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1 Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, 91904 Israel
2 Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
3 Howard Hughes Medical Institute, Departments of Physiology and Microbiology, Immunology, and Molecular Genetics, Molecular Biology Institute, University of California, Los Angeles, California 90095, USA
Reprint requests to: Shimon Schuldiner, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, 91904 Israel; e-mail: Shimon.Schuldiner{at}huji.ac.il; fax: 972-2-5634625.
(RECEIVED January 28, 2004; FINAL REVISION January 28, 2004; ACCEPTED April 7, 2004)
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Abstract |
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Keywords: membrane protein; ion-coupled transporters; drug resistance; NEM accessibility
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04657704.
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Introduction |
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The major facilitator superfamily (MFS) represents the largest group of ion-coupled transporters (Saier et al. 1999). They translocate substrates against their electrochemical gradient by coupling the movement of an ion or a second solute down its gradient (Kaback et al. 2001). Despite intense interest in these proteins and a large number of laboratories experimenting with several of them, structural information at the atomic level was nonexistent until very recently.
The first structural evidence approaching atomic resolution was supplied by the interpretation of the three-dimensional structure of the oxalate transporter, OxlT, at a resolution of 6.5 Å (Hirai et al. 2002, 2003). By combining the structural information for OxlT with biochemical and sequence information from other MFS proteins, Subramanian, Maloney, and collaborators (Hirai et al. 2003) proposed a structural model. In their model, 12 helices surround a central cavity and form a symmetrical structure formed by two, six-helical lobes.
High-resolution structures of two proteins from this superfamily recently became available: the lactose permease (LacY; Abramson et al. 2003) and the glycerol-3-phosphate transporter (GlpT; Huang et al. 2003) from Escherichia coli. The sequence identity between GlpT and LacY is only 21% and their mechanism of action appears to be different because the former uses the downhill gradient of phosphate for an obligatory exchange with glycerol-Pi (antiporter), whereas the latter cotransports 
-galactosides with protons (symporter). Despite these differences, both structures show a highly similar fold, which is, in general, similar to the overall low-resolution structure of OxlT, on the basis of electron microscopy (EM) images (Hirai et al. 2002). C
-RMSD between the structurally conserved regions of the two proteins is only 3.7 Å. Structural differences are observed in loop length and conformation. In addition, some of the trans membrane domains (TMs) of LacY are more kinked than in GlpT (Abramson et al. 2003; Huang et al. 2003).
The findings suggest the intriguing possibility that the fold of these transporters constitutes a scaffold for all MFS transporters with 12 helices. Although the fold is conserved, the specific function is obtained by varying sets of amino acids at the substrate binding and translocation domains. As observed earlier for other protein families, the fold appears to be better conserved than the sequence (Aronson et al. 1994; Rupasinghe et al. 2003).
In this work, we test this contention by simple comparative modeling of two experimentally characterized MFS proteins using the coordinates of the two known structures, LacY and GlpT, as templates. Both of the modeled proteins belong to the MFS. TetAB, the Tn10-encoded tetracycline/ H+ antiporter, has been exhaustively studied, and information about N-ethyl maleimide (NEM) accessibility of each one of the residues has been documented (Kimura et al. 1996, 1998; Konishi et al. 1999; Kimura-Someya et al. 2000; Tamura et al. 2001). The vesicular monoamine transporter (VMAT) is a mammalian transporter responsible for accumulation of monoamines in synaptic vesicles and chromaffin granules (Liu et al. 1992). VMAT is weakly but distinctly related to the multidrug transporters in the MFS (Liu et al. 1992; Schuldiner et al. 1995; Saier and Paulsen 2001).
The generated models are in excellent agreement with the available experimental data supporting the contention that structure is highly conserved in this superfamily.
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Results |
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). Although the fold of GlpT and LacY is highly similar (Fig. 2A), the low homology prevents the generation of a reliable alignment. Therefore, a manual optimization was performed as suggested by Sali, Marti-Renom, and collaborators (Marti-Renom et al. 2000). Cycles of computerized modeling followed by manual improvement of the alignment were done until a model that satisfies data and constraints was generated.
