Research Article

Structure of an EIIC sugar transporter trapped in an inward-facing conformation

Zhenning Ren, Jumin Lee, Mahdi Muhammad Moosa, Yin Nian, Liya Hu, Zhichun Xu, Jason G. McCoy, Allan Chris M. Ferreon, Wonpil Im, and Ming Zhou
PNAS June 5, 2018 115 (23) 5962-5967; first published May 21, 2018 https://doi.org/10.1073/pnas.1800647115

  1. Edited by Ernest M. Wright, David Geffen School of Medicine at UCLA, Los Angeles, CA, and approved April 30, 2018 (received for review January 12, 2018)

Significance

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) is a multiprotein system unique to bacteria. The PTS transports sugars into bacteria and then phosphorylates the sugars. Phosphorylation prevents sugars from escaping the cell and primes them for metabolic consumption. As a major component of the PTS, Enzyme IIC (EIIC) transports sugar across the membrane and assists the phosphorylation process, but the molecular mechanism of EIIC-mediated sugar transport is unclear. Results from this study allow visualization of conformational changes during sugar transport and establish the mechanism of transport at the atomic level. The knowledge will facilitate development of inhibitors against EIIC and provide a foundation for understanding the phosphorylation process.

Abstract

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) transports sugar into bacteria and phosphorylates the sugar for metabolic consumption. The PTS is important for the survival of bacteria and thus a potential target for antibiotics, but its mechanism of sugar uptake and phosphorylation remains unclear. The PTS is composed of multiple proteins, and the membrane-embedded Enzyme IIC (EIIC) component transports sugars across the membrane. Crystal structures of two members of the glucose superfamily of EIICs, bcChbC and bcMalT, were solved in the inward-facing and outward-facing conformations, and the structures suggest that sugar translocation could be achieved by movement of a structured domain that contains the sugar-binding site. However, different conformations have not been captured on the same transporter to allow precise description of the conformational changes. Here we present a crystal structure of bcMalT trapped in an inward-facing conformation by a mercury ion that bridges two strategically placed cysteine residues. The structure allows direct comparison of the outward- and inward-facing conformations and reveals a large rigid-body motion of the sugar-binding domain and other conformational changes that accompany the rigid-body motion. All-atom molecular dynamics simulations show that the inward-facing structure is stable with or without the cross-linking. The conformational changes were further validated by single-molecule Föster resonance energy transfer (smFRET). Combined, these results establish the elevator-type mechanism of transport in the glucose superfamily of EIIC transporters.

The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is ubiquitous in bacteria. The membrane-embedded component EIIC transports a sugar from the periplasmic side to the cytoplasmic side where the sugar is phosphorylated by the EIIB protein and then released into the cytoplasm. Phosphorylation occurs while the sugar is still bound to EIIC, and the phosphorylation facilitates the release of the sugar (1). The phosphate group originates from phosphoenolpyruvate and is transferred sequentially through four soluble phosphocarrier proteins, enzyme I (EI), the histidine phosphocarrier protein (HPr), enzyme IIA (EIIA), and enzyme IIB (EIIB) and eventually reaches the incoming sugar while it is still bound on EIIC (SI Appendix, Fig. S1). Phosphorylation of the incoming sugar helps maintain its concentration gradient to sustain the uptake, and the energy stored in the phosphate bond is recovered when the sugar is metabolized (2⇓⇓–5). Thus, EII systems are more efficient than other membrane-embedded transporters that either hydrolyze ATP or dissipate an ion gradient. EII systems have been categorized into four superfamilies, of which the largest is the glucose superfamily (6) and the focus of this research. Each EII has its own preference for a group of sugar molecules and is composed of three proteins, EIIA, B, and C. In some of the EIIs, EIIB and C, or EIIA, B, and C form a single polypeptide. Crystal structures of two EIICs from the glucose superfamily (7, 8) and one from the ascorbate and galactitol superfamily (9) have been reported. Several electron microscopy projection maps of two EIICs from the glucose superfamily have also been reported (10⇓–12). While members of the same superfamily share the same structural fold, those from different superfamilies do not.

