH) and either E. coli polar lipids or 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) (Avanti Polar Lipids), at lipid-to-protein ratios of either 0.2 to 0.4 (w/w) (tubelike crystals) or 0.15 to 0.25 (sheet-like crystals). Prior to dialysis, OxlT samples were combined with lipid solubilized in CYMAL-7 and incubated for 1hr at RT. Dialysis was performed in ÔSlide-a-lyzersÕ (Pierce) in a temperature controlled environment: 100 ll aliquots of OxlT-lipiddetergent mixtures were loaded and dialyzed for 3 days with daily buffer changes. Dialysis was carried out at either fixed temperature (27 °C; for tube-like crystals) or using a temperature profile (37 °C for 12 h followed by lowering temperature to RT by 1 °C every 3.5 h for sheetlike crystals) against a solution (pH 4.5) containing either 150 mM potassium acetate (tube-like crystals) or 50 mM potassium citrate (sheet-like crystals), 100 mM potassium oxalate, 6 mM b-mercaptoethanol, and 20% glycerol. The quality of crystals obtained with POPC was generally superior to those obtained with E. coli polar lipids as judged by visual inspection of computed diffraction patterns calculated from images of negatively stained specimen captured on CCD. 2.3. Microscopy and image processing Typically, 2 ll of a suspension of OxlT crystals in dialysis buffer was injected into 20 ll of 2–4% trehalose
322
J.A.W. Heymann et al. / Journal of Structural Biology 144 (2003) 320–326
Fig. 1. (A) Model for secondary structure of the oxalate–formate antiporter OxlT from O. formigenes. OxlT is an integral membrane protein with 12 membrane-spanning helices arranged with a topology such that both N and C termini face the cytoplasm. An extended cytoplasmic loop region connects two sets of six transmembrane helices. The transmembrane helices are color coded according to our previously published model (Hirai et al., 2003). Green indicates helices that are almost perpendicular to the membrane plane, yellow indicates helices that include bends, and purple indicates helices that include both a bend and a curve in the transmembrane region. The single lysine residue in transmembrane helix 11 that is believed to be involved in substrate transport (Fu and Maloney, 1998) is shown enlarged. A 9-histidine tag was added to the C terminus to facilitate purification resolution, superimposed with models using Ni2þ –NTA affinity chromatography. (B) Top view of the three-dimensional density map of OxlT at 6 A for the 12 helices, indicating the symmetrical arrangement of the helices and the central transport channel pathway. The color code is as described in (A). (C) Schematic depicting the topological relationship of the four sets of three-helix units, viewed from the cytoplasmic side. The units are related by three quasi-twofold symmetries and occupy structurally similar environments.
Fig. 2. Morphologies (A and B) and IQ plots (C and D) of tube-like or sheet-like two-dimensional crystals of OxlT visualized by negative staining with uranyl acetate. Scale bar is equivalent to 1 lm. The tube-like crystals are flattened into two bilayers upon deposition on the carbon surface (A), while the sheet-like crystals are deposited occasionally as single bilayer crystals (B). Insets: Idealized schematic cross-sections of the tube-like and sheet-like crystals are shown as insets in (A) and (B), respectively. Image-quality (IQ) plots of images recorded from tube-like (C) or sheet-like (D) plunge-frozen OxlT crystals recorded at liquid nitrogen temperatures on a Tecnai 30 (FEI) equipped with a field-emission gun, operated at 300 kV. ) at the indicated distances. Tube-like crystals show reflections up to about 6 A whereas The concentric circles indicate resolutions (3, 4, 6, 8, and 20 A . Periodic absence of reflections is due to the planar p221 21 symmetry present in both crystal types. sheet-like crystals display reflections up to 3.4 A
J.A.W. Heymann et al. / Journal of Structural Biology 144 (2003) 320–326
(in 50 mM citrate and 50 mM oxalate, pH 4.5) on continuous carbon. Excess liquid was removed by sideblotting using Whatman #1 filter paper upon which grids were flash-frozen in liquid nitrogen. Images were recorded using a Tecnai 30 electron microscope (FEI, OR) operating at 300 kV, equipped with a field-emission gun and a 626 single-tilt cryo-transfer system (Gatan, Pleasanton, CA), at a magnification of 59 000 and with measured specimen temperatures between )178 and )183 °C. Specimens were imaged under low-dose con2 ) either in the flood-beam ditions (10 electrons per A or in the spot-scan mode of illumination using SO-163 electron image film (Eastman Kodak, Rochester, NY). The exposed films were developed for 12 min in fullstrength D19 developer. Micrographs were screened using an optical diffractometer (Fujiyoshi Ironworks), to select for the best images. Useful images were obtained under both types of illumination conditions. Electron diffraction patterns from the sheet-like crystals were collected on a GATAN 2k by 2k CCD camera. The three best electron diffraction patterns used for analysis were obtained from a single specimen grid. Well-ordered
Table 1 Crystallographic data for sheet-like OxlT crystals Plane group Lattice constants Resolution limit for merging
p221 21 , b ¼ 67:3 A , c ¼ 90° a ¼ 102:7 A 3.4 A
No. of images Total No. of observations No. of unique reflections Completeness Overall phase residual
12 4224 (IQ 6 6) 463 99.4% 26.6°
No. of diffraction patterns Total No. of observations No. of unique reflections Completeness Friedel R-factor Merging R-factor
3 2011 390 82.8% 25.5% 29.2%
323
areas (0.7 lm 0.7 lm) were scanned and digitized using a Zeiss SCAI scanner (Z/I Imaging, Huntsville, AL). Images and diffraction patterns were processed using established methods (Henderson et al., 1986). Plane group symmetry of p221 21 was established by analysis of the images using ALLSPACE (Valpuesta et al., 1994). The phase residual values shown in Tables 1 and 2 reflect deviations determined before imposing p221 21 symmetry.