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In order to get a good reference model, a structural alignment of GlpT and LacY was used first. The use of structural alignment enabled the generation of two models: the LacY model based on the GlpT structure, and the GlpT model based on the LacY structure. Because structural alignment relates residues by minimizing the RMSD between the two aligned structures, it is appropriate for comparative modeling.
Each of these models was compared with the solved structure. C-RMSD for the structure versus the model was calculated for the two symmetrical halves of the protein (helices 1–6 and 7–12) and for the entire protein (Table 1). The position and orientation of membrane-embedded residues in the models were compared with the known structure (Fig. 2B,C
). Superimposition of the models and structures produced, as expected, similar differences to superimposition of LacY and GlpT (~3.7 Å). The differences between the two halves of the proteins are smaller, especially of the C-terminal half (Table 1). That is mostly attributed to the fact that these proteins consist of two structurally similar lobes. The relative positioning of the lobes may change in different proteins and even at different conditions for the same protein. This mechanistic feature is made possible by the long central loop between TM6 and TM7 (L6–7) and has been suggested to be necessary for protein function (Weinglass and Kaback 2000).
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-RMSD of the two helices, but nevertheless the side chains in the TM regions of the models are mostly oriented as in the structure. These two examples represent the expected differences between a comparative model for MFS and its real structure when an optimal alignment is provided.
Modeling of LacY
In order to explore the capability of TM prediction combined with NEM accessibility data to generate an alignment good enough for comparative modeling, we built a model for LacY in three stages (Fig. 1): (1) Multiple sequence alignment was done for 20 MFS sequences from which the pairwise alignment of LacY (target) and GlpT (template) was extracted; (2) because of low homology, manual adjustment according to TM prediction was necessary; and (3) after modeling, the alignment was readjusted again, so that charged residues and NEM-accessible residues face the center of the protein (Venkatesan et al. 2000a,b,c; Kwaw et al. 2001). Six cycles of improvement were performed until a model that fits the constraints was achieved. The resulting model was then compared with the model built according to the structural alignment. The general fold of the two models is almost identical (the same template was used for both models) and most of the residues are positioned similarly in both models (Fig. 2D). Residues in TMs 4, 5, 6, and 8 are shifted by one position between the two models (a phase shift), but still all NEM accessibility data are compatible to both models. The phase shift is most probably caused by a faulty alignment, but because all NEM data are in agreement with the model, it might also reflect a structural substate with a rotated helix. The data available neither allow us to rectify the alignment nor to assign a functional importance.
Modeling of TetAB
The fold of GlpT and LacY is more conserved than their sequences. Based on the assumption that helix packing and fold of MFS is conserved and using alignments based on experimental data, models of TetAB with either LacY or GlpT as template were built. The preliminary alignments, derived from multiple sequence alignment, were manually improved, as was done for LacY modeling. The improved alignments were then used to generate models, which were in accordance with NEM accessibility data (Fig. 3A; Tamura et al. 2001) and the common helix packing pattern of MFS proteins with 12 TMs (Fig. 3B).
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) and that most of the intervals between the sequences were three to four amino acids long, meaning that the differences between the models at these domains are of one helix turn (the helices are in phase), suggesting that helix orientation is similar. There are regions at the sequence alignment where a phase shift is observed inside the TM regions (± one or two residues). At those places (TM4, TM8, and TM11), structural differences between the helices of the two templates forced the phase shift and enabled compatibility of the experimental data. Thus, for example, the third turn in TM11 is of three residues in GlpT and four residues in LacY. The problem is overcome by a phase shift of the two models at that point.
As a consequence of our alignment procedure, all of the membrane-embedded charged residues in each of the models are facing the internal cavity. Because most of these residues are acidic, the cavity, according to the model, is mostly negative (Fig. 3C). The periphery of the models is mostly neutral, the cytoplasmic side is positively charged, and the periplasmic side is negatively charged. This is in accordance with the positive-inside rule (von Heijne and Gavel 1988).
