GMP adopts different conformations in the binding site

The difference observed between the transport of GDP-mannose and GMP raised the question of whether the nucleoside monophosphate was recognised in a different way within the binding site. To understand how GMP is recognised by Vrg4 we determined a co-crystal structure with GMP at 3.3 Å resolution (Fig. 1a and Supplementary Table 1). Vrg4 adopts a very similar pose as that for the GDP-mannose transporter (root mean square deviation ~0.47 Å between 276 C α atoms), with the binding site open to the luminal side of the Golgi membrane and sealed shut on the cytoplasmic side through the packing together of TM6 & TM7 against TM8 and TM9. Clear mF o -DF c difference electron density was observed for the GMP ligand in three of the eight transporters in the unit cell (Supplementary Fig. 1). Unexpectedly the three GMP ligands adopt slightly different positions within the three binding sites (Fig. 1b), despite a r.m.s.d of only 0.45 Å between the three structures. In two of the three positions the GMP ligand adopts an extended conformation (Fig. 1c, d). However, in the third position captured, the GMP adopts a bent configuration, with the phosphate group pointing down towards the Golgi lumen (Fig. 1e). In chain C, the phosphate group of the GMP molecule forms a salt bridge with a conserved lysine (K289) on TM9 and interacts with a conserved tyrosine on TM1 (Y28) through a hydrogen bond (Fig. 1c). Lysine 289 forms part of the previously identified GALNK289 motif, which plays an important role in discriminating between sugar groups of different nucleotide sugar molecules10. The interaction with Y28 is consistent with the essential role this side chain has in transport10. Moving along the GMP molecule, the ribose group also interacts with TM9 via another conserved tyrosine (Y281), which, although not strictly conserved, must be a bulky side chain to support transport10. Interestingly, the orientation of the ribose ring is significantly different to that observed in the GDP-mannose structure, resulting in an interaction between Y281 and the ring oxygen instead of the two hydroxyl oxygens (Supplementary Fig. 2). At the far end of the ligand the guanine ring is flipped 180° relative to that of GDP-mannose, but still sits within the previously observed nucleotide binding pocket. This pocket is characterised by the presence of a conserved FYNN221 motif on TM7, which is required for guanine recognition. Similar conserved motifs are observed in other families of nucleotide sugar transporters in this region of the binding site, consistent with a general importance of TM7 in nucleotide recognition11,13. However, unlike the GDP-mannose ligand, GMP does not make a direct interaction to the conserved asparagines of the FYNN motif, which are ~4.5 Å away. Instead the guanine interacts with a conserved serine (S266) on TM8, via an interaction with the ring nitrogen.

Fig. 1 Crystal structure of Vrg4 bound to GMP. a Crystal structure of Vrg4 with helices coloured blue (N-terminus) to red (C-terminus). The binding cavity is open to the Golgi lumen, and shown as a transparent surface (wheat). The three different positions observed for the GMP molecule are overlaid in blue, orange and pink. The position of the lipid bilayer is indicated by white dashed lines. b An overlay showing the three GMP molecules as presented in a, with arrows highlighting the changes in positions observed for the nucleoside and phosphate groups. c, d, e Zoomed in views of the binding site from chains C, D and E respectively, showing the different positions and interactions made to GMP in the crystal structure Full size image

In chain D the GMP ligand adopts a similar extended position to that observed in chain C, with the guanine ring again flipped 180° relative to that observed in the GDP-mannose structure (Fig. 1d and Supplementary Fig. 2). Overall the interactions between the GMP in chain D and those in chain C are similar despite the different orientation of the ribose ring (Fig. 1b). S266 interacts with the ring nitrogen on the guanine base, Y281 with the ribose ring oxygen, while K289 interacts with the phosphate group. However, a surprising finding from our analysis of the GMP co-crystal structures was the unusual position of the GMP ligand in chain E (Fig. 1b). Unlike the position of the GMP ligand in chains C and D, the molecule adopts a bent configuration, with the phosphate group now orientated towards the luminal entrance of the binding site (Fig. 1e). In this position the phosphate now interacts with a conserved serine on TM1 (S32), rather than K289, which adopts a different rotamer configuration compared to chains C and D (Supplementary Fig. 3). The ribose group sits in a similar location within the binding site to that observed in chains C and D, but now Y281 interacts with the O2 hydroxyl rather than the ring oxygen, similar to that observed previously with GDP-mannose (Supplementary Fig. 2). In this position the guanine ring now sits further into the nucleotide binding pocket compared with the carbonyl group positioned near to asparagine N221 of the FYNN221 motif. An additional interaction is also observed between the ring nitrogen of the nucleotide (N3) and a conserved serine on TM8 (S269), which was previously observed interacting with the ribose hydroxyl oxygen in the GDP-mannose structure. Taken together, the different positions of GMP within the crystal structure suggested that Vrg4 recognises GMP differently to GDP-mannose, which as we discuss below, is important for understanding how these transporters discriminate between nucleoside monophosphate and nucleotide sugar in the cell.