Structures of an N,N′-diacetylchitobiose EIIC transporter bcChbC (7) and a maltose EIIC transporter bcMalT (8), both from Bacillus cereus, have been reported. bcChbC and bcMalT share 19% sequence identity and 50% similarity, and yet both have the same structural fold with almost all of the secondary structural elements conserved (SI Appendix, Fig. S2 A–E). Both proteins are homodimers, and each protomer has 10 transmembrane (TM1-10) helices, two reentrant loops (HP1-2), and two amphipathic helices (AH1-2). These structural elements fold into two distinctive structural domains. The dimerization domain (also referred to as the interface domain), which consists of TM1-5 and AH1, forms an expansive dimer interface. The substrate-binding domain (also referred to as the transport domain), which is composed of TM6-10 and two reentrant loops (HP1-2), contains the sugar-binding site. In both structures, the sugar substrate is coordinated by residues from TM6, TM7, HP1, and HP2 (SI Appendix, Fig. S2B). The two domains are bridged by an amphipathic helix (AH2) (SI Appendix, Fig. S2 C and D).

The bcChbC and bcMalT structures represent different conformations required to complete a transport cycle (13). Based on the location of the substrate-binding site, bcChbC is in an inward-facing conformation, while bcMalT is in an outward-facing conformation (SI Appendix, Fig. S2A). When the two structures are aligned by their dimerization domains, it seems that the substrate-binding domain could carry the substrate across the membrane by a rigid-body motion. A similar elevator-type transport mechanism has also been reported in a number of secondary solute transporters including amino acid transporters (EAAT1 and GltPh) (14⇓–16), bile acid transporters (ASBT) (17), proton sodium exchangers (NhaA) (18, 19), concentrative nucleotide transporters (CNTNW) (20), and citrate transporters (vcINDY and SeCitS) (21, 22). These transporters have different structural folds, and yet they all transport substrates from one side of the cell membrane to the other by rigid-body motions of a substrate-binding domain.

Although by comparing the inward-facing bcChbC structure and the outward-facing bcMalT structure we can postulate that the glucose superfamily of EIICs have an elevator-type mechanism of transport, we need to visualize both conformations on the same transporter to reveal details of the conformational changes. To achieve this, we first generated a structural model of bcMalT in an inward-facing conformation by collective variable-based steered molecular dynamics (CVSMD) simulation (SI Appendix, Fig. S2F) using the bcChbC structure as a guide (23). During the simulation, the interface domain was kept static, and the substrate-binding domain was steered toward the inward-facing conformation. Since the substrate-binding domain moves relative to the interface domain, we expect distance changes between the two domains. Indeed, the CVSMD model shows that residues that are far away from each other in the outward-facing structure become closer, for example, residues T280 and D55 and residues N284 and E54 (SI Appendix, Fig. S2G). We then showed that the pairs of residues predicted to become closer to each other can be cross-linked by a mercury ion when mutated to cysteine residues and thus provide an experimental validation to the CVSMD model and the elevator-type mechanism of transport (23).

In this study, we solved the crystal structure of bcMalT cross-linked in an inward-facing conformation. The structure provides direct experimental evidence that the substrate-binding domain can undergo a rigid-body rotation toward the intracellular side. The structure also shows conformational changes in other regions of the transporter that accommodate the rigid-body movement of the substrate-binding domain. Since cross-linking of a pair of residues could potentially trap the structure into a conformation that may not be native, we did two experiments to examine whether the structure is distorted. First, we applied all-atom molecular dynamics simulations to the structure and found that the inward-facing conformation is stable even when the cross-linking constraint is released. Second, we estimated the distance between a pair of symmetry-related residues on the substrate-binding domain by single-molecule Föster resonance energy transfer (smFRET) and found that the distances between the two residues are consistent with those measured from the inward- and outward-facing structures.

Results

Trapping bcMalT in an Inward-Facing Conformation.