Tilting and bending of the helices in both models is compatible with the NEM accessibility profile of the protein (Fig. 4C). The periplasmic half of TM4 and TM10 is blocked from NEM molecules by TM1–TM2 and TM7–TM8, respectively (Fig. 4A), whereas TM1 and TM7 are NEM inaccessible at the cytoplasmic half, where they are blocked by TM4–TM5 and TM10–TM11, respectively (Fig. 4B). The two different models are very similar (C-RMSD 3.8 Å) in the sense of fold and orientation of the residues in the helices, even though there are differences that derive from the structural differences of the two templates and alignment of TetAB with each of them.
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). Examination of the model reveals a potential ion pair that connects TM2 and TM11 (Fig. 5B). Experimental evidence for the existence of this ion pair between Asp 427 and Lys 139 was described (Merickel et al. 1997).
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). According to the alignment, His 338 is conserved in VAChT and is aligned against a conserved Tyr in VMATs. That Tyr (Y342) is positioned <3 Å from the Asp in TM10 in our model (Fig. 5D). |
Discussion |
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To get a more realistic estimate of correctness of our models, we generated a model for LacY, based on a manually optimized alignment (according to multiple sequence alignment, TM prediction, and experimental data) without considering structural alignment. Comparison of this model to a model derived from an ideal alignment showed that 70% of the manually optimized alignment is identical to the structural alignment (Table 1). The remaining 30% is located mainly in loops but also derived from a phase shift between TMs 4, 5, 6, and 8 of the two models. The data fit both models and the residues are positioned similarly despite the phase shift. From the comparison of the first "ideal" model and the second model, it can be deduced that the manual optimization of a sequence alignment of MFS proteins may generate an alignment very close to optimum. Models based on such an alignment may serve as insightful tools for biochemists studying MFS proteins.
TetAB model
Once the alignments were optimized, models were built for TetAB according to GlpT and LacY. The two models are mostly in agreement with NEM accessibility data (Figs. 3A, 4A–C). Residues 20, 24, 136, and 311 in the TetAB model by GlpT and residues 24 and 253 in the TetAB model by LacY do not face the central cavity of the model, although they are NEM accessible according to biochemical data. Residues that do not agree with NEM accessibility data may suggest that those points are relevant to movement of the protein during the transport cycle or to a local difference in helix bending or stretching. TM7 may serve as an example for such a case because there are more possible NEM-accessible residues, according to the model, than there are in the data (Fig. 4B). The periplasmic half of TM4 and the cytoplasmic half of TM1 are NEM inaccessible, even though they are facing the central cavity of the protein. Those helices in the models are blocked from the cavity by two other helices; TM4 is blocked by TM1 and TM2, whereas TM1 is blocked in a similar way by TM4 and TM5 (Fig. 4). That pattern in the N-terminal lobe continues in the C-terminal lobe of the protein, where TM7 and TM10, which symmetrically correspond to TM1 and TM4, are blocked in the same way by the corresponding TMs (TM7 by TM10–TM11, TM10 by TM7–TM8; Fig. 4).
The charge distribution of the model suggests that the cavity is mostly negative. That feature is in accordance with the positive charge of the GlpT cavity (Huang et al. 2003). The Pi transported by GlpT is negatively charged, whereas Mg++-tetracycline is positively charged. The charge distribution of the GlpT and TetAB model seems to fit their substrate specificity.
The two models are not identical and residues in some places are shifted by one to two helix turns between the two models. The fact that the two models differ from each other indicates that the alignment achieved after manual optimization is not optimal and the exact positioning of the residues is questionable. Still, the accessibility pattern of the helices in both of the suggested models fits the data and the fold is shown to be suitable for this protein. Based on their extensive work on TetAB, Yamaguchi and collaborators (Tamura et al. 2001, 2003) postulated a model for helix packing different from the one shown here. This model proposes a symmetric structure with a central cavity. However, some of the peripheral helices are located at positions that are different from those suggested here.
