Modeling of glycerol-3-phosphate transporter suggests a potential 'tilt' mechanism involved in its function

Many major facilitator superfamily (MFS) transporters have similar 12-transmembrane alpha-helical topologies with two six-helix halves connected by a long loop. In humans, these transporters participate in key physiological processes and are also, as in the case of members of the organic anion transporter (OAT) family, of pharmaceutical interest. Recently, crystal structures of two bacterial representatives of the MFS family--the glycerol-3-phosphate transporter (GlpT) and lac-permease (LacY)--have been solved and, because of assumptions regarding the high structural conservation of this family, there is hope that the results can be applied to mammalian transporters as well. Based on crystallography, it has been suggested that a major conformational "switching" mechanism accounts for ligand transport by MFS proteins. This conformational switch would then allow periodic changes in the overall transporter configuration, resulting in its cyclic opening to the periplasm or cytoplasm. Following this lead, we have modeled a possible "switch" mechanism in GlpT, using the concept of rotation of protein domains as in the DynDom program17 and membranephilic constraints predicted by the MAPAS program.(23) We found that the minima of energies of intersubunit interactions support two alternate positions consistent with their transport properties. Thus, for GlpT, a "tilt" of 9 degrees -10 degrees rotation had the most favorable energetics of electrostatic interaction between the two halves of the transporter; moreover, this confirmation was sufficient to suggest transport of the ligand across the membrane. We conducted steered molecular dynamics simulations of the GlpT-ligand system to explore how glycerol-3-phosphate would be handled by the "tilted" structure, and obtained results generally consistent with experimental mutagenesis data. While biochemical data remain most consistent with a single-site alternating access model, our results raise the possibility that, while the "rocker switch" may apply to certain MFS transporters, intermediate "tilted" states may exist under certain circumstances or as transitional structures. Although wet lab experimental confirmation is required, our results suggest that transport mechanisms in this transporter family should probably not be assumed to be conserved simply based on standard structural homology considerations. Furthermore, steered molecular dynamics elucidating energetic interactions of ligands with amino acid residues in an appropriately modeled transporter may have predictive value in understanding the impact of mutations and/or polymorphisms on transporter function.     

MFS超家族转运蛋白结构与分子机制的研究

主要协助转运蛋白超家族（major facilitator superfamily,MFS）是一个主要的次级膜转运蛋白超家族。MFS超家族蛋白转运底物的多样性使得它们在细胞物质交换和能量代谢过程中起着重要作用。从2003年第一个高分辨率的LacY蛋白三维结构的解析到现在,已经有5个细菌MFS超家族的蛋白结构被解析出来,结合大量的生化研究结果,使得对其转运的分子机制有了更为深入的理解。将对MFS超家族蛋白的三维结构和转运机理进行阐述。     

3D model of the Escherichia coli multidrug transporter MdfA reveals an essential membrane-embedded positive charge

MdfA is an Escherichia coli multidrug transporter of the major facilitator superfamily (MFS) of secondary transporters. Although several aspects of multidrug recognition by MdfA have been characterized, better understanding the detailed mechanism of its function requires structural information. Previous studies have modeled the 3D structures of MFS proteins, based on the X-ray structure of LacY and GlpT. However, because of poor sequence homology, between LacY, GlpT, and MdfA additional constraints were required for a reliable homology modeling. Using an algorithm that predicts the angular orientation of each transmembrane helix (TM) (kPROT), we obtained a remarkably similar pattern for the 12 TMs of MdfA and those of GlpT and LacY, suggesting that they all have similar helix packing. Consequently, a 3D model was constructed for MdfA by structural alignment with LacY and GlpT, using the kPROT results as an additional constraint. Further refinement and a preliminary evaluation of the model were achieved by correlated mutation analysis and the available experimental data. Surprisingly, in addition to the previously characterized membrane-embedded glutamate at position 26, the model suggests that Asp34 and Arg112 are located within the membrane, on the same face of the cavity as Glu26. Importantly, Arg112 is evolutionarily conserved in secondary drug transporters, and here we show that a positive charge at this position is absolutely essential for multidrug transport by MdfA.     