CVSMD was used to show that residues T280 on the substrate binding domain and D55 on the dimerization domain can move to within 12.4 Å of each other as opposed to 23.7 Å in the outward facing crystal structure (SI Appendix, Fig. S2G). Similarly, N284 on the substrate-binding domain and E54 on the dimerization domain are 26.7 Å away in the crystal structure and 13.1 Å in the CVSMD model. Both pairs can be cross-linked by a mercury ion when mutated to cysteines, thus confirming their proximity (23). Although we were not able to crystallize the cross-linked proteins, we were encouraged by the predictive power of the inward-facing model and tested additional pairs in the vicinity of the previous successful ones. The Cα atoms of the T280-E54 pair are 19.3 Å apart in outward-facing bcMalT and 9.6 Å apart in the inward-facing model. When both residues were mutated to cysteines (T280C-E54C) they can be cross-linked at low (micromolar) concentrations of Hg2+ and at a protein-to-mercury molar ratio of ∼1:1. The cross-linked protein migrates faster on SDS/PAGE (Fig. 1A, lanes 2 and 3). The cross-linking is complete within seconds (Fig. 1B). These results suggest that the two residues have a high probability of reaching proximity. We then showed that the cross-linking is specific to the two cysteine mutations because the reaction can be reversed by addition of reducing reagent β-mercaptoethanol (Fig. 1A, lane 6), prevented by pretreatment with N-ethyl maleimide that alkylates free cysteines (Fig. 1A, lane 4), and did not happen on single-cysteine mutations (SI Appendix, Fig. S3A).

Fig. 1.
Fig. 1.

Cross-linking of bcMalT(T280C-E54C). (A) SDS/PAGE analysis of bcMalT(T280C-E54C) under various conditions. Lanes: 1, molecular weight standard; 2, bcMalT(T280C-E54C) without any treatment; 3, bcMalT(T280C-E54C) treated with Hg2+. Cross-linked bcMalT migrates faster, and the band is marked as X-linked; 4, bcMalT(T280C-E54C), first treated with 2 mM N-ethyl maleimide (NEM) and then incubated with 1:1 molar ratio of Hg2+; 5, bcMalT(T280C-E54C) in the presence of 2 mM β-mercaptoethanol (β-ME); 6, bcMalT(T280C-E54C) treated with Hg2+ and then incubated with 2 mM β-ME. (B) Time course of Hg2+-induced cross-linking of bcMalT(T280C-E54C).

We then assessed the functions of the T280C-E54C double-cysteine mutation in bcMalT. The double mutant transports maltose through facilitated diffusion when reconstituted into liposomes, suggesting that the cysteine mutations do not affect the function of the protein significantly (SI Appendix, Fig. S3B). We also found that the double-cysteine mutant binds to maltose with similar affinity before and after cross-linking (SI Appendix, Fig. S3C), suggesting that cross-linking the two residues does not significantly distort the sugar-binding site.

Crystal Structure of the Cross-Linked bcMalT EIIC.

The T280C-E54C bcMalT EIIC cross-linked by Hg2+ (termed bcMalT-X hereafter) can be crystallized. After extensive refinement of crystallization conditions, the crystals diffracted to a resolution of 3.2 Å, and a full dataset was collected and processed (SI Appendix, Table S1). Molecular replacement using individual interface and transport domains led to a clear solution (SI Appendix, Fig. S4 A and B). A nonprotein density is resolved at the substrate-binding site into which we can unambiguously build a maltose (SI Appendix, Fig. S4C). Since the dataset was collected at a wavelength close to the mercury edge, we calculated an anomalous difference Fourier map and it shows a strong peak (>9σ) that identifies the Hg2+ position between cysteines 280 and 54 (SI Appendix, Fig. S4D). The distance between the Cα atoms of cysteine 54 and 280 is 7.7 Å, and the distances between the Hg and the two sulfur atoms are ∼2.3 Å. These distances are consistent with those reported in previous crystal structures of Hg2+-mediated cysteine cross-links (14, 24). The final refined model contains a homodimer of bcMalT, and within each protomer, residues 5–451, one Hg2+ and one maltose are resolved.