rVMAT2 model
The structural data concerning VMAT are not as comprehensive as for TetAB. Because of the lack of experimental data, the structural model of VMAT is less reliable than the model of TetAB. Additional data describing the closely related VAChT were used for further validation. The model was built only to satisfy VMAT data, meaning that all membrane-embedded charged residues are facing the central cavity and the Lys 139 in TM2 is facing Asp 427 in TM11 to enable a salt bridge. The 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) reaction with Cys 439 in human VMAT2 is inhibited by tetrabenazine (TBZ) binding, suggesting that this residue is close to the TBZ binding site (Thiriot and Ruoho 2001). In the rVMAT2 model, the corresponding residue, Cys 431, is positioned toward the central cavity, one helix turn away from the functionally important Asp 427. Asp 461 in TM12 is related to the protein’s apparent affinity to several substances (Peter et al. 1996; Finn and Edwards 1997). In the model, Asp 461 is close to the cytoplasmic side of the transporter and is hidden from the central cavity. Phe 464 in the same helix is related to the apparent affinity of the protein to serotonin (Peter et al. 1996; Finn and Edwards 1997). According to the model, Phe 464 is located in the interface with TM7 in close proximity to Phe 304. The role of those residues in TM12, according to the data, is likely structural and that concept is supported by the model.
VAChT is an MFS protein closely related to VMAT, and biochemical data from VAChT research are often used to support data from VMAT studies (Parsons 2000). Kim et al. (2000) suggested that there is a salt bridge between His 338 in TM8 and Asp 398 in TM10. There is a His in TM8 in the rVMAT model, but it is too far from the Asp in TM10 and cannot interact directly with it. A multiple sequence alignment of VAChT and VMAT revealed that His 338 of VAChT corresponds to Tyr 342 of VMAT. The distance between Tyr 342 and Asp 400 is ~3 Å in the model of VMAT (Fig. 5D), a distance that enables the formation of a salt bridge between two opposite charges.
We demonstrated, with the aid of comparative modeling, that the fold observed for LacY and GlpT also satisfies the experimental and evolutionary data for two other members of the superfamily, VMAT and TetAB. This work supports the line of reasoning presented by Hirai et al. (2003) and the idea that structural conservation of the MFS proteins is high despite the low sequence homology. The simple modeling approach used here may provide a good starting point for research of new MFS proteins. Three-dimensional models, such as those discussed here, may be helpful for biochemical research because they may direct work toward educated guesses. Because the substrate binding site is localized in the cavity between the two lobes, residues facing the cavity are obvious candidates for mutagenesis and biophysical studies. Although the information supplied by the models will simplify the processing and understanding of biochemical data, the limitation and low accuracy of these models must be taken into consideration as well.
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Materials and methods |
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Generating an alignment for modeling
To achieve an alignment as close to optimal as possible, we generated a sequence alignment of target and template and manipulated it basically as suggested by Sali, Marti-Renom, and collaborators (Marti-Renom et al. 2000): Twenty randomly chosen MFS sequences of 20% to 40% homology with TetAB or rVMAT2 were aligned with ClustalW (Thompson et al. 1994) against TetAB, rVMAT2, LacY, and GlpT. A desired pairwise alignment of a template protein—LacY or GlpT—with a target protein—TetAB or VMAT—was extracted from the multiple sequence alignment (LacY was also modeled, in order to test the modeling approach). Because of the poor sequence homology, two additional steps were required to optimize the initial alignment for each pair (Fig. 1): (1) overlapping of TM regions predicted for the modeled protein and TM regions of the known structure and (2) fine-tuning of the alignment using experimental data on NEM accessibility or based on the assumption that charged residues in membrane domains should face the hydrophilic cavity. During each modeling cycle, the alignments were modified so that no buried charged residues occur. For TetAB and LacY models, NEM-accessible residues were realigned in a manner that enabled them to face the water-filled cavity of the model.
Prediction of TM regions of MFS proteins was performed with the TMpred (Hofmann and Stoffel 1993) and the TMHMM (Krogh et al. 2001) algorithms. During the optimization process, no gaps were allowed in the predicted or known TM regions.
The long loop between TM1 and TM2 (L1–2; residues 57 to 101 in rVMAT2) in VMATs was not modeled because it is unique to VMAT and VAChT (no homology with other MFSs).
In order to validate the modeling approach, an additional model for LacY was constructed, on the basis of sequence alignment rather than structural alignment, using GlpT as a template. The optimization steps described earlier were used to improve the alignment. The sequence alignment was tuned without taking into consideration the known structure of LacY. The model, produced from adjusted sequence alignment, was compared with the model based on structural alignment.