Specific residues discriminate between ligands

Although GMP adopts three different positions within the binding site, it is notable that in all three poses an interaction with Y281 on TM9 is observed. Previously we showed that a conservative mutation to phenylalanine retained activity whereas an alanine variant was non-functional10. However, given the prominent role that Y281 plays in GMP recognition we performed a more in-depth IC 50 analysis on the Y281F variant (Fig. 2a). We observed that the affinity of this variant for GDP-mannose remains the same as wild type (WT; 7.6 μM10), however the IC 50 for GMP increased to 23 μM, indicating a hydroxyl at this position aids recognition of the GMP ligand. Given that Y281 appears to discriminate between the two ligands, we sought to identify additional side chains that are important for GMP transport. Tyrosine 28 is essential, and its replacement with alanine abolishes transport, and K289 is important for sugar recognition and transport10. However, our structures also identified serines 266 and 269 as interacting with the guanine base. Interestingly, we observed a similar result to that of Y281 with S266, with an alanine variant resulting in a significant increase in the IC 50 value for GMP transport, whilst the affinity for GDP-mannose was only slightly reduced (Fig. 2b). The similar affinity for GDP-mannose as WT in this mutant is most likely due to the additional interactions formed between the GALNK motif and the mannose moiety compensating for the loss of interaction sites on the guanine base. Removal of S269 on the other hand did not result in any significant change in the IC 50 for either GMP or GDP-mannose (Fig. 2c). Our analysis of the binding site shows that Vrg4 is able to discriminate between GMP and GDP-mannose ligands and that different sets of residues are important for transport of either ligand.

Fig. 2 Characterisation of the GMP binding site. a, b, c Representative IC 50 curves determined for both GMP and GDP-mannose in the Y281F, S266A, and S269A variants, respectively. Insets show the calculated IC 50 value from three independent experiments, errors shown are standard deviations (s.d) of the mean. d Transport assays showing the highest level of transport occurs when GDP-mannose is present on both sides of the liposome membrane. n = 4 independent experiments, error bars s.d. Source data are provided as a Source Data file Full size image

Structural explanation for transport rate differences

NSTs are obligate antiporters, requiring the movement of one ligand in exchange for another. In the cell NSTs function to shuttle nucleotide sugars into the lumen of the ER or Golgi, in exchange for the cognate nucleoside monophosphate7. An important question has been how these systems ensure the direction of transport for their ligands. Previously we showed that Vrg4 functions at different rates depending on the ligands it is moving across the membrane, with GDP-mannose:GMP being more efficient than GMP:GMP in liposome-based assays10. The GMP co-crystal structures now provides an explanation for this phenomenon. It appears that GMP is simply less efficient at docking into the binding site and triggering transport compared to the larger GDP-mannose, which interacts with more sites in the protein. However, if this structural hypothesis is correct and the observed rate difference is caused by a more flexible binding position, we would predict that GDP-mannose:GDP-mannose antiport via Vrg4 would have a faster transport rate due to the lack of alternative interaction positions available with this larger ligand. Similarly, a ligand that makes fewer interactions than GMP, such as AMP (discussed below), would be expected to have a slower rate. We tested this hypothesis using a liposome-based assay and monitoring transport of both radiolabelled GMP and GDP-mannose. We observed the fastest transport rate when GDP-mannose is transported against itself and the slowest for GMP:AMP antiport (Fig. 2d and Supplementary Fig. 4), confirming that the transport rate of Vrg4 correlates with the number of interactions made between the transporter and substrate.

