Structural basis of nucleotide sugar transport across the Golgi membrane

Abstract

Glycosylation is a fundamental cellular process that, in eukaryotes, occurs in the lumen of both the Golgi apparatus and the endoplasmic reticulum1. Nucleotide sugar transporters (NSTs) are an essential component of the glycosylation pathway, providing the diverse range of substrates required for the glycosyltransferases2,3. NSTs are linked to several developmental and immune disorders in humans, and in pathogenic microbes they have an important role in virulence4,5,6,7,8. How NSTs recognize and transport activated monosaccharides, however, is currently unclear. Here we present the crystal structure of an NST, the GDP–mannose transporter Vrg4, in both the substrate-free and the bound states. A hitherto unobserved requirement of short-chain lipids in activating the transporter supports a model for regulation within the highly dynamic membranes of the Golgi apparatus. Our results provide a structural basis for understanding nucleotide sugar recognition, and provide insights into the transport and regulatory mechanism of this family of intracellular transporters.

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Figure 1: Crystal structure and functional analysis of Vrg4.
Figure 2: Recognition of GDP–mannose by Vrg4.
Figure 3: Molecular determinants of substrate specificity in Vrg4.
Figure 4: Alternating-access transport mechanism of nucleotide sugar transporters.

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Acknowledgements

We thank the staff of beamlines I24 Diamond Light Source, UK, Proxima-2a, Soleil, France and BL1-A, Photon Factory, Japan for assistance. We thank O. Nureki and R. Ishitani for assistance in accessing BL1-A and G. Kuteyi for technical support. This work was supported by Wellcome awards (102890/Z/13/Z and 102890/Z/13/A).

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J.L.P. and S.N. designed and carried out the experiments, interpreted the data and wrote the paper.

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Correspondence to Joanne L. Parker or Simon Newstead.

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Reviewer Information Nature thanks M. Wild, J. Zimmer 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 Figure 1 Analysis of anomalous difference electron density and substrate specificity and recognition by Vrg4.

a, Anomalous difference peaks for the mercury (orange mesh) and sulfur (green mesh) atoms calculated using AnoDe and contoured at 3σ, and overlaid on the structure of Vrg4. b, Final refined 2mFo − DFc maps for Vrg4, contoured at 1σ. TM10 is highlighted along with side-chain density. c, Transport cannot be driven by GTP in the presence or absence of CCCP (C) or valinomycin (v). n = 4 independent experiments, data are mean ± s.d. d, Competition assays demonstrate that GMP and GDP–mannose act as the most effective competitors of transport, but other guanosine-containing substrates (GDP, GDP–fucose, GDP–glucose) can also compete and be recognized by the binding site. However, the free base guanosine is very poorly recognized, as is cGMP. Pyrophosphate, which consists of a diphosphate moiety and is an inhibitor of ATPases, cannot inhibit the transport activity of Vrg4. n = 4 independent experiments, data are mean ± s.d.

Extended Data Figure 2 Lipid molecules are found at the dimer interface of Vrg4.

a, The eight molecules in the P1 unit cell are shown, four of which adopt a dimer arrangement with the monomers arranged in a physiological orientation. Several lipid molecules could be identified and are shown in blue. b, Zoomed-in view of the Vrg4 dimer showing the electrostatic surface of the dimer interface between helices 5 and 10 and the two monoolein lipids bound. The hydrophobic belt is noticeably shorter (~30 Å) than that observed for plasma or inner membrane transporters (~40 Å). Within the dimer interface, the two well-ordered monoolein molecules contribute ~60% to the total buried surface area of 1,514 Å2. In the remaining four monomeric molecules in the unit cell we do not observe any density for lipid molecules at these sites, suggesting an important role for lipid-mediated dimerization. c, Refined 2mFo − DFc electron density for the lipid molecules shown in b, present in the apo crystal structure. d, The dimer-interface lipids were more ordered in the GDP–mannose soaked crystals. All electron density maps are contoured at 1σ.

Extended Data Figure 3 Vrg4 recognizes both GMP and GDP–mannose with the same apparent affinity.

a, Representative IC50 curves calculated for wild-type Vrg4. ‘GDP–M in’ denotes GDP–mannose-loaded liposomes; ‘GMP in’ denotes GMP-loaded liposomes. b, Calculated IC50 values are the mean of three independent experiments, errors shown are s.d. c, Calculated IC50 values for Fig. 2a, c. n = 3 independent experiments, data are mean ± s.d. d, Calculated IC50 values for Fig. 3c. n = 3 independent experiments, data are mean ± s.d.

Extended Data Figure 4 Electron density maps for bound GDP–mannose.

a, Crystal structure of Vrg4 bound to GDP–mannose in the plane of the membrane. GDP–mannose is shown as sticks with refined 2mFo − DFc electron density contoured at 1σ. b, Zoomed-in view of the binding site showing key polar interactions. c, Stereo view of bound GDP–mannose shown in b.

Extended Data Figure 5 Alignment of Vrg4 with GDP–mannose and GDP–fucose transporters.

Amino acid alignment of S. cerevisiae Vrg4 with GDP–mannose transporters from various organisms (_GMT or _NST) or GDP–fucose transporters (SLC35C1). Identical residues are highlighted in red. The nucleotide-binding, FYNN, and sugar-recognition motifs are indicated as sequence logos. The positions of the 10 transmembrane domains are indicated and coloured from blue to red as in the crystal structure representation of Vrg4 shown on the bottom right.