Comparative modeling
Comparative modeling was performed with the Modeller 6.2 software (Sali and Blundell 1993). Modeling was performed with the default "model" routine using LacY and GlpT (PDB ID-1PV6 and 4PW4, respectively) as templates and the alignment of each of them with rVMAT2 or TetAB as input. The first five residues and residues 227 to 239 in the L6–7 region of GlpT are not resolved in the structure (Huang et al. 2003) and were therefore omitted from the sequence in the alignments (replaced with gaps).
Analysis of the models
The final alignments were used to generate two models for each of the proteins TetAB and rVMAT2. Similarity of the models and the differences between them were examined by a C-RMSD calculation and by visual inspection of the overlapping models, using Swiss pdb viewer and VMD programs (Humphrey et al. 1996; Guex and Peitsch 1997).
The accuracy potential of the MFS models was assessed by comparing optimal models, based on structural alignment, of GlpT and LacY to their solved structures.
To test the ability to reproduce a modified sequence alignment as similar as possible to the structural alignment, we compared the two earlier mentioned LacY models.
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Acknowledgments |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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References |
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 J. Holyoake, V. Caulfeild, S. A. Baldwin, and M. S. P. Sansom Modeling, Docking, and Simulation of the Major Facilitator Superfamily Biophys. J., November 15, 2006; 91(10): L84 - L86. [Abstract] [Full Text] [PDF]
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Z. Hou, J. Ye, C. L. Haska, and L. H. Matherly Transmembrane Domains 4, 5, 7, 8, and 10 of the Human Reduced Folate Carrier Are Important Structural or Functional Components of the Transmembrane Channel for Folate Substrates J. Biol. Chem., November 3, 2006; 281(44): 33588 - 33596. [Abstract] [Full Text] [PDF]
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 T. Beuming, L. Shi, J. A. Javitch, and H. Weinstein A Comprehensive Structure-Based Alignment of Prokaryotic and Eukaryotic Neurotransmitter/Na+ Symporters (NSS) Aids in the Use of the LeuT Structure to Probe NSS Structure and Function Mol. Pharmacol., November 1, 2006; 70(5): 1630 - 1642. [Abstract] [Full Text] [PDF]
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T. Kasahara, M. Ishiguro, and M. Kasahara Eight Amino Acid Residues in Transmembrane Segments of Yeast Glucose Transporter Hxt2 Are Required for High Affinity Transport J. Biol. Chem., July 7, 2006; 281(27): 18532 - 18538. [Abstract] [Full Text] [PDF]
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 E. Vardy, S. Steiner-Mordoch, and S. Schuldiner Characterization of Bacterial Drug Antiporters Homologous to Mammalian Neurotransmitter Transporters J. Bacteriol., November 1, 2005; 187(21): 7518 - 7525. [Abstract] [Full Text] [PDF]
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Z. Hou, S. E. Stapels, C. L. Haska, and L. H. Matherly Localization of a Substrate Binding Domain of the Human Reduced Folate Carrier to Transmembrane Domain 11 by Radioaffinity Labeling and Cysteine-substituted Accessibility Methods J. Biol. Chem., October 28, 2005; 280(43): 36206 - 36213. [Abstract] [Full Text] [PDF]
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X. Zhang, N. V. Shirahatti, D. Mahadevan, and S. H. Wright A Conserved Glutamate Residue in Transmembrane Helix 10 Influences Substrate Specificity of Rabbit OCT2 (SLC22A2) J. Biol. Chem., October 14, 2005; 280(41): 34813 - 34822. [Abstract] [Full Text] [PDF]
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M. Sharoni, S. Steiner-Mordoch, and S. Schuldiner Exploring the Binding Domain of EmrE, the Smallest Multidrug Transporter J. Biol. Chem., September 23, 2005; 280(38): 32849 - 32855. [Abstract] [Full Text] [PDF]
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 Q. Yang, X. Wang, L. Ye, M. Mentrikoski, E. Mohammadi, Y.-M. Kim, and P. C. Maloney Experimental tests of a homology model for OxlT, the oxalate transporter of Oxalobacter formigenes PNAS, June 14, 2005; 102(24): 8513 - 8518. [Abstract] [Full Text] [PDF]
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