3. Results and discussion 3.1. Optimization of crystallization conditions We obtained two-dimensional crystals of the transporter OxlT from protein-detergent-lipid micelles by removal of detergent using dialysis. Transmission electron microscopy of negative stained crystals showed vesicular crystals of an elongated tube-like shape (Fig. 2A). Typically, such crystals were 0.4–0.7 lm wide and up to several micrometers long. Upon deposition on continuous carbon, crystalline OxlT vesicles flatten out with the crystallinity often being preserved in both bilayers. These tube-like crystals displayed p221 21 planar symmetry and had lattice constants of 100.3 A (sd ¼ 1.0 A) by 79.0 A (sd ¼ 0.4 A), but generally did not display order that extended much beyond about 6 A (Fig. 2C). To increase the size, as well as to improve the quality of the crystals, we carried out a systematic analysis of the effect of lipid-to-protein ratios, detergent concentration, and temperature on crystal quality. Lowering the lipid-to-protein ratio (LPR) in the crystallization reaction to a range of 0.15–0.25 (weight lipid to weight protein), and using higher initial temperatures (37 °C) led to the formation of a different crystal morphology, that we refer to as Ôsheet-likeÕ based on its appearance in negatively stained specimens (Fig. 2B). Both parameters appear to be important:
Table 2 Analysis of images and diffraction pattern Resolution range ) (A
Image analysis
Diffraction pattern analysis
No. of unique reflections
Completeness (%)
Phase residual
No. of unique reflections
Completeness (%)
110–11.2 11.2–7.9 7.9–6.5 6.5–5.6 5.6–5.0 5.0–4.6 4.6–4.2 4.2–4.0 4.0–3.7 3.7–3.5 3.5–3.4
42 45 43 43 45 43 41 41 44 41 35
100.0 100.0 100.0 100.0 100.0 100.0 100.0 95.3 100.0 100.0 97.2
9.3 8.6 11.4 21.6 25.8 26.8 34.7 33.8 37.3 43.8 47.3
0 8 43 43 44 43 41 43 44 41 36
0 17.8 100.0 100.0 97.8 100.0 100.0 100.0 100.0 100.0 100.0
R-Friedel
R-merge
—
—
0.2281 0.2224 0.3237 0.2591 0.1980 0.1874 0.2208 0.3354 0.3686 0.3561
0.2858 0.2661 0.3216 0.2920 0.2775 0.2790 0.2780 0.3013 0.3527 0.3297
324
J.A.W. Heymann et al. / Journal of Structural Biology 144 (2003) 320–326
lowering the LPR without the use of higher temperatures led to precipitation of most of the protein, while increasing the temperature without lowering the LPR led to the formation of non-crystalline vesicles and occasionally tube-like crystals. Sheet-like crystals formed preferentially when a slow ÔcoolingÕ process followed the initial high temperature treatment as described in Section 2. Increasing the concentration of the detergent CYMAL-7 from 0.3% (w/v) to about 0.4% (w/v) in the crystallization reaction improved the yield of crystals, possibly by keeping the protein more soluble at a later stage of the dialysis. Low salt concentrations (<150 mM) promoted the formation of sheet-like crystals whereas higher salt concentrations (>200 mM) were beneficial for the formation of tube-like crystals. Replacement of acetate buffer with citrate improved the overall appearance of OxlT crystals, possibly by chelating Niþ -ions that co-eluted from the Ni–NTA matrix used in the purification step. Acidic buffer conditions (pH 4.5–5.5) favored the formation of both tube- and sheet-like crystals. At neutral or slightly alkaline conditions, vesicles were obtained, but these were rarely found to be crystalline. 3.2. Analysis of sheet-like crystals Under the most favorable conditions for sheet formation, about 10% of the protein was found in crystalline sheets, with the remainder in non-crystalline vesicles, tube-like crystals, and protein aggregates. The reconstitution of OxlT into two-dimensional crystals was about 2.5 times less efficient for the formation of sheet-like crystals than for the formation of tube-like crystals, i.e., there was much more material present in aggregates under conditions that were optimal for the formation of sheet-like crystals. This is likely due to the decreased stability of OxlT at lower lipid concentrations. The largest sheets ranged in size from about 0.5 lm to about 2 lm, and in some instances, sheets with sizes of up to 2 lm 5 lm could be observed. Inspection of the larger crystals by TEM revealed that they contained several smaller crystalline areas of varying sizes, while smaller crystals tended to be single crystals with welldefined order. Sheet-like crystals were sometimes present as single bilayers on the carbon film, but also as double bilayers, as seen in the case of the flattened tube-like crystals. Analysis of images from sheet-like crystals in (Fig. 2D), in dicated a resolution limit at about 3.4 A observed with the best contrast to the value of about 6 A tube-like crystals (Fig. 2C). These sheet-like crystals were of the same planar symmetry (p221 21 ) as the tubelike crystals previously described (Heymann et al., 2001). Lattice constants observed here (102.7 A ) by 67.3 A (sd ¼ 0.4 A )) were, however, sig(sd ¼ 0.9 A nificantly shorter in one direction than in the tube-like