Structural basis of nucleotide specificity

The features that underpin substrate selectivity in the SLC35 family are unclear, with sequence identity being a poor predictor of ligand recognition9. However, previously identified putative sequence motifs in Vrg4 appear to correlate with substrate specificity, which may facilitate sequence based functional assignment within the SLC35 family11,13. Specifically, in Vrg4 the FYNN221 motif located on TM7 was linked to recognition of the guanine base. Vrg4 shows strict substrate specificity with respect to the nucleotide moiety, being able to recognise only purine bases (Supplementary Fig. 5). Within the purine bases, the native substrate GMP is transported with a much higher affinity (IC 50 7 μM) compared to AMP (IC 50 50 μM). It is known that the asparagines in the FYNN221 motif are important for ligand recognition, but it is unclear what role they play10. To further understand this role, we analysed their ability to discriminate between the different purine bases. Alanine variants of both N220 and N221 resulted in a higher IC 50 for GMP than the WT protein; N220A 12 μM and N221A 22 μM (Fig. 3a). This result suggests that both asparagines are used to recognise and position the amino and carbonyl groups on the GMP ligand, and explains why AMP, with only one amino group is recognised at a much lower affinity than the WT protein. Analysis of the IC 50 of the mutants for AMP however, shows that for the N220A variant the affinity for AMP is markedly reduced, suggesting that the remaining asparagine, N221 cannot interact as well with the amine of AMP as it can with the carbonyl of GMP. However, the N221A variant has IC 50 values for AMP and GMP which are very similar to each other, 27 μM and 22 μM respectively, thereby no longer discriminating between these nucleotides (Fig. 3b). This result thus demonstrates that within the FYNN221 motif, N221 plays a more significant role in purine selectivity. These biochemical data are supported by the structural comparison between the GMP and the GDP-mannose structures (Supplementary Fig. 2), which show that a similar feature observed in all conformations captured is the position of the carbonyl group close to N221.

Fig. 3 The FYNN motif mediates nucleotide discrimination. a Representative IC 50 curves determined for GMP in wild type (WT), N220A and N221A variants. b Representative IC 50 curves determined for AMP in wild type (WT), N220A and N221A variants. The chemical structures of GMP and AMP are shown for reference. Insets show the calculated IC 50 value from three independent experiments, errors shown are s.d. of the mean. Source data are provided as a Source Data file Full size image

Lipids play an important role in mediating dimerisation

NSTs involved in the transport of UDP-GalNac, GDP-fucose, PAPS (3′phosphoadenosine-5′-phosphosulphate) and GDP-mannose are reported to form homo-oligomers ranging from dimers to hexamers17,18,19. However, the significance of oligomerisation and the role of lipids in regulating oligomeric state within the NST family remain unknown. Analysis of the apo, GMP and GDP-mannose bound structures of Vrg4 show the presence of well−ordered monoolein lipid molecules at a potential dimer interface. The dimer interface is formed between TM5 and 10 and contains two well-ordered monoolein lipid molecules, which contribute ~60% to the total buried surface area of 1514 Å2 (Fig. 4a)20. Although Vrg4 crystallises as a dimer in monoolein, in detergent the protein is monomeric10, raising the question of whether lipids induce dimer formation in Vrg4 and what implication dimerisation may have for function. To test the impact of the lipid environment on stabilisation of the protein we used a thermal shift assay, which has been used to test membrane protein lipid interactions21. We noticed that in a lipid environment Vrg4 has a significantly higher melting temperature (~55 °C) than with either the detergents decylmaltoside (DM) (33 °C) or dodecylmaltoside (DDM) (38 °C) (Supplementary Fig. 6a). This large increase in melting temperature in the presence of lipid could be indicative of further stabilisation due to oligomerisation. We also observe possible dimer formation in SDS PAGE analysis of Vrg4 reconstituted into liposomes, which is not seen when the protein is in detergent, even at high concentrations of protein (Fig. 4b and Supplementary Fig. 6b). This raised the question of whether Vrg4 is a functional dimer in the membrane. To investigate whether lipids can induce higher order oligomer formation we performed glutaraldehyde crosslinking using protein purified under lipid rich conditions. A dimer band can be observed in SDS–PAGE which increases in intensity in the presence of both crosslinking agent and additional yeast polar lipids, indicating that Vrg4 can form higher order oligomers in the presence of lipid (Supplementary Fig. 6c).