When bcMalT and bcMalT-X structures were shown side-by-side with their AH1 and AH2 aligned to either side of the membrane (Fig. 2 A and B), the bound substrate on bcMalT-X is closer to the intracellular side, and its substrate-binding domain assumes an inward-facing conformation (Fig. 2A). The distance between residues 280 and 54 is smaller in the bcMalT-X structure due to the cross-linking (Fig. 2C). However, the root-mean-square deviation (rmsd) of all Cα atoms in the dimerization domain between the two structures is 0.64 Å and that of the substrate-binding domain is 0.43 Å, indicating that the cross-linking does not change the arrangement of the secondary structural elements within each domain (Fig. 2D). The structure provides direct evidence that a rigid-body elevator-type motion of the substrate-binding domain carries a substrate from one side of the membrane to another (Movie S1).

Fig. 2.
Fig. 2.

Side-by-side comparison of bcMalT and bcMalT-X. Crystal structures of bcMalT wild type (Left, PDB accession number 5IWS) and bcMalT-X (Right) are shown in cartoon (A) and surface (B) representations. In A, the two protomers are colored blue and green, respectively. Within each protomer, the dimerization domain is colored darker than the substrate-binding domain. In B, the electrostatic surface was calculated by the Adaptive Poisson–Boltzmann Solver plugin in Pymol (34); the bar represents electrostatic potential from −61.1 to +61.1 kBT/eC. (C) Location of T280 and E54 on a single protomer in the outward-facing conformation (Left) and inward-facing conformation (Right). TM1 is removed for clarity. Cβ of T280 (magenta) and E54 (orange) are shown as spheres. (D) Superposition of the dimerization domain (Left) and substrate-binding domain (Right). Green, bcMalT wild type; blue, bcMalT-X.

Substrate-Binding Site.

Similar to the bcMalT structure, the bcMalT-X structure also has a nonprotein electron density corresponding to a bound maltose on each protomer (Fig. 3 and SI Appendix, Fig. S4C). Since there is very little change in the substrate-binding domain, the coordination of the bound maltose is essentially identical in the two structures. The C6 hydroxyl on the nonreducing end is known to be phosphorylated by EIIB, and the hydroxyl is coordinated by E355 and H240 from HP1b and TM7, respectively (Fig. 3). These two residues are implicated in the phosphotransfer reaction and are highly conserved in the glucose EIIC superfamily (25, 26) (SI Appendix, Fig. S5). The bound maltose has one additional interaction with the protein in the bcMalT-X structure. Y249 on TM7 likely forms a weak hydrogen bond with the C1 hydroxyl on the reducing end (Fig. 3). This additional hydrogen bond does not seem to affect binding affinity (SI Appendix, Fig. S3B). The distance between the hydroxyl on Y249 and the hydroxyl on the C1 is 3.1 Å in bcMalT-X and 4.5 Å in bcMalT, and the difference is due to slight movement of TM7. Coincidentally, in a previous molecular dynamics simulation study performed on the outward-facing bcMalT, Y249 and TM7 were found to be more mobile than the neighboring structural element, moving away from the substrate to open the entrance to the substrate-binding site (8).

Fig. 3.
Fig. 3.

Stereoview of the maltose-binding site in bcMalT and bcMalT-X. (A) bcMalT wild type; (B) bcMalT-X. Maltose is shown as sticks with carbon in yellow and oxygen in red. Protein–maltose interactions are marked as dotted lines between the sugar and residues from HP1 (blue), HP2 (raspberry), and TM6,7,8 (light green). The C6-OH on the nonreducing end of maltose where phosphorylation happens is marked with a star.

Major Differences Between the Inward- and Outward-Facing Structures.

The rigid-body motion of the transport domain results in different interactions between the dimerization domain and transport domain. In the previous bcMalT structure, the two domains have a buried surface area of 1,317 Å2, while in the current structure of bcMalT-X the buried surface area is slightly less at 1,217 Å2 (SI Appendix, Fig. S6 A and B). The buried surface is largely hydrophobic, with several hydrogen bonds. Of particular interest are residues S343 and T351 on the HP1 motif. The backbone carbonyl oxygen of A169 from the interface domain forms a hydrogen bond with the side-chain hydroxyl of S343 in the outward-facing conformation, and the same carbonyl oxygen atom forms a hydrogen bond with the hydroxyl from T351 in the inward-facing structure (SI Appendix, Fig. S6C). We also found similar exchange of hydrogen bond partners at the extracellular side, for example, K34-E397 in the outward-facing conformation and K34-N284 in the inward-facing conformation (SI Appendix, Fig. S6D). These interactions between the interface and substrate-binding domain likely stabilize the relative position of the two domains. Residues that form hydrogen bonds in the inward-facing state, S343 and T351, are both highly conserved in the glucose subfamily of EIICs such as MalT and PtsG (SI Appendix, Fig. S5). These observations also suggest that the cross-linking likely stabilizes an inward-facing state that is not significantly different from the native state.