79.0 A ). The tighter packing, likely crystals (100.3 A due to reduction in the number of lipid molecules in the lattice, appears to be responsible for the higher resolution achieved. Because the sizes of the crystalline areas were frequently larger than 1 lm, electron diffraction patterns could be recorded, and in the best cases, they displayed detectable reflections to a resolution of about (Figs. 3A and B). Quantitative aspects of the im3.2 A age and diffraction pattern analysis are summarized in Tables 1 and 2. Using the images collected from the sheet-like crystals we have calculated new projection maps of OxlT at resolution in which the amplitudes are derived 3.4 A either from images (Fig. 3D) or from electron diffraction patterns (Fig. 3E). The higher resolution projection map is more featured as expected, but its overall appearance is very similar to the map obtained previously at 6 A resolution (Fig. 3C). In both cases, the projections of the monomers occupy an elliptical area in which the 12 transmembrane helices are tightly packed, although subtle differences in the position of each of the densities corresponding to the transmembrane helices can be found. The origins of these differences are likely to be due to the differences in order between tube and sheetlike crystals, and the different intermolecular interactions of monomers in the two crystal lattices. It is also plausible that the different lipid packing in the two crystal morphologies contributes to these differences. Closer inspection of the crystal packing in the tube and sheet-like crystals (Fig. 4) provides useful insights into specific differences in the molecular arrangements. In tube-like crystals, monomers align such that alternating OxlT molecules are Ôout-of-registerÕ by half the width of the molecule along both axes leading to a ÔzigzagÕ arrangement (Fig. 4A). In contrast, in sheet-like crystals, monomers arrange in almost parallel rows (Fig. 4B). Thus, they are in register with each other along the long axis, and Ôout-of-registerÕ by half the width of the molecule along the short axis. As a result, the distances and therefore the contacts between the monomers are different in the two crystal forms. The superposition of the 3D model of OxlT on the projection structure (Figs. 4A and B) further clarifies the nature of the interaction between monomers in the lattice: different regions of the protein are involved in the intermolecular contacts in the two crystal forms, arguing against the presence of a preferred interface for dimer formation. The better order in the sheet-like crystals may simply arise form the fact that a larger surface area of the protein is involved in protein–protein contacts. Our observations are consistent with findings for several transporters of the MFS that identified monomers and not dimers as the functional unit (Auer et al., 2001; Burant and Bell, 1992; Sahin-Toth et al., 1994). However, there are also examples such as the lactose transport protein LacS, that can undergo reversible
J.A.W. Heymann et al. / Journal of Structural Biology 144 (2003) 320–326
325
Fig. 3. Electron diffraction experiment on OxlT sheet-like crystals and comparison of monomers from OxlT projection density maps. (A), diffraction ) at the indicated distances with the highest resolution pattern, (B) indexed diffraction pattern. The concentric circles indicate resolutions (3, 6, and 8 A being at the outer circle. Diffraction patterns of trehalose-embedded OxlT crystals were recorded at liquid nitrogen temperatures with a 2k 2k cooled CCD camera (Gatan) on a Tecnai 30 electron microscope (FEI) equipped with a field-emission gun, operated at 300 kV. Lattice dimensions derived from the electron diffraction patterns were identical to the values calculated from the computed diffraction patterns of images with ; (0, 20), 3.37 A ; and (30, 0), 3.24 A . (C) Contour a =b ¼ 0:655 in both cases. Spots at the highest resolutions were determined to be: ()11, 20), 3.17 A resolution using merged data from from tube-like crystals as reported previously (Heymann et al., 2001). (D) map of OxlT monomer calculated at 6 A resolution using merged data from 10 sheet-like crystals. (E) Diffraction amplitude-corrected Contour map of OxlT monomer calculated at 3.4 A resolution. Amplitudes derived from diffraction patterns were used to contour map of OxlT monomer (from sheet-like crystals) calculated at 3.4 A correct the contour map obtained from sheet-like crystals leading to a noticeable sharpening of helix contours (E) when compared with the map calculated using OxlT image amplitudes (D).