Integration of Evolutionary Features for the Identification of Functionally Important Residues in Major Facilitator Superfamily Transporters

The identification of functionally important residues is an important challenge for understanding the molecular mechanisms of proteins. Membrane protein transporters operate two-state allosteric conformational changes using functionally important cooperative residues that mediate long-range communication from the substrate binding site to the translocation pathway. In this study, we identified functionally important cooperative residues of membrane protein transporters by integrating sequence conservation and co-evolutionary information. A newly derived evolutionary feature, the co-evolutionary coupling number, was introduced to measure the connectivity of co-evolving residue pairs and was integrated with the sequence conservation score. We tested this method on three Major Facilitator Superfamily (MFS) transporters, LacY, GlpT, and EmrD. MFS transporters are an important family of membrane protein transporters, which utilize diverse substrates, catalyze different modes of transport using unique combinations of functional residues, and have enough characterized functional residues to validate the performance of our method. We found that the conserved cores of evolutionarily coupled residues are involved in specific substrate recognition and translocation of MFS transporters. Furthermore, a subset of the residues forms an interaction network connecting functional sites in the protein structure. We also confirmed that our method is effective on other membrane protein transporters. Our results provide insight into the location of functional residues important for the molecular mechanisms of membrane protein transporters.     

Structure and transport mechanism of the bacterial oxalate transporter OxlT

Membrane proteins that belong to the major facilitator superfamily (MFS) are found in organisms across the evolutionary spectrum and mediate the transport of a variety of substrates ranging from small metabolites to neurotransmitters. The oxalate transporter (OxlT) is a representative MFS protein, and exchanges formate for oxalate across the cytoplasmic membrane of the organism Oxalobacter formigenes. Here, we present a structural model for the protein conformational changes that occur during oxalate transport by combining a three-dimensional map of the oxalate-bound, "closed" state of OxlT at 6.5 A determined by cryo-electron microscopy with a model of the "open" state of OxlT based on the atomic structures of the related transporters, glycerol-3-phosphate transporter (GlpT) and lactose permease (LacY). We demonstrate that the principal structural change associated with substrate transport is a concerted rocking movement of the two structurally similar halves of the protein relative to each other. Our structural model places two positively charged residues, Arg-272 and Lys-355 in the central cavity, suggesting that electrostatic interactions between these residues and the oxalate anion is a key step in generating the conformational change between the open and closed states of the transporter.     

Eukaryotic major facilitator superfamily transporter modeling based on the prokaryotic GlpT crystal structure

The major facilitator superfamily (MFS) of transporters represents the largest family of secondary active transporters and has a diverse range of substrates. With structural information for four MFS transporters, we can see a strong structural commonality suggesting, as predicted, a common architecture for MFS transporters. The rate for crystal structure determination of MFS transporters is slow, making modeling of both prokaryotic and eukaryotic transporters more enticing. In this review, models of eukaryotic transporters Glut1, G6PT, OCT1, OCT2 and Pho84, based on the crystal structures of the prokaryotic GlpT, based on the crystal structure of LacY are discussed. The techniques used to generate the different models are compared. In addition, the validity of these models and the strategy of using prokaryotic crystal structures to model eukaryotic proteins are discussed. For comparison, E. coli GlpT was modeled based on the E. coli LacY structure and compared to the crystal structure of GlpT demonstrating that experimental evidence is essential for accurate modeling of membrane proteins.     

