The decoration of secretory glycoproteins and glycolipids with sialic acid is critical to many physiological and pathological processes. Sialyation is dependent on a continuous supply of sialic acid into Golgi organelles in the form of CMP-sialic acid. Translocation of CMP-sialic acid into Golgi is carried out by the CMP-sialic acid transporter (CST). Mutations in human CST are linked to glycosylation disorders, and CST is important for glycopathway engineering, as it is critical for sialyation efficiency of therapeutic glycoproteins. The mechanism of how CMP-sialic acid is recognized and translocated across Golgi membranes in exchange for CMP is poorly understood. Here we have determined the crystal structure of a Zea mays CST in complex with CMP. We conclude that the specificity of CST for CMP-sialic acid is established by the recognition of the nucleotide CMP to such an extent that they are mechanistically capable of both passive and coupled antiporter activity.
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Nature Communications Open Access 27 November 2020
Structural basis for substrate specificity and regulation of nucleotide sugar transporters in the lipid bilayer
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The coordinates and the structure factors for CSTZM have been deposited in the Protein Data Bank with accession numbers PDB 6I1R (CMP-bound CSTZM structure) and PDB 6I1Z (apo CSTZM structure). Data supporting the findings of this manuscript and requests for resources and reagents are available upon reasonable request to D.D. (firstname.lastname@example.org).
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We are grateful to Y. Chatzikyriakidou for generation of Fig. 1a and M. Claesson for assistance with data collection and manuscript preparation. We wish to especially thank A. McCarthy for extra assistance at the European Synchrotron Radiation Facility and the Diamond Light Source synchrotron beamline scientists for their excellent assistance. This work was funded by the Knut and Alice Wallenberg Foundation (to D.D.). D.D. acknowledges support from the Wenner-Gren foundation and EMBO through the Young Investigator Program (YIP).
The authors declare no competing interests.
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Integrated supplementary information
a, Size-exclusion chromatogram of CSTZM and corresponding SDS-PAGE-gel with DDM purified CSTZM migrating predominantly as a dimer. b, Determination of the Michaelis constant (KM) for [3H]-CMP/CMP-Sia exchange for CSTZM and hCST. Kinetic curves were fitted from data points recorded over a range of increasing CMP concentrations (internal liposomes containing steady-state levels of CMP-Sia) at 90 s time points and fitted by non-linear regression using data from 3 independent experiments (the values reported are the mean ± s.e.m. of the fit) c, The GFP-TS melting curves using GFP-fusions for CSTZM in crude-detergent solubilized membranes (black open circles) and for hCST (cyan filled squares); errors bars, s.e.m.; n = 3 and the values reported for the apparent Tm are the mean ± s.e.m. of the data fitting. d, The apparent Tm for CSTZM and hCST were determined as in c, and further in the presence of 1 mM CMP, 1 mM UMP, 1 mM GMP, 1 mM TMP, 10 mM CMP-sialic acid or 10 mM sialic acid. The difference in melting temperature ΔTm is shown for CSTZM (filled bars) and hCST (open bars); errors bars are the s.e.m. 3 independent experiments (cyan-filled circles). Markedly, absolute ΔTm differences between CMP and UMP are equivalent to that measured using the CPM assay to estimate binding differences between ATP and AMP in the ADP/ATP exchanger (Majd, H. et al., Elife. 7, 2018). e, FSEC traces of DDM-solubilised membranes of CSTZM and hCST constructs assessed for substrate-binding by GFP-TS (see Supplementary Table 1).
Supplementary Fig. 2 Superimposition of apo- and substrate-bound outward-facing CSTZM structures to Vrg4 and other structural homologues.
a, Representative portions of the 2mFo-DFc electron density map (1.4σ), which is shown here for the TM segments involved in CMP binding. b, Cartoon representation of outward-facing 2.8 Å resolution structure of CSTZM in complex with CMP (colored as in Fig. 1d) and the 3.6 Å outward-facing structure of Vrg4 in complex with GDP-mannose (PDB id: 5OGK; light grey), as viewed from the top of the membrane. The CMP and GDP-mannose moieties from CSTZM and Vrg4 respectively, are shown as sticks. The polder (OMIT) map (Liebschner, D. et al., Acta Crystallogr D Struct Biol. 73, 148-157, 2017) for CMP contoured at 3σ is also shown. c, As in b., the apo 3.4Å outward-facing structure of CSTZM (colored in wheat) and the apo 3.2 Å resolution outward-facing Vrg4 (PDB id: 50GE; light grey) structures. d, Structural comparison of CMP bound (in wheat) and apo (in light grey) CST. The structures superposed with a very low r.m.s.d. of 0.3 Å, indicating no significant conformation difference between the two isoforms. e, Cartoon representation of outward facing bacterial amino acid transporter YddG (PDB id: 5I20; light grey) and CSTZM (wheat). A lipid molecule (in grey sticks) is present at the putative binding pocket of YddG. Both the structures differ with an r.m.s.d. of 4.1 Å, owing to the large evolutionary gap between the two DMT superfamily members. f, Cartoon representation of outward open CSTZM and occluded model of plant triose-phosphate/phosphate translocator (PDB id: 5Y78; light grey). Large inward motion of helix 3 and 4 during transport and conformational switching is consistent with the observations made using structural inverted repeats of CSTZM (see Fig. 3a). Dimerization helices 5 and 10 are not shown for clarity.