Fig. 4. Comparison of intermolecular helix–helix interactions (proximities) in the two OxlT crystal morphologies. (A) Projection density map from resolution overlaid with model from three-dimensional structure derived from tube-like OxlT crystals. The tube-like OxlT crystals calculated at 6 A transmembrane helices of an OxlT monomer are depicted as idealized cylinders as in Fig. 1(B). (B) Amplitude corrected projection density map from resolution. The OxlT monomers are arranged in a ‘‘zig-zag’’ fashion in the map derived from tube-like crystals sheet-like crystals calculated at 3.4 A (A), and arranged in rows parallel to each other along the long axis in the map derived from sheet-like crystals (B). The 3D-model of OxlT obtained from tube-like crystals was overlayed on the projection map from sheet-like crystals for the sole purpose of indicating the approximate positions of the peripheral (green) transmembrane segments and their proximity to the neighboring monomers (pale blue shaded area).
self-association, and may function in the membrane as a cooperative dimer with two sugar translocation pathways (Veenhoff et al., 2001).
The transporter OxlT belongs to the MFS, the largest family of transporters in the class of porters (Pao and Paulsen, 1998; Saier et al., 1999). Although there are no
326
J.A.W. Heymann et al. / Journal of Structural Biology 144 (2003) 320–326
atomic structures that have been published yet of proteins in this family, rapid progress is being made, and progress towards three-dimensional crystallization of GlpT, a bacterial sn-glycerol-3-phosphate transporter has been recently reported (Auer et al., 2001). Interestingly, the architecture of OxlT does not bear a resem blance to the three-dimensional structure at 7 A resolution (Williams, 2000) of the sodium-proton exchanger NhaA, also a 12-helix transporter. The progress in obtaining larger, and well-ordered, sheet-like crystals may provide an opportunity to extend our current analysis of the three-dimensional structure of OxlT in a lipid bilayer membrane towards atomic resolution, and to obtaining new biological insights into this family of membrane proteins under physiologically relevant conditions. After submission of this manuscript, atomic models for the structures of two other MFS proteins have been reported (lactose permease (Abramson et al., 2003) and glycerol-3-phosphate transporter (Huang et al., 2003)). Inspection of these structures reveals that the relative arrangement of transmembrane helices in both transporters is identical with the model that we previously reported for OxlT (Hirai et al., 2002; Hirai et al., 2003), although the proteins appear to have been crystallized in a different conformation. Acknowledgments We thank Drs. Rafiquel Sarker, Liwen Ye, and Peter Maloney for providing purified OxlT during the initial stages of this project, and Drs. Jacqueline Milne and Mario Borgnia for valuable advice and helpful discussions. References Abramson, J., Smirnova, I., Kasho, V., Verner, G., Kaback, H.R., Iwata, S., 2003. Structure and mechanism of the lactose permease of Escherichia coli. Science 301, 610–615. Allison, M.J., Dawson, K.A., Mayberry, W.R., Foss, J.G., 1985. Oxalobacter formigenes gen. nov., sp. nov.: oxalate-degrading anaerobes that inhabit the gastrointestinal tract. Arch. Microbiol. 141, 1–7. Anantharam, V., Allison, M.J., Maloney, P.C., 1989. Oxalate: formate exchange. The basis for energy coupling in Oxalobacter. J. Biol. Chem. 264, 7244–7250. Auer, M., Kim, M.J., Lemieux, M.J., Villa, A., Song, J., Li, X.D., Wang, D.N., 2001. High-yield expression and functional analysis of Escherichia coli glycerol-3-phosphate transporter. Biochemistry 40, 6628–6635. Burant, C.F., Bell, G.I., 1992. Mammalian facilitative glucose transporters: evidence for similar substrate recognition sites in functionally monomeric proteins. Biochemistry 31, 10414–10420.
