Elucidating the mechanism of sugar import requires a molecular understanding of how transporters couple sugar binding and gating events. Whereas mammalian glucose transporters (GLUTs) are specialists1, the hexose transporter from the malaria parasite Plasmodium falciparum PfHT12,3 has acquired the ability to transport both glucose and fructose sugars as efficiently as the dedicated glucose (GLUT3) and fructose (GLUT5) transporters. Here, to establish the molecular basis of sugar promiscuity in malaria parasites, we determined the crystal structure of PfHT1 in complex with d-glucose at a resolution of 3.6 Å. We found that the sugar-binding site in PfHT1 is very similar to those of the distantly related GLUT3 and GLUT5 structures4,5. Nevertheless, engineered PfHT1 mutations made to match GLUT sugar-binding sites did not shift sugar preferences. The extracellular substrate-gating helix TM7b in PfHT1 was positioned in a fully occluded conformation, providing a unique glimpse into how sugar binding and gating are coupled. We determined that polar contacts between TM7b and TM1 (located about 15 Å from d-glucose) are just as critical for transport as the residues that directly coordinate d-glucose, which demonstrates a strong allosteric coupling between sugar binding and gating. We conclude that PfHT1 has achieved substrate promiscuity not by modifying its sugar-binding site, but instead by evolving substrate-gating dynamics.
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We thank D. Daley for advice on PfHT1 overexpression optimization, and G. von Heijne and S. Newstead for critical reading of the manuscript. X-ray diffraction data were collected at the European Synchrotron Radiation Facility beamlines, the Diamond Light Source beamlines and the MaxIV BioMax beamline with assistance from beamline scientists. This work was funded by the Knut and Alice Wallenberg Foundation (D.D.) and, the Science for Life Laboratory (L.D.); D.D. acknowledges support from EMBO through the Young Investigator Program (YIP).
The authors declare no competing interests.
Peer review information Nature thanks Jeff Abramson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Fig. 1 PfHT1 is distantly related to both the human GLUT transporters and the bacterial homologue XylE.
a, Unrooted phylogenetic tree of human GLUT1, GLUT2, GLUT3, GLUT4 and GLUT5 (GLUT1–5), rat GLUT5, E. coli XylE and PfHT1 b, Table of protein sequence identity of proteins shown in a. (only rat (and not human) GLUT5 is shown). c, Sequence alignment of PfHT1, human GLUT1–5, rat GLUT5 and E. coli XylE. Secondary structure elements of PfHT1 are indicated above the alignment, and coloured as in Fig. 1c. Residues conserved in at least 80% of the alignment are highlighted by red boxes, and gating residues are highlighted by blue boxes. Conserved binding-site residues between PfHT1 and human GLUT3 are indicated with purple filled dots and non-conserved residues are indicated with non-filled purple dots. Conserved residues close to the binding site are indicated with yellow filled dots and non-conserved residues with non-filled yellow dots. Black bars beneath the alignment indicate residues in the sugar-porter motifs15,16. The Uniprot reference numbers of the alignment proteins are: PfHT1 (Q7KWJ5), human GLUT1 (P11166), human GLUT2 (Q102R8), human GLUT3 (P11169), human GLUT4 (P14672), human GLUT5 (P22732), rat GLUT5 (P43427) and XylE (P0AGF4). For the sake of clarity, residues 109–121 from XylE and residues 54–86 from human GLUT2 were omitted.
a, Size-exclusion chromatogram of DDM-purified PfHT1 showing PfHT1 migrates as two oligomeric species (dimer and monomer); the sample migrates as a monomer during SDS–PAGE. b, Time-dependent uptake of [14C]d-mannose (black circles) by PfHT1 in proteoliposomes. Inset, PfHT1 uptake of radiolabelled sugar (cyan trace) compared with non-specific uptake estimated from radioactivity measured from liposomes incubated without protein (black trace). Error bars represent the mean ± s.e.m. of n = 3 biologically independent experiments. c, As in b, for [14C]d-galactose. d, As in b, for [14C]d-fructose. e, As in b, for [3H]d-xylose. f, As in b, for [3H]d-glucosamine.
a, Zero trans kinetics of PfHT1 d-glucose transport. Kinetic curves were fitted from data points recorded at increasing d-glucose concentrations after 20 s and fitted by nonlinear regression. Error bars represent mean ± s.e.m. of n = 3 biologically independent experiments. b. As in a, for d-mannose. c, As in a, for d-galactose and except that time points were recorded after 60 s. d, As in a, for d-fructose and except that time points were recorded after 60 s. e, As in a, for d-glucosamine. f, Bars represent specificity constant (kcat/KM) values of PfHT1 for different sugars as tabulated and described in g. g, The fitted values reported for the Michaelis constant (KM) and Vmax of PfHT1 for different transported sugars are mean ± s.e.m. of n = 3 biologically independent experiments. Turnover (kcat) and specificity constant (kcat/KM) values are derived from these kinetic parameters and adjusted to the amount of protein reconstituted into liposomes (Methods).