Sequence alignment and homology threading reveals prokaryotic and eukaryotic proteins similar to lactose permease

Certain prokaryotic transport proteins similar to the lactose permease of Escherichia coli (LacY) have been identified by BLAST searches from available genomic databanks. These proteins exhibit conservation of amino acid residues that participate in sugar binding and H(+) translocation in LacY. Homology threading of prokaryotic transporters based on the X-ray structure of LacY (PDB ID: 1PV7) and sequence similarities reveals a common overall fold for sugar transporters belonging to the Major Facilitator Superfamily (MFS) and suggest new targets for study. Evolution-based searches for sequence similarities also identify eukaryotic proteins bearing striking resemblance to MFS sugar transporters. Like LacY, the eukaryotic proteins are predicted to have 12 transmembrane domains (TMDs), and many of the irreplaceable residues for sugar binding and H(+) translocation in LacY appear to be largely conserved. The overall size of the eukaryotic homologs is about twice that of prokaryotic permeases with longer N and C termini and loops between TMDs III-IV and VI-VII. The human gene encoding protein FLJ20160 consists of six exons located on more than 60,000 bp of DNA sequences and requires splicing to produce mature mRNA. Cellular localization predictions suggest membrane insertion with possible proteolysis at the N terminus, and expression studies with the human protein FJL20160 demonstrate membrane insertion in both E.coli and Pichia pastoris. Widespread expression of the eukaryotic sugar transport candidates suggests an important role in cellular metabolism, particularly in brain and tumors. Homology is observed in the TMDs of both the eukaryotic and prokaryotic proteins that contain residues involved in sugar binding and H(+) translocation in LacY.     

Eukaryotic major facilitator superfamily transporter modeling based on the prokaryotic GlpT crystal structure (Review)

The major facilitator superfamily (MFS) of transporters represents the largest family of secondary active transporters and has a diverse range of substrates. With structural information for four MFS transporters, we can see a strong structural commonality suggesting, as predicted, a common architecture for MFS transporters. The rate for crystal structure determination of MFS transporters is slow, making modeling of both prokaryotic and eukaryotic transporters more enticing. In this review, models of eukaryotic transporters Glut1, G6PT, OCT1, OCT2 and Pho84, based on the crystal structures of the prokaryotic GlpT, based on the crystal structure of LacY are discussed. The techniques used to generate the different models are compared. In addition, the validity of these models and the strategy of using prokaryotic crystal structures to model eukaryotic proteins are discussed. For comparison, E. coli GlpT was modeled based on the E. coli LacY structure and compared to the crystal structure of GlpT demonstrating that experimental evidence is essential for accurate modeling of membrane proteins.     

Lactose permease and the alternating access mechanism

Crystal structures of the lactose permease of Escherichia coli (LacY) reveal 12, mostly irregular transmembrane α-helices surrounding a large cavity open to the cytoplasm and a tightly sealed periplasmic side (inward-facing conformation) with the sugar-binding site at the apex of the cavity and inaccessible from the periplasm. However, LacY is highly dynamic, and binding of a galactopyranoside causes closing of the inward-facing cavity with opening of a complementary outward-facing cavity. Therefore, the coupled, electrogenic translocation of a sugar and a proton across the cytoplasmic membrane via LacY very likely involves a global conformational change that allows alternating access of sugar- and H(+)-binding sites to either side of the membrane. Here the various biochemical and biophysical approaches that provide strong support for the alternating access mechanism are reviewed. Evidence is also presented indicating that opening of the periplasmic cavity is probably the limiting step for binding and perhaps transport.     

Conserved cytoplasmic loops are important for both the transport and chemotaxis functions of PcaK, a protein from Pseudomonas putida with 12 membrane-spanning regions.