Fu, D., Maloney, P.C., 1998. Structure–function relationships in OxlT, the oxalate/formate transporter of Oxalobacter formigenes. Topological features of transmembrane helix 11 as visualized by sitedirected fluorescent labeling. J. Biol. Chem. 273, 17962–17967. Goswitz, V.C., Brooker, R.J., 1995. Structural features of the uniporter/symporter/antiporter superfamily. Protein Sci. 4, 534– 537. Henderson, R.H., Baldwin, J.H., Downing, K.H., Lepault, J., Zemlin, F., 1986. Structure of purple membrane from Halobacterium halobium: recording, measurement and evaluation of electron resolution. Ultramicroscopy 19, 174–178. micrographs at 3.5 A Heymann, J.A., Sarker, R., Hirai, T., Shi, D., Milne, J.L., Maloney, P.C., Subramaniam, S., 2001. Projection structure and molecular architecture of OxlT, a bacterial membrane transporter. EMBO J. 20, 4408–4413. Hirai, T., Heymann, J.A., Shi, D., Sarker, R., Maloney, P.C., Subramaniam, S., 2002. Three-dimensional structure of a bacterial oxalate transporter. Nat. Struct. Biol. 9, 597–600. Hirai, T., Heymann, J.A., Maloney, P.C., Subramaniam, S., 2003. Structural model for 12-helix transporters belonging to the major facilitator superfamily. J. Bacteriol. 185, 1712–1718. Huang, Y., Lemieux, M.J., Song, J., Auer, M., Wang, D.N., 2003. Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli. Science 301, 616–620. Kuhner, C.H., Hartman, P.A., Allison, M.J., 1996. Generation of a proton motive force by the anaerobic oxalate-degrading bacterium Oxalobacter formigenes. Appl. Environ. Microbiol. 62, 2494– 2500. Maloney, P.C., Anantharam, V., Allison, M.J., 1992. Measurement of the substrate dissociation constant of a solubilized membrane carrier. Substrate stabilization of OxlT, the anion exchange protein of Oxalobacter formigenes. J. Biol. Chem. 267, 10531–10536. Marger, M.D., Saier Jr., M.H., 1993. A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport. Trends Biochem. Sci. 18, 13–20. Pao, S.S., Paulsen, I.T., Saier Jr., M.H., 1998. Major facilitator superfamily. Microbiol. Mol. Biol. Rev. 62, 1–34. Paulsen, I.T., Sliwinski, M.K., Saier Jr., M.H., 1998. Microbial genome analyses: global comparisons of transport capabilities based on phylogenies, bioenergetics and substrate specificities. J. Mol. Biol. 277, 573–592. Ruan, Z.S., Anantharam, V., Crawford, I.T., Ambudkar, S.V., Rhee, S.Y., Allison, M.J., Maloney, P.C., 1992. Identification, purification, and reconstitution of OxlT, the oxalate: formate antiport protein of Oxalobacter formigenes. J. Biol. Chem. 267, 10537– 10543. Sahin-Toth, M., Lawrence, M.C., Kaback, H.R., 1994. Properties of permease dimer a fusion protein containing two lactose permease molecules from Escherichia coli. Proc. Natl. Acad. Sci. USA 91, 5421–5425. Saier Jr., M.H., Beatty, J.T., Goffeau, A., Harley, K.T., Heijne, W.H., Huang, S.C., Jack, D.L., Jahn, P.S., Lew, K., Liu, J., Pao, S.S., Paulsen, I.T., Tseng, T.T., Virk, P.S., 1999. The major facilitator superfamily. J. Mol. Microbiol. Biotechnol. 1, 257–279. Valpuesta, J.M., Carrascosa, J.L., Henderson, R., 1994. Analysis of electron microscope images and electron diffraction patterns of thin crystals of phi 29 connectors in ice. J. Mol. Biol. 240, 281– 287. Veenhoff, L.M., Heuberger, E.H., Poolman, B., 2001. The lactose transport protein is a cooperative dimer with two sugar translocation pathways. EMBO J. 20, 3056–3062. Williams, K.A., 2000. Three-dimensional structure of the ion-coupled transport protein NhaA. Nature 403, 112–115.