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Structural basis for substrate transport in the GLUT-homology family of monosaccharide transporters
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Esben M Quistgaard1,3, Christian Löw1,3, Per Moberg1, Lionel Trésaugues1 & Pär Nordlund1,2 Here we present two structures of the major facilitator (MFS) xylose transporter XylE from Escherichia coli in inward open and partially occluded inward open conformations. These structures provide key information about the transport cycle of XylE and the closely related human GLUT transporters. This is, to our knowledge, the first MFS transporter structure determined in more than one conformational state, which may establish XylE as an important MFS model protein. Members of the GLUT family are mammalian monosaccharide transporters belonging to the ubiquitous MFS superfamily. The best characterized of the 14 human GLUT members are GLUT1 and GLUT4, which are essential for glucose homeostasis and are among the most intensely studied of all membrane proteins1. Structural and functional homologs of the family are found in all branches of life including bacteria (Supplementary Fig. 1). The E. coli d-xylose transporter XylE2 shares 20–29% pairwise identities with the 14 human GLUT transporters and possesses all the characteristic GLUT signature motifs (Fig. 1 and Supplementary Fig. 1). MFS transporters function by an alternate access mechanism involving at least three conformational states: inward open, occluded and outward open3,4. With the aim of obtaining a better understanding of the transport mechanism of MFS transporters in general and of GLUT transporters in particular, we undertook crystallization of XylE. While this work was in progress, a partially occluded outward-facing structure of XylE in complex with d-xylose (PDB 4GBY) was reported5 (Fig. 2a) along with two highly similar structures cocrystallized with d-glucose and brominated d-glucose5. Here we report two crystal structures of XylE in different conformational states: inward open and partially occluded inward open (Fig. 2b,c). The structure of XylE was determined in two different space groups (P61 and C2) using experimental data to 4.2 and 3.8 Å, respectively (Supplementary Table 1). Phase information was improved by using three-fold and two-fold averaging, respectively (Online Methods). Each crystal form corresponds to different conformational states of XylE: inward open (P61) and partially occluded inward open (C2).
In the inward open state, the N and C subdomains tightly interact at the periplasmic side to form an overall V-shaped structure with a wide opening extending from the cytoplasm to the binding site in the center of the protein (Fig. 2c). Despite the low level of sequence identity (10–12%), the structure is considerably more similar to the other three known structures of MFS transporters in inward open conformation6–8 (LacY, GlpT and PepTSt; r.m.s. deviation Cα 3.0–3.3 Å over 340–372 residues) than to the partially occluded outwardfacing conformation of XylE (3.7 Å over 415 residues). The substantial conformational change as compared to the latter is mainly due to rigid body movement of the two subdomains with respect to each other, but additional differences are also found within the C subdomain (Supplementary Fig. 2). The structure of the partially occluded inward open state (Fig. 2b) is overall similar to the inward open state, but the C-terminal half of the interrupted transmembrane helix 10 (TM10b) is much closer in position to the N subdomain and thus partially blocks the cytoplasmic opening. A number of signature motifs are present in the cytoplasmic loops and helices of the GLUT-homology family1,5 (Fig. 1). In the partially occluded outward-facing conformation, these motifs form numerous interactions including three intersubdomain salt bridges, Glu153-Arg404, Arg160-Glu397 and Arg225-Glu472 (Fig. 3a). These interactions appear to be functionally important because previous mutations of Glu153, Arg160, Arg225, Glu397 or Arg404 to alanine abolished XylE transport activity in cellbased uptake assays and, generally, also in proteoliposome-based Motif
Sequence XylE
GLUT1
GLUT2
GLUT3
GLUT4
GRR/K
GRR 83–85
GRR 91–93
GRIK 123–126
GRR 90–92
GRKR 108–111
E(6X)RG
E(6X)RG 153–161
E(6X)RG 146–154
E(6X)RG 178–186
E(6X)RG 144–152
E(6X)RG 162–170
PESPR
PESPR 221–225
PESPR 208–212
PESPR 240–244
PESPR 206–210
PESPR 224–228
E/DRAGRR
DKFGRK 337–342
ERAGRR 329–334
EKAGRR 361–366
ERAGRR 327–332
ERAGRR 345–350
E(6X)R/K
E(6X)R 397–404
E(6X)R 393–402
E(6X)R 425–432
E(6X)R 391–398
E(6X)R 409–416
PETKG
PETKG 464–468
PETRG 453–457
PETKG 485–489
PETRG 431–435
PETKG 469–473
D/E in C terminus
Glu472
Asp461
Glu493
Glu439
Asp477
Figure 1 Overview of conserved cytoplasmic motifs in selected members of the GLUT-homology family. Residues colored red are mutated in cases of GLUT1-deficiency syndromes 1 and 2.