Chemotaxis to the aromatic acid 4-hydroxybenzoate (4-HBA) by Pseudomonas putida is mediated by PcaK, a membrane-bound protein that also functions as a 4-HBA transporter. PcaK belongs to the major facilitator superfamily (MFS) of transport proteins, none of which have so far been implicated in chemotaxis. Work with two well-studied MFS transporters, LacY (the lactose permease) and TetA (a tetracycline efflux protein), has revealed two stretches of amino acids located between the second and third (2-3 loop) and the eighth and ninth (8-9 loop) transmembrane regions that are required for substrate transport. These sequences are conserved among most MFS transporters, including PcaK. To determine if PcaK has functional requirements similar to those of other MFS transport proteins and to analyze the relationship between the transport and chemotaxis functions of PcaK, we generated strains with mutations in amino acid residues located in the 2-3 and 8-9 loops of PcaK. The mutant proteins were analyzed in 4-HBA transport and chemotaxis assays. Cells expressing mutant PcaK proteins had a range of phenotypes. Some transported at wild-type levels, while others were partially or completely defective in 4-HBA transport. An aspartate residue in the 8-9 loop that has no counterpart in LacY and TetA, but is conserved among members of the aromatic acid/H(+) symporter family of the MFS, was found to be critical for 4-HBA transport. These results indicate that conserved amino acids in the 2-3 and 8-9 loops of PcaK are required for 4-HBA transport. Amino acid changes that decreased 4-HBA transport also caused a decrease in 4-HBA chemotaxis, but the effect on chemotaxis was sometimes slightly more severe. The requirement of PcaK for both 4-HBA transport and chemotaxis demonstrates that P. putida has a chemoreceptor that differs from the classical chemoreceptors described for Escherichia coli and Salmonella typhimurium.     

Substrate-bound structure of the E. coli multidrug resistance transporter MdfA

Multidrug resistance is a serious threat to public health. Proton motive force-driven antiporters from the major facilitator superfamily (MFS) constitute a major group of multidrug-resistance transporters. Currently, no reports on crystal structures of MFS antiporters in complex with their substrates exist. The E. coli MdfA transporter is a well-studied model system for biochemical analyses of multidrug-resistance MFS antiporters. Here, we report three crystal structures of MdfA-ligand complexes at resolutions up to 2.0 Å, all in the inward-facing conformation. The substrate-binding site sits proximal to the conserved acidic residue, D34. Our mutagenesis studies support the structural observations of the substrate-binding mode and the notion that D34 responds to substrate binding by adjusting its protonation status. Taken together, our data unveil the substrate-binding mode of MFS antiporters and suggest a mechanism of transport via this group of transporters.     

Lactose permease as a paradigm for membrane transport proteins (Review)

Our structural knowledge of the major facilitator superfamily (MFS) has dramatically increased in the past year with three structures of proteins from the MFS (oxalate/formate antiporter; lactose/proton symporter and the P(i)/glycerol-3-phosphate antiporter). All three structures revealed 12 transmembrane helices forming two distinct domains and could imply that members of the MFS have preserved both secondary as well as tertiary structural elements during evolution. Lactose permease, a particularly well-studied member of the MFS, has been extensively explored by a number of molecular biological, biochemical and biophysical approaches. In this review, we take a closer look at the structure of LacY and incorporate a wealth of biochemical and biophysical data in order to propose a possible mechanism for lactose/proton symport. In addition, we make some brief comparisons between the structures of LacY and GlpT.     

Lactose permease as a paradigm for membrane transport proteins (Review)

Our structural knowledge of the major facilitator superfamily (MFS) has dramatically increased in the past year with three structures of proteins from the MFS (oxalate/formate antiporter; lactose/proton symporter and the P_i/glycerol-3-phosphate antiporter). All three structures revealed 12 transmembrane helices forming two distinct domains and could imply that members of the MFS have preserved both secondary as well as tertiary structural elements during evolution. Lactose permease, a particularly well-studied member of the MFS, has been extensively explored by a number of molecular biological, biochemical and biophysical approaches. In this review, we take a closer look at the structure of LacY and incorporate a wealth of biochemical and biophysical data in order to propose a possible mechanism for lactose/proton symport. In addition, we make some brief comparisons between the structures of LacY and GlpT.     