Fig. 4 Lipid mediated dimerisation of Vrg4. a Vrg4 dimer observed in the crystal structure with two monoolein lipid molecules (magenta), packed within the dimer interface (dashed line). b SDS–PAGE analysis of Vrg4 reconstituted at different ratios of protein to lipid, showing the presence of a dimer band at ∼ 55 kDa. c Density plot of DPPC lipids around the Vrg4 dimer, in a slice perpendicular to the membrane. Data were gathered using ca. 40 µs of CG MD simulation, and calculated with the VMD volmap plugin. Specific green regions highlight density present at the primary lipid binding region in the dimer interface. The Vrg4 dimer is shown in cartoon and overlaid for reference d Vrg4 dimer observed in the simulation shown in c with four DPPC lipids (green) within the dimer interface shown. e Dominant-negative assay showing the functional unit of Vrg4 is monomeric, and that Vrg4 forms dimers in a liposome. n = 3 (upper panel) or 5 (lower panel) independent experiments, errors shown are s.d. Source data are provided as a Source Data file Full size image

To further understand the role of lipids within the dimer interface of Vrg4 we used molecular dynamics to embed Vrg4 in a membrane composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). These simulations confirm that phospholipids accumulate within the dimer interface observed in the crystal structure (Fig. 4c, d). Interestingly, the MD simulations show that the dimer interface can accommodate four DPPC lipids, arranged as a bilayer (Supplementary Fig. 6d). This indicates that in the native membrane environment the Vrg4 dimer will be held together through stronger interactions, than in a detergent micelle, consistent with our cross-linking analysis.

To test the functional significance of dimerisation we developed a dominant negative biochemical assay (Fig. 4e and Supplementary Fig. 6e). We reasoned that if equimolar amounts of functional and non-functional Vrg4 were reconstituted into liposomes, then random mixed dimers would form, containing both active and inactivate transporters. If Vrg4 is a functional dimer we would expect to see a greater than 50% reduction in transport. However, we observed that mixing fully functional WT protein with an inactive mutant, Y281A, discussed above, resulted in exactly 50% transport activity. This result implies that each monomer of Vrg4 acts as an independent functional unit, similar to other dimers of SLC transporters22,23. We then repeated this experiment, but instead used a K118A variant, which is transport inactive but binding competent (Supplementary Fig. 6f), and observed ~75% activity (Fig. 4e). Reconstituting at different ratios (75 and 25% WT) also gave a higher than expected transport rate, confirming that Vrg4 forms oligomers within the liposome membrane (Fig. 4e). Our interpretation of this data is that when Vrg4 is in a mixed dimer, where one subunit is unable to transport and in a locked conformation, the WT subunit is able to cycle faster. This effect is most likely due to the increased stability being imparted by an immobile partner, which provides a stable platform against which the active transporter can move. This phenomenon was also recently shown for the SLC26 anion transporters, which despite being a different family also form structural oligomers in the membrane24. In this study a similar effect was observed, where the mixing of active and inactive monomers created dimers with great than 50% activity. Together with the structural, cross linking and MD data, we propose that Vrg4 dimerises through an interface mediated by lipid molecules, which form an integral part of the dimer.

Structural basis for short chain lipid dependence

The Golgi membrane is known to have a different lipid composition in comparison to the plasma membrane, which gives rise to a thinner bilayer thickness25. Vrg4 transport activity is dependent on short chain lipids to function, with a severe drop in activity observed in longer chain lipids, such as 1-palmitoyl-2-oleoyl (PO) lipids, which consist of one 16 and one 18 carbon fatty acid chain10. However, the structural basis for this observation remains obscure. This phenomenon could be due to hydrophobic matching, especially given the short length of the hydrophobic region of Vrg4. Indeed, when Vrg4 is inserted into a coarse grained DPPC membrane, it results in the thinning of the bilayer to more resemble the thickness of a DMPC bilayer (Supplementary Fig. 7). Our simulation data from the mixed DMPC/DPPC bilayers also showed the accumulation of lipid at an additional site on the transporter (Fig. 5a, b). This second lipid binding site only accommodates DMPC and not DPPC, and occurs in a shallow groove between TMs 1, 9, and 10 (Fig. 5c and Supplementary Fig. 8). The preference for DMPC over DPPC at this site suggests lipids of 16 carbon chain lengths and above are excluded, whereas the shorter DMPC lipid, with only a 14 carbon acyl chain, can be accommodated. Based on a repeat swapped model of the cytoplasmic facing state of Vrg4, we previously suggested that TM9 is likely to play an important role in the alternating access mechanism10. TM9 was also identified as being important for the transition between luminal and cytoplasmic facing conformations of the plant triose phosphate antiporter, TpT, a distant homologue of the NST family26,27. These results suggest that short chain lipids may allow TM9 to undergo the conformational changes necessary to cycle between inward and outward facing states during transport.