1Department
of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. 2School of Biological Sciences, Nanyang Technological University, Singapore. 3These authors contributed equally to this work. Correspondence should be addressed to P.N. (
[email protected]). Received 19 December 2012; accepted 15 March 2013; published online 28 April 2013; doi:10.1038/nsmb.2569
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Figure 2 Overall structure of XylE. (a) Partially occluded outward open conformation (PDB 4GBY), shown for reference. The structure is shown in both cartoon representation with the color ramped from blue over green to yellow and in surface representation colored light blue (N subdomain) and pink (C subdomain). Transmembrane helix 10b (TM10b) is labeled. (b) Partially occluded inward open conformation, shown with N subdomain in the same orientation as in a. (c) Inward open conformation, shown with N subdomain in the same orientation as in a.
Cytoplasm TM10b C
N
60° D-xylose
90°
90°
TM10b Periplasm
b
N subdomain C subdomain
C
60° TM10b
N
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90°
c ounterflow assays (in which R225A 90° retained 30% activity) 5. On the basis of TM10b the distance between the subdomains, it is clear that these salt bridges are all broken 60° c D-xylose in the inward open state (Fig. 3a), which binding site C together with the mutational data suggests N TM10b that they have a key role in concert with the 90° cytoplasmic helices in controlling access to the binding site from the cytoplasm. In the 90° case of the partially occluded inward open TM10b state, we infer from the position of TM10b that Glu397 forms a new interaction with the N terminus of TM5, Arg341, in the C subdomain of both the partially occluded outwardpossibly involving the nearby Arg160 (Fig. 3a). Conversely, the facing conformation and the inward open conformation is absent. salt bridge formed between Glu397 and another signature residue, This salt bridge is important, because mutating Arg341 to alanine
a
E472 R84 E153 R404 E222 R85
R225
E222
R84
E153
E397
R225 E472 R160
R84
R85
E153
E222
R160
E465
E397
R341
R225 R404
R341
E397
R160
R85
R341
E465
E337
E337
E465 K467
E337
Partially occluded outward open (4GBY)
Inward open
Partially occluded inward open
c
b TM2 TM10a
TM7
TM7
TM11
TM3 TM10
Tyr298 Asn294
TM12
Asn325
TM6 TM4
Trp392
N subdomain
TM10b TM1
Gln289
Gln415
TM9 TM5
Phe24
Trp416
Gln288
TM1
Gln168
200°
TM8 TM8
Trp392
TM11
TM5
TM10
d
Figure 3 Structural changes during the transport cycle. (a) Conserved cytoplasmic salt bridges in the three TM5 TM8 different states. Gray, N subdomain; hot pink, C subdomain. Residues 231–277 of the cytoplasmic TM6 TM10 intersubdomain linker were removed for clarity. Sticks denote side chains of conserved cytoplasmic motifs TM7 (colored as in Fig. 1). Residues labeled in pink form intersubdomain interactions. (b) Structural differences TM3 NG at the cytoplasmic side between the inward open state and partially occluded outward-facing state (PDB 4GBY), shown both from the side highlighting the movement of TM10 (left) and from the cytoplasm, highlighting the TM4 overall rigid body movement of the subdomains (right). The 4GBY structure is superimposed on the N subdomain TM9 TM1 of the inward open state. N and C subdomains are pink and light blue, respectively, in the inward open state and TM12 brick red and violet-blue in 4GBY. Arrows denote movements of helices upon opening. In the side view, only the N subdomain and TM10 are shown for the inward open state, and only the bound d-xylose and TM10 are shown for 4GBY. TM2 TM11 (c) Changes in the substrate-binding site. Gray, inward open state; light brown, 4GBY. Sticks, residues involved in binding; transparent yellow spheres, bound d-xylose in 4GBY. (d) Structural differences between the inward open state and 4GBY, as viewed from the periplasm. A β-nonylglucoside (NG) detergent molecule present in 4GBY is shown in orange.