Helix dynamics in a membrane transport protein: comparative simulations of the glycerol-3-phosphate transporter and its constituent helices

The glycerol-3-phosphate transporter (GlpT) is a member of the major facilitator superfamily (MFS). GlpT is an organic phosphate/inorganic phosphate antiporter. It shares a similar fold with other MFS transporters (e.g. LacY and EmrD) consisting of 12 transmembrane (TM) helices which form two domains (each of six TM helices) surrounding a central ligand-binding cavity. The TM helices (especially the cavity-lining helices) contain a large number of proline and glycine residues, which may aid in the conformational changes believed to underline the transport mechanism. Molecular dynamics simulations in a phospholipid bilayer have been used to compare the conformational properties of the isolated TM helices with those in the intact GlpT protein. Analysis of these simulations focuses on the role of proline-induced flexibility in the TM helices. Our results are consistent with the proposed rocker switch mechanism for transport by GlpT. In particular, the simulations highlight the cavity-lining helices (H4, H5, H10 and H11) as being significantly flexible, suggesting that the transport mechanism may involve intra-helix motions in addition to pseudo-rigid body motions of the N- and C-terminal domains relative to one another.     

Flexible Gates Generate Occluded Intermediates in the Transport Cycle of LacY

The major facilitator superfamily (MFS) transporter lactose permease (LacY) alternates between cytoplasmic and periplasmic open conformations to co-transport a sugar molecule together with a proton across the plasma membrane. Indirect experimental evidence suggested the existence of an occluded transition intermediate of LacY, which would prevent leaking of the proton gradient. As no experimental structure is known, the conformational transition is not fully understood in atomic detail. We simulated transition events from a cytoplasmic open conformation to a periplasmic open conformation with the dynamic importance sampling molecular dynamics method and observed occluded intermediates. Analysis of water permeation pathways and the electrostatic free-energy landscape of a solvated proton indicated that the occluded state contains a solvated central cavity inaccessible from either side of the membrane. We propose a pair of geometric order parameters that capture the state of the pathway through the MFS transporters as shown by a survey of available crystal structures and models. We present a model for the occluded state of apo-LacY, which is similar to the occluded crystal structures of the MFS transporters EmrD, PepT_So, NarU, PiPT and XylE. Our simulations are consistent with experimental double electron spin–spin distance measurements that have been interpreted to show occluded conformations. During the simulations, a salt bridge that has been postulated to be involved in driving the conformational transition formed. Our results argue against a simple rigid-body domain motion as implied by a strict “rocker-switch mechanism” and instead hint at an intricate coupling between two flexible gates.     

Protonation of Glu135 Facilitates the Outward-to-Inward Structural Transition of Fucose Transporter

Major facilitator superfamily (MFS) transporters typically need to alternatingly sample the outward-facing and inward-facing conformations, in order to transport the substrate across membrane. To understand the mechanism, in this work, we focused on one MFS member, the L-fucose/H⁺ symporter (FucP), whose crystal structure exhibits an outward-open conformation. Previous experiments imply several residues critical to the substrate/proton binding and structural transition of FucP, among which Glu¹³⁵, located in the periplasm-accessible vestibule, is supposed as being involved in both proton translocation and conformational change of the protein. Here, the structural transition of FucP in presence of substrate was investigated using molecular-dynamics simulations. By combining the equilibrium and accelerated simulations as well as thermodynamic calculations, not only was the large-scale conformational change from the outward-facing to inward-facing state directly observed, but also the free energy change during the structural transition was calculated. The simulations confirm the critical role of Glu¹³⁵, whose protonation facilitates the outward-to-inward structural transition both by energetically favoring the inward-facing conformation in thermodynamics and by reducing the free energy barrier along the reaction pathway in kinetics. Our results may help the mechanistic studies of both FucP and other MFS transporters.     

Probing the Periplasmic-Open State of Lactose Permease in Response to Sugar Binding and Proton Translocation