The rigid-body motion of the substrate-binding domain is accompanied by conformational changes at both the N- and C-terminal ends of the amphipathic helix AH2 (Fig. 4). In the bcMalT outward-facing structure, AH2 becomes an extension of TM5, extending 10 Å into the extracellular space. The angle between AH2 and TM5 is 137.5°. In the bcMalT-X inward-facing structure, AH2 and TM5 forms a bend, and the angle between these two helices becomes 117.0°, and, as a result, AH2 resides horizontally at the membrane surface (Fig. 4). The bend occurs at Pro199 at the N-terminal end of AH2, which is highly conserved in the glucose subfamily of EIIs (SI Appendix, Fig. S5). This movement allows the substrate-binding domain to descend vertically toward the intracellular side. Conformational changes at the C-terminal end of AH2 are equally important for accommodating movements of the substrate-binding domain. In the bcMalT structure, six residues (215–220) at the C-terminal end of AH2 form a loop that connects TM6, and the two helices form an angle of 25°. In the bcMalT-X structure, residues S215, K216, and D217 wind into a helical turn, and the angle between AH2 and TM6 is 70° (Fig. 4). The length of AH2 changes from 17.1 Å in the outward-facing bcMalT to 20.3 Å in bcMalT-X. These changes seem to allow the rotation of the substrate-binding domain.

Fig. 4.
Fig. 4.

Conformational changes of AH2. One protomer of bcMalT (A) and bcMalT-X (B) is shown as cartoon representation. The dimerization domain is colored dark blue and the substrate-binding domain light blue. TM5 is colored green; AH2 is colored orange and TM6 is colored red. The hinge point between TM5 and AH2, P199, is marked as a magenta sphere.

Structure of bcMalT-X (T280C-E54CHg) Resembles the Native Inward-Facing State.

Although evidence from the cross-linking experiment suggests that the two cysteine residues are in close proximity, and hence the cross-linked bcMalT is likely not distorted into a nonnative state, we further examined the effect of cross-linking on bcMalT. We examined the stability of the bcMalT-X structure using molecular dynamics (MD) simulations. MD simulations of bcMalT-X embedded in a fully hydrated lipid bilayer showed that the structure is stable for 1 µs with or without cross-linking the two cysteines. The inward-facing conformation was well maintained in all of the simulations with backbone rmsd of 2–3 Å against the bcMalT-X crystal structure (Fig. 5A) and backbone rmsd of 7–8 Å against the outward-facing bcMalT crystal structure (Fig. 5B). The backbone rmsd of each domain is also stable, fluctuating around 1–3 Å (SI Appendix, Fig. S7 A–C). These results indicate that bcMalT-X is close to an energy minimum and likely not significantly distorted from the native conformation. In addition, we compared the bcMalT-X structure with the previous CVSMD model of bcMalT. We performed 1-µs MD simulation using the final structure from our previous bcMalT CVSMD simulation (23). The 1-µs equilibrated bcMalT CVSMD model is well aligned to the bcMalT-X structure with a backbone rmsd of 2.41 Å (SI Appendix, Fig. S7D), reinforcing the predictive power of the previous computational approach.

Fig. 5.
Fig. 5.

Backbone rmsd of bcMalT-X structure during all-atom MD simulations. (A) Average backbone rmsd between the simulated structure and bcMalT-X structure. (B) Average backbone rmsd between the simulated structure and bcMalT structure. Each line is the average of three independent simulations. Red, bcMalT-X; blue, bcMalT-X with the cross-linking between T280 and E54 removed; black, bcMalT CVSMD (23).