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BRIE F COMMUNICATIONS completely abolishes activity5. We propose that closure toward the cytoplasm is facilitated by the break in TM10 that probably renders TM10b highly dynamic, which in turn facilitates partial occlusion through interaction with TM5. Subsequent full closure would then require formation of the full complement of intersubdomain salt bridges and re-formation of the Arg341-Glu397 interaction. In the partially occluded outward-facing state, TM10b shields the substrate from the cytoplasm, with the conserved Trp392 situated right beneath the bound d-xylose and contributing a hydrogen bond to its coordination5. However, upon breakage of the intersubdomain salt bridges and subsequent transition to the inward open state through rigid body–like tilting of the subdomains, TM10b moves away and thereby permits release of the substrate (Fig. 3b). The d-xylose–binding site is composed of residues from both subdomains (Fig. 3c)5. However, in the inward open state the two half-sites move apart (Fig. 3c), which together with the substantial movement of Trp392, probably results in considerably lowered affinity in the inward open state, thus facilitating substrate release. This may also explain why GLUT1 displays asymmetric transport with higher affinity for binding at the extracellular than the intracellular side9. At the periplasmic side, interactions between the N and C subdomains are tighter in the inward open conformation than in the partially occluded outward open conformation, but the differences are rather modest (Fig. 3d). This is, however, not surprising because the partially occluded outward-facing conformation is almost fully occluded also from this side5. Salt-bridge formation and breakage at the periplasmic side seems to be a common feature of the transport mechanism of a variety of MFS transporters8,10. However, in the inward open state of XylE the periplasmic interface is dominated by van der Waals and hydrophobic interactions, and we find no evidence for the presence of conserved salt bridges (Supplementary Fig. 3). This suggests that a different mechanistic model is needed to explain the transition between the occluded and outward open states of XylE. A fully outward open structure of XylE may be necessary to further elucidate this issue. In conclusion, the XylE structures presented here represent two snapshots along the transport cycle that together with the recently reported structure5 provide a solid foundation for further studies on GLUT-homology proteins. In addition, they may provide new inroads for the understanding of MFS transporters in general, because XylE
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now represents what is, to our knowledge, the first MFS transporter for which structures are known for more than one conformational state. Methods Methods and any associated references are available in the online version of the paper. Accession codes. Coordinates and structure factors for the XylE structures have been deposited in the Protein Data Bank under accession codes 4JA3 (partially occluded inward open) and 4JA4 (inward open). Note: Supplementary information is available in the online version of the paper. Acknowledgments E.M.Q. was supported by The Danish Council for Independent Research (Medical Sciences; grant 271-09-0187). C.L. was supported by a European Molecular Biology Organization postdoctoral fellowship. This research was further supported by grants from the Swedish Research council, the Swedish Cancer Society and the integrated EU project European drug initiative on channels and transporters (EDICT), as well as a Singapore National Research Foundation Competitive Research Programme grant (NRF2008NRF-CRP002-067). We thank Diamond Light Source for access to beamline I02 (MX5873 and MX6603) that contributed to the results presented here and acknowledge the SOLEIL synchrotron for provision of synchrotron radiation facilities at beamline PROXIMA1 (proposal 20110314). We also thank the Protein Science Facility at the Karolinska Institute for provision of crystallization infrastructure. AUTHOR CONTRIBUTIONS E.M.Q., C.L. and P.M. conducted experiments. L.T. assisted in data collection and analysis. E.M.Q. and C.L. wrote the initial manuscript, and L.T. and P.N. contributed with revisions. P.N. supervised the project. All authors contributed to experimental design and have read and approved the final manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. Augustin, R. IUBMB Life 62, 315–333 (2010). 2. Davis, E.O. & Henderson, P.J. J. Biol. Chem. 262, 13928–13932 (1987). 3. Newstead, S. et al. EMBO J. 30, 417–426 (2011). 4. Jardetzky, O. Nature 211, 969–970 (1966). 5. Sun, L. et al. Nature 490, 361–366 (2012). 6. Abramson, J. et al. Science 301, 610–615 (2003). 7. Huang, Y. et al. Science 301, 616–620 (2003). 8. Solcan, N. et al. EMBO J. 31, 3411–3421 (2012). 9. Carruthers, A. et al. Am. J. Physiol. Endocrinol. Metab. 297, E836–E848 (2009). 10. Law, C.J. et al. Annu. Rev. Microbiol. 62, 289–305 (2008).