a, PfHT1 crystallized as a dimer with four molecules in the asymmetric unit. The shared dimer interface was formed between the respective N-terminal domains (blue) that—although not extensive (522 Å buried surface area)—are consistent with the fact that a fraction of purified PfHT1 migrates as a dimer by size-exclusion chromatography (Extended Data Fig. 2a). Notably, the gating helix TM7b is not making any crystal contacts. b, Superposition of the outward-occluded GLUT3 (PDB 4ZWB) (grey) and the occluded PfHT1 structures. The r.m.s.d. is 1.4 Å for 446 pairs of Cα atoms (Methods). c, Cartoon representation of PfHT1 as viewed from the cytoplasm. Blue, NTD; magenta, CTD. ICHs are not shown for clarity. Interdomain salt-bridge-forming residues are shown as sticks, and labelled. d, Cartoon representation of TM7b of human GLUT1 (PDB 4PYP) (orange) and bovine GLUT5 (PDB 4YB9) (purple) in the inward-open conformation, and PfHT1 in the d-glucose-bound (yellow sticks) occluded conformation (magenta). e, Electron density map 2Fo − Fc (1.5σ) (blue mesh) for the PfHT1 structure (left) and the d-glucose residues in the sugar-binding pocket in cyan (right). The Fo – Fc (3.0σ) (green mesh) maps before addition of and refinement in the presence of d-glucose are also shown. Despite the high quality of the maps, we observed no electron density for ICH5 (location in human GLUT3 shown as a dashed ellipse).
a, Chemical structures of the investigated sugars used in competitive-uptake assays. Differences in hydroxyl-group position of the respective d-glucose epimers are coloured red. Sugars labelled green were also tested as radiolabelled substrates in time-course experiments (Fig. 1b, Extended Data Fig. 2b–f). b, The competitive uptake of [14C]d-glucose by PfHT1 in proteoliposomes in the absence (white bars) and presence of non-labelled sugars (black bars). Non-specific uptake was estimated from radioactivity measured from liposomes incubated without protein (red bar). Error bars represent mean and s.e.m. of n = 3 biologically independent experiments.
Extended Data Fig. 6 Assessed quality of purified PfHT1–GFP fusions and analysis of the sugar-binding pocket of PfHT1.
a, FSEC traces of DDM-purified PfHT1–GFP wild type and mutants of the sugar-binding pocket; PfHT1–GFP migrates as two species (dimer and monomer), consistent with purified PfHT1 (Extended Data Fig. 2a). FSEC traces were recorded at least twice for wild type and each respective mutant. b. As in a, for mutants peripheral to the sugar-binding pocket. c, As in a, for mutants located in TM1 and TM7b. d, Cartoon representation of PfHT1 with d-glucose and interacting residues labelled, shown as yellow sticks. The position of d-glucose in E. coli XylE (green) (PDB 4JA3) and d-glucose in human GLUT3 (grey) (PDB 4ZW9) are shown as sticks, after protein superimposition. e, Determination of the Michaelis constant (KM) for d-glucose by the PfHT1 mutant Ala404Glu, constructed to mimic the human GLUT3 binding site. Kinetic curves were fitted from data points recorded over a range of increasing d-glucose concentrations after 90 s, and fitted by nonlinear regression using data from n = 3 biologically independent experiments (values reported are mean ± s.e.m. of the fit). f, As in e, for d-fructose. g, Sugar-binding-site comparison between PfHT1 side chains (yellow sticks) and rat GLUT5 side chains (conserved side chains, grey sticks; non-conserved side chains, cyan sticks). h, As in e, for the PfHT1 mutant Trp412Ala constructed to mimic the rat GLUT5 binding site.
a, Surface transversal cross-sections through the membrane of the PfHT1 structure in the occluded conformation with d-glucose shown as sticks, and a side vestibule accessible to the C3- and C4-hydoxyl groups that—in human GLUT3—was occupied by a monoolein lipid. b, The competitive uptake of [14C]d-glucose (black bars) by PfHT1 wild type and the mutant W412A in proteoliposomes in the absence and presence of cytochalasin B (Cyb) (120 μM), MMV009085 (MMV) (100 μM) or C3361 (100 μM). Error bars represent s.e.m. of n = 3 biologically independent experiments (left). Cytochalasin B and the inhibitor C3361 (which is d-glucose with a undec-10-en chain at the C3-hydroxyl position) are shown (right). c, IC50 curves for MMV009085 (blue filled circles) and derivative lacking the butanol tails (grey filled circles). Error bars represent s.e.m. of n = 3 biologically independent experiments (left). Structures of MMV009085 and derivative are shown on the right.