The proposed large motion of the substrate-binding domain would cause large distance changes between certain residues. Single-molecule Föster resonance energy transfer (smFRET) provides us a tool to visualize individual protein conformations without the need for ensemble averaging, therefore providing a method to directly observe minor protein populations that are in dynamic equilibrium. We made a mutation N288C at the extracellular side on the substrate-binding domain of bcMalT and labeled it with Alexa Fluor 488 and Alexa Fluor 594 maleimide (Fig. 6A). Similarly, M340 at the intracellular side of the substrate-binding domain was mutated and labeled with the two dyes as well. Since bcMalT is a homodimer, only the ones labeled with both dyes will register a FRET signal. The labeled proteins, when reconstituted into liposomes, transport maltose with similar efficiency as wild-type bcMalT (SI Appendix, Fig. S3D), suggesting that the mutation and the subsequent labeling do not significantly affect the motions of the protein. smFRET histograms of each mutant show two populations. For N288C, we observed one population of low FRET efficiency (EFRET) at 0.22 and one of high EFRET at 0.50 (Fig. 6B). The estimated distances for these two populations are 74.1 and 60.0 Å, respectively (SI Appendix, Table S2). For M340, EFRET segregates into two well-resolved populations centered around 0.37 and 0.84 (Fig. 6C), which correspond to an estimated distance of 65.4 and 45.3 Å, respectively (SI Appendix, Table S2). As a negative control, we mutated and labeled a residue (D123) on the dimerization domain, which is predicted to remain anchored in the membrane during substrate translocation. Its EFRET shows a uniform population at 0.59 (SI Appendix, Fig. S8), which corresponds to a distance of 56.5 Å. These distances are consistent with the two structures within the error of smFRET measurements (SI Appendix, Fig. S8 and Table S2).

Fig. 6.
Fig. 6.

smFRET on bcMalT. (A) bcMalT (Left) and bcMalT-X (Right) are shown as cartoon and colored according to Fig. 2A. Cβ of N288 and M340 are shown as spheres and colored red and purple respectively. The distances between the two symmetry-related residues are marked on the dotted lines. Histograms of smFRET efficiency are shown for N288C (B) and M340C (C). Solid lines are fitting of the histograms with two Gaussian functions, and the mean value of each Gaussian function (appEFRET) is tabulated in SI Appendix, Table S2.

smFRET also provides an estimate to the dynamics of bcMalT (27). In both N288C and M340C, bcMalT seems to prefer the outward- to the inward-facing conformation by a ratio of 5:1 and 7:3, respectively. This fractional population ratio corresponds to a free energy difference of approximately 2–4 kJ/mol. Similar energy difference was observed in conformational changes of Gltph measured by the atomic force microscopy (28). A more stable outward-facing conformation seems consistent with a slightly more extensive buried interface between the dimerization and substrate-binding domains and may explain why it was difficult to crystallize the wild-type bcMalT in an inward-facing conformation.

Summary and Discussion

The structures of bcMalT and bcMalT-X provide direct visualization of the outward- and inward-facing conformations of an EIIC from the glucose superfamily (Fig. 2A and Movie S1). The structures show that the substrate-binding domain moves as a rigid body that keeps the substrate-binding site intact while moving it to face the alternate side of the membrane. An amphipathic helix, AH2, which connects the dimerization and the substrate-binding domains, bends at a conserved hinge point and changes its length to accommodate the movement of the substrate-binding domain.

The bcMalT-X structure shows that the substrate-binding domain has a 9-Å vertical translation and a 45° rotation. Such large-scale motions are common among transporters using the elevator-type mechanism (14⇓⇓⇓⇓⇓⇓⇓–22). In at least three of the multimeric transporters that have an elevator-type mechanism, the substrate-binding domains can move independently of each other. Both smFRET and atomic force microscopy studies have visualized the unsynchronized subunit motion in a single trimeric Gltph (19, 28). In crystal structures of CNTNW, one transport domain of the trimeric transporter was found in different states than the other two (20). In addition, the structure of sodium citrate symporter SeCitS captured an asymmetric state of the dimer, with one protomer in the outward-facing state and the other in the inward-facing state (22). It is not clear whether in bcMalT the two substrate-binding domains can move independently. If there exists a conformation in bcMalT where one protomer is outward-facing and the other inward-facing, a population of EFRET at 0.34 for N288 and 0.63 for M340 would be expected on the smFRET histogram. However, we were not able to resolve either of these states under the current experimental conditions.