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ONLINE METHODS
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Chemicals. Terrific broth (TB) was from Formedium, isopropyl-β-dthiogalactopyranoside (IPTG) was from Saveen Werner, Complete EDTA-free Protease Inhibitor Cocktail was from Roche Applied Science, detergents were from Affymetrix, and crystallization reagents were from Qiagen. All other chemicals were of analytical grade and obtained from Sigma-Aldrich. Gene construction, protein expression and purification. The cDNA of fulllength XylE was cloned into a modified pTH27 vector11 containing a tobacco etch virus (TEV) protease–cleavable N-terminal histidine tag. The gene construct was then transformed into the E. coli C41(DE3) expression strain. For the native protein, cells were grown in TB medium at 220 r.p.m. and 37 °C, and overexpression was induced by the addition of 0.2 mM IPTG when the cell density had reached an optical density at 600 nm (OD600 nm) of 0.7–1.0. Cells were harvested after induction for 16–20 h at 20 °C and stored frozen at –80 °C. For resuspension, 5 ml lysis buffer was used per 1 g of cells. The lysis buffer was composed of 20 mM sodium phosphate (Na-P), pH 7.5, 300 mM NaCl, 0.5 mM Tris(2-carboxyethyl)phosphine (TCEP), 5% glycerol, 1 mg/ml lysozyme, 5 U/ml DNaseI and EDTA-free Complete Protease Inhibitor Cocktail (1 tablet per 100 ml lysis buffer). Resuspension was carried out with stirring at 4 °C for 45 min. Cells were then lysed by using an Emulsiflex microfluidizer (Avestin C3) at 15,000 p.s.i. chamber pressure. Cell debris were removed by centrifugation at 10,000g for 10 min at 4 °C, and the membranes were collected by ultracentrifugation at 104,000g (Beckman Coulter, Ti45 rotor) at 4 °C for 50 min. Membranes obtained from 200 OD600 nm units were resuspended in 3 ml solubilization buffer (20 mM Na-P, pH 7.5, 300 mM NaCl, 15 mM imidazole, 5% glycerol, 0.5 mM TCEP and Complete Protease Inhibitor Cocktail) and solubilized by the addition of 1% decyl-β-d-maltoside (DM). After 60 min at 4 °C, the solubilized membranes were clarified by ultracentrifugation at 104,000g for 30 min at 4 °C and loaded on immobilized metal ion affinity chromatography (IMAC) nickel-nitrilotriacetate beads. The beads were incubated with the supernatant for 1 h, loaded on a plastic column (BioRad) and washed with wash buffer (20 mM Na-P, pH 7.5, 300 mM NaCl, 15–30 mM imidazole, 5% glycerol, 0.5 mM TCEP and 0.2% DM). XylE was eluted by cleaving it from the beads by the addition of 1 mg TEV per milliliter of IMAC beads and incubation overnight at 4 °C (typically, 4 ml of resin was used per 2 liters of cell culture). Cleavage was usually >95% complete. The flow-through fraction with highly pure tag-free XylE was concentrated in 50-kDa–cutoff concentrators (Sartorius Stedim Biotech, VIVASPIN 20) and further purified on a HiLoad Superdex 200 16/60 GL column by using an ÄKTAexplorer 10 chromatography system in a buffer consisting of 20 mM HEPES, 150 mM NaCl, 5% glycerol, 0.5 mM TCEP and 0.2% DM. Peak fractions were collected, concentrated to 8–18 mg/ml and directly used for crystallization or flash frozen in liquid nitrogen for long-term storage. For the selenomethioninelabeled protein, cells were grown in minimal medium supplemented with selenomethionine according to standard protocols. Cell lysis, membrane preparation, purification and concentration procedures were as described above for the native protein. Crystallization. Crystallization trials were performed by using a Mosquito robot (TTP LabTech). Drops of a total volume of 0.2–0.3 µl were dispensed in 96-well sitting-drop plates (Corning), and the plates were subsequently incubated at 4 °C. It was not found beneficial to scale up the crystallization experiments to 24-well plates and 4-µl drops. The crystal for which the P61 data set was collected had dimensions of ~170 × 170 × 15 µm and was grown in 40% PEG 400, 500 mM NaCl, 100 mM MES, pH 6.5, and 2 mM CdCl2. X-ray diffraction data were collected at the PROXIMA1 beamline at the Soleil synchrotron in France (λ = 0.937). The crystal for which the C2 data set was collected had dimensions of ~150 × 150 × 25 µm. Here, the protein was pre incubated with 5 mM lutetium acetate for 1–2 h before dispensing the plate. The crystallization condition was 32% PEG 400, 100 mM MES, pH 6.0, 5 mM CdCl2, 4% 2-methyl-2,4-pentane diol (MPD) and 0.2% (w/v) octyl glucose neopentyl glycol (OGNG) detergent. The data set was collected at beamline I02 at the Diamond synchrotron in England (λ = 1.000). The selenomethionine-labeled
