Functional architecture of MFS D-glucose transporters - PubMed (original) (raw)

Functional architecture of MFS D-glucose transporters

M Gregor Madej et al. Proc Natl Acad Sci U S A. 2014.

Abstract

The Major Facilitator Superfamily (MFS) is a diverse group of secondary transporters with over 10,000 members, found in all kingdoms of life, including Homo sapiens. One objective of determining crystallographic models of the bacterial representatives is identification and physical localization of residues important for catalysis in transporters with medical relevance. The recently solved crystallographic models of the D-xylose permease XylE from Escherichia coli and GlcP from Staphylococcus epidermidus, homologs of the human D-glucose transporters, the GLUTs (SLC2), provide information about the structure of these transporters. The goal of this work is to examine general concepts derived from the bacterial XylE, GlcP, and other MFS transporters for their relevance to the GLUTs by comparing conservation of functionally critical residues. An energy landscape for symport and uniport is presented. Furthermore, the substrate selectivity of XylE is compared with GLUT1 and GLUT5, as well as a XylE mutant that transports D-glucose.

Keywords: glucose transport; ligand docking; membrane proteins; sequence analysis; solute carrier transporter.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Alignment. (A) Helices represented by colored boxes are shown in consecutive order in the sequence and colored according to helix-triplets. Arrows of the same color indicate the positions of correlating residues. (B) Helix-triplets from XylE and LacY are aligned (helices 1–3, blue; helices 4–6, green; helices 7–9, orange; helices 10–12, yellow). The flags indicate the loops within triple-helix motifs. Helix-triplets from LacY and XylE are colored as in A. The alignments are oriented with the LacY cytoplasmic side at the top. The flags indicate loops within triple-helix motifs (white, cytoplasmic loop; gray, periplasmic loop). The numbers on the flags indicate the two helices that are connected by the respective loop. (C) Schematic superposition. Helix-triplets are colored as in A. Overlapping side-chain positions are shown in the same color for corresponding helices. Contacts between side-chains are indicated as broken lines. Red boxes indicate positions lowering the transport activity of GLUT1 Cys-mutants; gray boxes indicate positions not tested by Cys-scanning mutagenesis in GLUT1. Yellow background indicates positions implicated in a medical condition.

Fig. 2.

Transport activity of XylE mutants. (A) Superposition of helix-triplet D from LacY (beige) with helix-triplet A from XylE (blue) and GLUT1 (magenta). The position of Glu325, Asp27, and Asn29 is indicated with stick models colored according to the model. The helices are indicated with roman numbers, for LacY on a yellow circle and for XylE and GLUT1 on a magenta circle. (B) Levels of active transport (blue) and counterflow (green) of

-xylose by WT and mutants D27N, D27A, and E206Q. The transport activities of the mutants are normalized against the WT. “Control” refers to uptake by XylE-deficient E. coli transformed with empty vector in the active transport assay or liposomes without protein in the counterflow assay.

Fig. 3.

Counterflow of

-xylose or

-glucose by XylE WT or mutants. (A) Counterflow of

-glucose with XylE WT and given mutants. (B) Counterflow of

-xylose with WT and given mutants. “Control” refers to liposomes without protein. (C) Crystallographic binding pose of

-glucose (blue) compared with predicted binding pose in the homology model of XylE mutant Q175I/L297F (violet). Green lines indicate potential H-bonding. Importantly, interactions with Gly388 in the mutant, fulfilling potential H-bonding criteria, are not found for the

-glucose docking poses to the theoretical model of the Q175I/L297F mutant.

Fig. 4.

Comparison of binding sites in MFS sugar transporters. (A) The X-ray structures of XylE (PDB ID code 4GBY, green, and 4GBZ, yellow), as well as the comparative models of (B) GLUT1 (orange) and (C) GLUT5 (blue), in the ligand-bound occluded conformation are shown. (A) Crystallographic coordinates of

-xylose (yellow) and

-glucose (green) are compared. Labels indicate polar contacts of the protein to the ligand. (B) The predicted binding pose for

-glucose in GLUT1 (orange) is compared with the binding conformation in XylE (same as in A; green). Labels are shown for GLUT1, bold labels indicate important differences. (C) The predicted poses for

-glucose in GLUT1 (same as in B; orange) and

-fructose in GLUT5 (blue) are compared. Labels are shown for GLUT5; bold labels indicate important differences to GLUT1. Broken lines indicate polar contacts to the ligand. See

Fig. S2

for comparison of GlcP_Se_ with XylE and

Fig. S3

presenting the general orientation of the ligand-binding site.

Fig. 5.

Docking of STF-31 to GLUT1 homology model. (A) Cut-away representation of binding position of the inhibitor in GLUT1. Two docking poses are shown in the cavity leading to the extracellular space (outside). (B) STF-31 interactions with specific side-chains. Residues making polar interactions with the ligand are illustrated as sticks; substrate binding residues are colored light blue, Thr30 and Thr295 are colored orange, Thr292 is colored gray; hydrogen atoms are colored in white, nitrogen atoms in blue, and oxygen atoms in red; H-bonds are represented by red lines. (C) Chemical structure of STF-31.

Fig. 6.

Transport cycle of MFS symporter. (A) Overview of the postulated steps in the transport model. Inward-facing (green) and outward-facing (blue) conformations are separated by the apo-intermediate conformations (orange) or by the occluded-intermediate conformations (gray). Steps are numbered consecutively: 1: Opening of the H+ site; 2: Deprotonation to inside and reorientation to the apo-intermediate with a central cavity closed to either side of the membrane; 3: Opening of the outward-facing cavity and reprotonation from the outside; 4: Formation of outward-open, substrate-free conformation; 5 and 6: Substrate binding and induced fit to the occluded conformation; 7: Opening of the inward-facing cavity and release of the sugar; 8: Formation of the protonated, substrate-free conformation. (B) Hypothetical energy profile for the transport cycle. Conformational shown in A are translated into relative energy states (indicated by the icons of conformations defined in A with the cytoplasmic side of the symporter facing up). The schemes are cycles read by following the arrowheads. The red part of the cycle represents the transitions between steps 1 and 4 of the empty pathway. The blue line corresponds to steps 5–8 for net sugar transporting steps (and for the exchange reaction). The free energy of the putative rate-limiting step in absence of ∆ formula image H+ (opening of the periplasmic cavity) is indicated by the vertical red arrow (ΔGn*). The hypothetical effect of an imposed ∆H+ is shown as a broken black vertical arrow, and the broken red lines show the resulting energy profile. (C) Postulated steps in the transport cycle of a uniporter. Steps 1–4 are similar to steps 1–4 in A but without release of a H+. Steps 5–8 are the same as in A. (D) Hypothetical energy profile for the transport cycle of a uniporter. The colors of the lines correspond to parts of the transport reaction equivalent to A. The energy trap for binding an inhibitor is indicated by the broken blue line.

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Functional architecture of MFS D-glucose transporters - PubMed (original) (raw)