The substrate in bcMalT-X is now accessible to solvent from the cytoplasm. Among six independent MD simulations, we have observed two sugar release events: one in the bcMalT CVSMD system and the other in the bcMalT-X without the disulfide bond system (SI Appendix, Fig. S9A). The sugar molecules were released without any significant conformational change during the simulations, suggesting that the bcMalT-X structure has an inward-facing open conformation. The comparison of the bcMalT-X with bcChbC (inward-occluded) crystal structures also supports this observation, as the intracellular exit of the sugar-binding site is blocked by the TM4-5 loop in bcChbC, while the same loop has a different orientation in bcMalT-X and no longer covers the sugar-binding site (SI Appendix, Fig. S9 B and C). Thus, bcMalT-X structure likely represents a conformation that is compatible for phosphate transfer from the EIIB. These observations also provide an explanation for facilitated sugar transport activity of bcMalT when no phosphorylation occurs.

In summary, the combined approach of structural biology, MD simulations, and smFRET revealed conformational changes required for substrate translocation in an elevator-type mechanism of transport. The inward-facing open conformation also provides a starting point for understanding how EIIB could interact with EIIC and how a phosphate group is transferred to the bound maltose.

Methods

Detailed materials and methods are found in SI Appendix. Protein purification and cross-linking experiments followed established protocols reported previously (8, 23). bcMalT-X was crystallized using the sitting-drop vapor diffusion method. The dataset was collected close to the mercury edge, and the phase problem was solved by molecular replacement. Maltose binding and transport were measured using the scintillation proximity assay and liposome uptake assay, respectively, as described previously (8, 23). MD simulations were conducted in CHARMM-GUI (29, 30), following optimized protocols (31, 32). For smFRET experiments, labeling conditions were optimized to maximize the amount of protein that contained both dyes. Diffusion-based smFRET was conducted using a custom-built ISS Alba confocal laser microscopy system, as described previously (33).

Acknowledgments

We thank K. Rajashankar for advice on X-ray crystallography and K. Rajashankar, L. Keefe, E. Zoellner, K. Battaile, and A. Mulichak for beamline support. We thank R. Bruni and B. Kloss, both from the Center on Membrane Protein Production and Analysis in New York City, for the cloning and initial screen of EIIC genes. This work was supported by National Key Basic Research Program of China 2014CB910301 (to M.Z.); National Institutes of Health Grants U54GM087519 (to W.I. and M.Z.) and GM098878, DK088057, and HL086392 (to M.Z.); and Grant 12EIA8850017 from the American Heart Association and Cancer Prevention and Research Institute of Texas Grant R12MZ (both to M.Z.).

Footnotes

  • ↵1Z.R. and J.L. contributed equally to this work.

  • ↵2To whom correspondence may be addressed. Email: mzhou{at}bcm.edu, allan.ferreon{at}bcm.edu, or woi216{at}lehigh.edu.

  • Author contributions: Z.R., J.L., M.M.M., Y.N., L.H., Z.X., J.G.M., A.C.M.F., W.I., and M.Z. designed research, collected and analyzed data, and wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID code 6BVG).

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1800647115/-/DCSupplemental.

Published under the PNAS license.

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Structure of an EIIC sugar transporter trapped in an inward-facing conformation
Zhenning Ren, Jumin Lee, Mahdi Muhammad Moosa, Yin Nian, Liya Hu, Zhichun Xu, Jason G. McCoy, Allan Chris M. Ferreon, Wonpil Im, Ming Zhou
Proceedings of the National Academy of Sciences Jun 2018, 115 (23) 5962-5967; DOI: 10.1073/pnas.1800647115
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Proceedings of the National Academy of Sciences: 115 (23)
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