doi:10.1038/nsmb.2569
C2 crystal was obtained in an overall similar crystallization condition, and the data set was collected at the same beamline (λ = 0.9795). Structure determination. Data processing was carried out by using XDS and XSCALE12. Both crystal forms diffracted X-rays anisotropically, and the pro cessed data sets were therefore subjected to ellipsoidal truncation by using the diffraction anisotropy server13 (http://services.mbi.ucla.edu/anisoscale/). As final resolution limits along the a, b and c axes, we used 4.2, 4.2 and 6.0 Å for the P61 form and 4.7, 3.9 and 3.8 Å for the C2 form. These limits were chosen on the basis of the F/σ versus resolution plot generated by the anisotropy server and the outershell I/σI values. Statistics for the truncated data are shown in Supplementary Table 1. Phasing was achieved by molecular replacement by using the N and C subdomains of the partially occluded outward-facing structure of XylE (PDB 4GBY) as separate search models in Phaser14. Three XylE molecules were present in the P61 form (77% solvent) and two in the C2 form (75% solvent). The models were completed by using iterative cycles of manual rebuilding in Coot15 and maximum-likelihood refinement. Sequence assignment was relatively trivial in spite of the modest resolution of our data, as a 2.6-Å structure with 100% sequence similarity is available (PDB 4GC0), for which the individual subdomains exhibit very high structural similarity to our structures (Supplementary Fig. 2). We are therefore confident that the transmembrane helices are in correct register. However, to further underline this point, we have also mapped the methionine positions, using selenomethionine labeling (Supplementary Fig. 4). The selenomethionine data set was like the other data sets corrected for anisotropy and processed with XDS and XSCALE (Supplementary Table 1). For the P61 form, we used both autoBUSTER (http://www.globalphasing.com/buster/) and PHENIX for refinement16. The final round of refinement was carried out by using PHENIX with noncrystallographic symmetry (NCS) and secondary-structure restraints. Target restraints were found not to be beneficial. For the C2 form, refinement was carried out with autoBUSTER alone. For this refinement, we used translation libration screw (TLS) as well as NCS and target restraints (using the 2.6-Å structure of XylE in complex with brominated d-glucose, PDB 4GC0, as target). B-factor sharpening of map coefficients was not used in the case of the P61 form but was found to greatly improve the C2 maps. Refinement statistics are shown in Supplementary Table 1, and samples of the final electron-density maps for both structures are shown in Supplementary Figure 5. After refinement, we used MolProbity v3.19 (http://molprobity.biochem.duke.edu/) to validate that the models have sound geometry. For the P61 form, there were 93.69% favored and 1.64% outliers in the Ramachandran plot. Clash score was 9.07 (97th percentile for structures at 3–9,999 Å), and the overall score was 2.31 (99th percentile for structures at 3.25–4.45 Å). For the C2 form, there were 94.08% favored and 0.71% outliers in the Ramachandran plot, and the clash and overall scores were 17.24 (97th percentile) and 3.02 (88th percentile), respectively. Sequence and structural analysis. Clustal Omega (http://www.ebi.ac.uk/Tools/ msa/clustalo/) was used to generate an initial sequence alignment, which was subsequently edited, colored and annotated with secondary structure by using JalView17. Structural alignments were generated by using either the DALI server (http://ekhidna.biocenter.helsinki.fi/dali_server/) or PyMol (http://www.pymol. org/). PyMol was also used for presentation of the structures. Buried and partially buried residues in the interface between the two subdomains were identified by using the PISA server (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html).
11. Woestenenk, E.A., Hammarstrom, M., van den Berg, S., Hard, T. & Berglund, H. J. Struct. Funct. Genomics 5, 217–229 (2004). 12. Kabsch, W. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010). 13. Strong, M. et al. Proc. Natl. Acad. Sci. USA 103, 8060–8065 (2006). 14. McCoy, A.J. et al. J. Appl. Crystallogr. 40, 658–674 (2007). 15. Emsley, P. & Cowtan, K. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004). 16. Adams, P.D. et al. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010). 17. Waterhouse, A.M., Procter, J.B., Martin, D.M., Clamp, M. & Barton, G.J. Bioinformatics 25, 1189–1191 (2009).
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