a, Cartoon representation of TM7b of human GLUT3 in the outward-open (green) (PDB 4ZWC) and outward-occluded d-glucose-bound conformation (light-brown) (PDB 4ZW9) and of PfHT1 in the d-glucose-bound occluded conformation (magenta). Arrows indicate that inward movement of TM7b is coupled to coordination of the C3- and C4- hydroxyl groups of d-glucose (shown as yellow sticks) by the strictly conserved asparagine residue corresponding to Asn311 in PfHT1. Only in PfHT1 does TM7b break into two perpendicular segments as observed in the in inward-facing structures of GLUT1 and GLUT5 (Extended Data Fig. 4d). The asterisk highlights the highly conserved tyrosine residues that occlude the substrate from exiting in the outward-occluded conformations, and which—in PfHT1—are replaced by serine (S315) and asparagine (N316). b, Molecular dynamics simulations of TM1–TM7 gating interactions: d-glucose-bound outward-occluded gating interactions by human GLUT3 in the presence (yellow) and absence (grey) of d-glucose. The distribution of conformations, shown as the gating distance, from three independent 1-μs molecular dynamics simulations, as described in the Methods. c, As in b, for d-glucose-bound outward-occluded gating interactions by PfHT1 in the presence (magenta) and absence (yellow) of d-glucose. The distribution of conformations, shown as the gating distance, from three independent 1-μs molecular dynamics simulations, as described in the Methods. d, Snapshots of the distance between residues Lys51 and Asn316 at 0-ns, 500-ns and 1,000-ns time points.
a, Aligned core of near-intact MFS transporter structures from 17 structures, including PfHT1, mammalian (GLUT1, GLUT3 and GLUT5) and prokaryotic (XylE) systems. The ‘core’ structural elements conserved among the structures are shown in different colours (Methods). b, Motions along the first principal component (PC1) derived from the core ensemble, tracking the rocker-switch motion. c, Residue fluctuations computed from PC1, with the core helix fragments shown as shadowed areas at the base. d, Schematic of the structural basis of the sugar-porter alternating-access mechanism. To summarize, in the outward and outward-occluded conformations (PDB 4YBQ and 4ZW9) the substrate-gating helix TM7b (magenta and transparent) is mobile and samples either state, as seen in molecular dynamics simulation of human GLUT3; spontaneous gate closure is further consistent with the fact that—even in the presence of maltose—GLUT3 crystallizes in both outward-open and outward-occluded conformations4. Substrate binding conformationally stabilizes the outward-occluded state, thus increasing the likelihood for TM7b to break in the middle, completely close the substrate pocket and form contacts with TM1. In the occluded state, the salt-bridge interactions between ICH5 in the C-terminal bundle and ICH1, ICH2 ICH3 and ICH4 are lost, which indirectly destabilizes the highly conserved intrabundle salt-bridge network. Breakage of the intrabundle salt-bridge network catalyses global rocker-switch rearrangements of the N- and C-terminal bundles. In the inward-occluded conformation (PDB 4JA3), the intracellular gating helix TM10b (cyan)—which is related by inverted symmetry to TM7b—spontaneously moves outward to the inward-open conformation (PDB 4YB9). After sugar release, the sugar porter spontaneously resets itself to the outward-facing conformation through an ‘empty’ occluded state22. Spontaneous resetting means that the energetic barriers separating opposite-facing states must be low enough that the occluded state can form in the absence of sugar binding. Nevertheless, consistent with a conformational-selection-driven rocker-switch mechanism, substrate binding catalyses transport as rates are substantially faster through ‘substrate-bound’ versus ‘empty’ occluded-state transitions1.
This file contains Supplementary Table 1: Molecular dynamics simulation details of replicates and Supplementary Figure 1: Uncropped gel of Extended Data Fig. 2a.
Video morphing between the open outward-facing, outward-occluded, occluded, inward-occluded and open inward-facing PfHT1 conformations as viewed parallel to the membrane. The open outward- and inward-facing conformations are based on the GLUT5 structures and occluded states were modelled based on the structures of human GLUT3 and E. coli XylE. (Methods). Morphing between conformations was performed using PyMol.
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Qureshi, A.A., Suades, A., Matsuoka, R. et al. The molecular basis for sugar import in malaria parasites. Nature 578, 321–325 (2020). https://doi.org/10.1038/s41586-020-1963-z
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