a, Cartoon representation illustrating the pronounced differences of the substrate-binding site residues when comparing the outward-facing CSTZM structure in complex with CMP (colored cyan) and the outward-facing Vrg4 structure in complex with GDP-mannose (colored light-grey). The position of the bound CMP is shown as an oval and residues as sticks. b, Cartoon representation illustrating the pronounced differences of the substrate-binding site residues in the Vrg4 structure in complex with GDP-mannose (colored grey) when compared to the structure of CSTZM in complex with CMP (colored cyan). The position of the GDP-mannose is shown as an oval and residues as sticks. c, Slab through the outward-facing CSTZM as viewed within the plane of membrane with CMP (shown as green sticks). All cavity waters in CSTZM are shown as red spheres, which was generated by overlaying separately to the sliced surface. To visualise structural sequence conservation, the ConSurf server (Landau, M. et al., Nucleic Acids Res. 33, W299-302, 2005) was used with default settings (colored based on sequence conservation; low-cyan to high-dark-red). The sequence alignment for conservation analysis was generated using Clustal Omega by comparing 63 CSTZM homologues with sequence identity ranging from 23 to 91 %. d, Modelled positions and 2mFO-DFC electron density map (1.0 σ) for the observed waters and CMP in the CSTZM complex and the residues involved in water coordination. Residues and CMP are shown as sticks and waters as red spheres.
a, CMP binding to CSTZM using GFP-fusions as estimated by a shift in ΔTm after addition of 1 mM CMP to WT (open bars), and to mutants of residues not interacting with crystallographic waters (N106A and S267A) in the cavity, but are present in close proximity to the water network (filled bars). Residues W209 and N206 are located in the CMP binding pocket of CSTZM, but are not conserved in hCST. Non-specific binding was estimated with addition of UMP (red bars). b, The GFP-TS assay was used to determine binding of CMP to purified GFP-fusions of CSTZM (black squares) and hCST (cyan squares). Binding affinities (Kd) were calculated from data points recorded over a range of CMP concentrations, and these were fitted by non-linear regression using data from 3 independent experiments. As has been observed in other transporters (Shukla, S. et al., J Biol Chem. 292, 7066-7076, 2017), the absolute apparent binding affinities in detergent, although consistently different, were significantly weaker than IC50 and KM values determined in membranes. c, as in b for the CSTZM Ser79Ala and hCST Ser95Ala mutants shown to have enhanced CMP binding in (Fig. 2b, c). The binding affinities were estimated using the solubilized membranes. d, as in b for CMP-Sia; see Methods). e, Time-dependent uptake of [3H]-CMP by CSTZM (open circles). Non-specific uptake was estimated with empty liposomes (filled squares). f, Time-dependent uptake of [3H]-CMP by hCST (open circles). Non-specific uptake was estimated with empty liposomes (filled squares). In all experiments errors bars, s.e.m.; n = 3.
a, The substrate-binding site in the outward facing CSTZM structure with modelled CMP-sialic acid (TMs in light brown). The CMP-sialic acid is shown as green sticks, and residues coordinating the modelled CMP-sialic acid as sticks colored in rainbow (as in Fig. 1d). b, View of the CSTZM surface (colored according to the calculated electrostatic potential, presented in blue-positive to red-negative) as viewed from the top with the modelled CMP-sialic acid (shown as green sticks). c-e, Cartoon representation of the structural comparison of CMP bound CSTZM (brown), GDP-mannose bound Vrg4 (cyan; PDB id: 5OGK) and 3-Phosphoglyceric acid (3PG) TPT (pink; PDB id: 5Y78) showing the position of the substrate relative to structural conserved TM4- and TM9-embedded lysine residues (dashed lines represent direct electrostatic interactions between lysine and terminal phosphate moiety), f, Structures of the α-keto acids, mentioned in the text. 9-carbon α-keto acids; N-acetylneuraminic acid (Neu5Ac) and 2-keto-3-deoxy-d-glycero-d-galacto-nonulosonic acid (Kdn), with the 8-carbon α-keto acid 2-keto-3-deoxy-d-manno-octulosonic acid (Kdo).
Cartoon representation showing the residues which by polar interactions stabilize the two transport bundles (colored cyan and wheat, respectively) in the outward-facing conformation. CSTZM E181 is equivalent to hCST E196, which when mutated to lysine (E196K) causes a congenital disorder of glycosylation as it abolishes CST transport activity (Fig. 3d).
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Nji, E., Gulati, A., Qureshi, A.A. et al. Structural basis for the delivery of activated sialic acid into Golgi for sialyation. Nat Struct Mol Biol 26, 415–423 (2019). https://doi.org/10.1038/s41594-019-0225-y
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