Extended Data Figure 6 Effect of Tyr114Phe mutation on substrate affinity.

a, Representative IC50 curves calculated for wild-type Vrg4 (WT) and Tyr114Phe (Y114F) for GMP and GDP–mannose. b, Bar chart showing the IC50 values calculated from a. n = 3 independent experiments, data are mean ± s.d. Wild-type Vrg4 recognizes both GMP and GDP–mannose with the same affinity. Mutating Tyr114 to phenylalanine reduced the affinity for both substrates, demonstrating the importance of the hydroxyl group on this residue for high-affinity binding. Unlike wild-type Vrg4, the Tyr114Phe mutant now recognizes GDP–mannose with a reduced affinity compared to GMP, supporting the interaction we observed in the crystal structure (Fig. 3a).

Extended Data Figure 7 Homology models of the human GDP–fucose transporter (SLC35C1) and CMP–sialic acid transporter (SLC35A1) conserved sequence features across the SLC35 family.

a, View of the binding of Vrg4 with bound GDP–mannose (grey) overlaid with the equivalent residues from the GDP–fucose homology model (blue). Residue numbers are shown first for Vrg4, then for the equivalent residue in the human transporter. The consensus motifs for mannose (GALNK) and fucose (GTAKA) binding are shown, as is the FYNN motif that dictates guanine binding. b, The equivalent analysis was carried out for the human CMP–sialic acid transporter (SLC35A1) which is shown in yellow. Our homology model reveals important insights into the human CMP–sialic acid transporter (CST), which is mutated in the congenital disorder of glycosylation IIf and results in a lack of sialic acid on the surface of cells5. The model of SLC35A1 reveals that the nucleotide-binding pocket has changed from the FYNN motif to the NIQM motif, and now contains a glutamine (Gln212) instead of the asparagine. The extension of the glutamine side chain over the asparagine may help to facilitate the recognition of cytosine, which is smaller than guanine. Previous studies have identified Tyr214 as being important for ligand recognition in the CST40. In our model, this residue faces the membrane and probably anchors Glu212 to stabilize its interaction with the cytosine. Another important difference between the CST and Vrg4 is the replacement of Tyr28 and Tyr281 with lysine side chains (Lys13 and Lys272). Indeed, the mutation of Lys272 results in complete inactivation of CST41. The GALNK motif, by contrast, is replaced by a stretch of alanine side chains. These changes suggest that the binding site is both larger and more positively charged than Vrg4, which would be required to accommodate the nine-carbon sialic acid sugar group; unlike fucose or mannose, it contains an N-acetyl extension at the C5 position in addition to an extended hydroxyl arm at C6.

Extended Data Figure 8 Repeat-swap modelling of the open-to-cytoplasm state of Vrg4.

a, Vrg4 contains two structural repeat units consisting of TM1–TM5 and TM6–TM10, which are related to each other via a two-fold rotation in the plane of the membrane. b, AlignMe42 sequence alignment between the two structural repeats reveals a high degree of sequence conservation, making repeat-swap modelling more accurate. Highlighted in magenta are the equivalent residues in each repeat that form polar interactions in the crystal structure. c, The two repeats align with a root mean square deviation of 5 Å, with the most notable deviation being between helices 4 and 9 and helices 1 and 6, which deviate from each other by 20° and 30°, respectively. It is notable that the axis on which these repeats deviate is where the residues highlighted in magenta overlap. d, The repeat-swapped model of Vrg4 in the open-to-cytoplasm state, which is achieved via a rotation of the structural repeats around an axis that runs through the residues highlighted in magenta.

Extended Data Figure 9 Analysis of purified Vrg4 in both detergent and lipid environments.

a, Representative gel filtration trace of the purified Vrg4 in the detergent decylmaltoside which was used for reconstitution, n = 20. The protein elutes as a single peak and is purified to homogeneity as judged by Coomassie-stained SDS–PAGE, in which it runs as a single band at ~30 kDa. b, Size-exclusion chromatography with multi-angle static light scattering analysis indicates that Vrg4 is monomeric in decylmaltoside with an expected mass of 37 kDa (the refractive index increment (dn/dc) values for protein and decylmaltoside detergent used were 0.185 and 0.147, respectively). n = 3 independent experiments, producing similar results. c, Representative SDS–PAGE gel showing reconstitution of Vrg4 into different lipid environments. The same amount of protein was used for each lipid type tested: YPL, POPE:POPG (in a 3:1 ratio), soy PE:egg PG (in a 3:1 ratio), POPE:POPG and POPC (a 3:1 ratio of POPE:POPG with 12% POPC added), POPE:POPG and DMPC (a 3:1 ratio of POPE:POPG with 12% DMPC added). n = 5 independent experiments, producing similar results. The asterisk shows the presence of a protein band (a dimer in size) observed upon reconstitution into liposomes, which is not seen when the protein is in detergent (a, inset) but is observed in the unit cell of the crystal structure. d, Representative gel-filtration profiles of all the mutants used in this study, n = 2 independent experiments, producing similar results. The traces show that all mutant variants behave similarly to wild-type protein during purification, showing monodisperse peaks with little aggregation. With relation to the differing amounts of protein shown in these graphs, this is owing to the different volumes used to express the protein (either >10 l for the upper two panels or 5 l for the lower panel). e, Representative SDS–PAGE gel showing reconstitution of Vrg4 and the mutant variants used in this study, showing that the same amount of protein was used in the assays. n = 3 independent experiments, producing similar results.

Extended Data Table 1 Data collection and refinement statistics for Vrg4

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Parker, J., Newstead, S. Structural basis of nucleotide sugar transport across the Golgi membrane. Nature 551, 521–524 (2017). https://doi.org/10.1038/nature24464

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