Based on the crystal structure of lactose permease (LacY) open to the cytoplasm, a hybrid molecular simulation approach with self-guided Langevin dynamics is used to describe conformational changes that lead to a periplasmic-open state. This hybrid approach consists of implicit (IM) and explicit (EX) membrane simulations and requires self-guided Langevin dynamics to enhance protein motions during the IM simulations. The pore radius of the lumen increases by 3.5 Å on the periplasmic side and decreases by 2.5 Å on the cytoplasmic side (relative to the crystal structure), suggesting a lumen that is fully open to the periplasm to allow for extracellular sugar transport and closed to the cytoplasm. Based on our simulations, the mechanism that triggers this conformational change to the periplasmic-open state is the protonation of Glu269 and binding of the disaccharide. Then, helix packing is destabilized by breaking of several side chains involved in hydrogen bonding (Asn245, Ser41, Glu374, Lys42, and Gln242). For the periplasmic-open conformations obtained from our simulations, helix-helix distances agree well with experimental measurements using double electron-electron resonance, fluorescence resonance energy transfer, and varying sized cross-linkers. The periplasmic-open conformations are also in compliance with various substrate accessibility/reactivity measurements that indicate an opening of the protein lumen on the periplasmic side on sugar binding. The comparison with these measurements suggests a possible incomplete closure of the cytoplasmic half in our simulations. However, the closure is sufficient to prevent the disaccharide from transporting to the cytoplasm, which is in accordance with the well-established alternating access model. Ser53, Gln60, and Phe354 are determined to be important in sugar transport during the periplasmic-open stage of the sugar transport cycle and the sugar is found to undergo an orientational change in order to escape the protein lumen.     

Cytoplasmic permeation pathway of neurotransmitter transporters

Ion-coupled solute transporters are responsible for transporting nutrients, ions, and signaling molecules across a variety of biological membranes. Recent high-resolution crystal structures of several transporters from protein families that were previously thought to be unrelated show common structural features indicating a large structural family representing transporters from all kingdoms of life. This review describes studies that led to an understanding of the conformational changes required for solute transport in this family. The first structure in this family showed the bacterial amino acid transporter LeuT, which is homologous to neurotransmitter transporters, in an extracellularly oriented conformation with a molecule of leucine occluded at the substrate site. Studies with the mammalian serotonin transporter identified positions, buried in the LeuT structure, that defined a potential pathway leading from the cytoplasm to the substrate binding site. Modeling studies utilized an inverted structural repeat within the LeuT crystal structure to predict the conformation of LeuT in which the cytoplasmic permeation pathway, consisting of positions identified in SERT, was open for diffusion of the substrate to the cytoplasm. From the difference between the model and the crystal structures, a simple "rocking bundle" mechanism was proposed, in which a four-helix bundle changed its orientation with respect to the rest of the protein to close the extracellular pathway and open the cytoplasmic one. Subsequent crystal structures from structurally related proteins provide evidence supporting this model for transport.     

Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2

PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di‐ and tri‐peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepT_So, a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 Å resolution, reveals a ligand‐bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide‐binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepT_So represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.     

A structural model for the osmosensor, transporter, and osmoregulator ProP of Escherichia coli

Transporter ProP of Escherichia coli, a member of the major facilitator superfamily (MFS), acts as an osmosensor and an osmoregulator in cells and after purification and reconstitution in proteoliposomes. H(+)-osmoprotectant symport via ProP is activated when medium osmolality is elevated with membrane impermeant osmolytes. The three-dimensional structure of ProP was modeled with the crystal structure of MFS member GlpT as a template. This GlpT structure represents the inward (or cytoplasm)-facing conformation predicted by the alternating access model for transport. LacZ-PhoA fusion analysis and site-directed fluorescence labeling substantiated the membrane topology and orientation predicted by this model and most hydropathy analyses. The model predicts the presence of a proton pathway within the N-terminal six-helix bundle of ProP (as opposed to the corresponding pathway found within the C-terminal helix bundle of its paralogue, LacY). Replacement of residues within the N-terminal helix bundle impaired the osmotic activation of ProP, providing the first indication that residues outside the C-terminal domain are involved in osmosensing. Some residues that were accessible from the periplasmic side, as predicted by the structural model, were more susceptible to covalent labeling in permeabilized membrane fractions than in intact bacteria. These residues may be accessible from the cytoplasmic side in structures not represented by our current model, or their limited exposure in vivo may reflect constraints on transporter structure that are related to its osmosensory mechanism.