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Structure of a prokaryotic fumarate transporter reveals the architecture of the SLC26 family

A Corrigendum to this article was published on 04 May 2016

This article has been updated

Abstract

The SLC26 family of membrane proteins combines a variety of functions within a conserved molecular scaffold. Its members, besides coupled anion transporters and channels, include the motor protein Prestin, which confers electromotility to cochlear outer hair cells. To gain insight into the architecture of this protein family, we characterized the structure and function of SLC26Dg, a facilitator of proton-coupled fumarate symport, from the bacterium Deinococcus geothermalis. Its modular structure combines a transmembrane unit and a cytoplasmic STAS domain. The membrane-inserted domain consists of two intertwined inverted repeats of seven transmembrane segments each and resembles the fold of the unrelated transporter UraA. It shows an inward-facing, ligand-free conformation with a potential substrate-binding site at the interface between two helix termini at the center of the membrane. This structure defines the common framework for the diverse functional behavior of the SLC26 family.

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Figure 1: Transport properties of SLC26Dg.
Figure 2: SLC26Dg structure.
Figure 3: TM domain and intracellular cavity.
Figure 4: Comparison of SLC26Dg and UraA.
Figure 5: Potential substrate interactions.
Figure 6: Potential transport mechanism.

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  • 12 April 2016

    In the version of this article initially published, the Protein Data Bank accession code for the coordinates and structure factors for SLC26DgΔSTAS (PDB 5IOF) was not included. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

This research was supported by a grant of the Swiss National Science Foundation through the National Center of Competence in Research (NCCR) Structural Biology (R.D.), a long-term fellowship from the Human Frontier Science Program (LT-00899/2008) (E.R.G.), the German Research Foundation through the Cluster of Excellence Frankfurt 'Macromolecular Complexes' (E.R.G.), INSTRUCT as part of the European Strategy Forum on Research Infrastructures (ESFRI) (J.S.) and the Hercules Foundation Flanders (J.S.). We thank B. Fakler and R.J.C. Hilf for input at initial stages of the project; the staff of the X06SA beamline for support during data collection; B. Blattman and C. Stutz-Ducommun (Protein Crystallization Center at University of Zurich) for their support with crystallization; B. Dreier for help with MALS experiments; and N. Buys and K. Willibal for help with nanobody selection. All members of the Dutzler laboratory are acknowledged for help in all stages of the project.

Author information

Authors and Affiliations

Authors

Contributions

F.R.S. and E.R.G. screened homologs, established purification of SLC26Dg and crystallized SLC26DgΔSTAS. E.P. performed immunization, cloned and expressed nanobodies, and guided E.R.G. during the initial selections. J.S. supervised nanobody production. E.R.G. and Y.N. established nanobody overexpression and purification for crystallization. Y.-N.C. and E.R.G. crystallized the SLC26Dg–nanobody complex and carried out crystallographic experiments and transport assays. R.D. assisted E.R.G. in structure determination. E.R.G. and R.D. jointly planned the experiments, analyzed the data and wrote the manuscript.

Corresponding authors

Correspondence to Eric R Geertsma or Raimund Dutzler.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Sequence alignment.

Sequence alignment of SLC26Dg (GI:499845065), the E. coli transporter DauA (GI:115512548) and human Prestin (SLC26A5, GI:30348882). The Alignment was improved based on the SLC26Dg structure and the structures of the STAS domains of Prestin and DauA. Secondary structure elements of SLC26Dg are shown below. The two halves of the transmembrane domain are colored in green and beige respectively. The STAS domain is colored in red. The numbering corresponds to SLC26Dg. Selected residues in the aqueous cavity in proximity to the potential substrate binding site are indicated as blue or beige dots, the start of the STAS domain as red dot.

Supplementary Figure 2 Functional properties.

Gel filtration and light scattering results for different protein constructs purified in the detergent DM. Continuous black traces correspond to the UV280 elution profiles. The respective molecular weight of the protein component obtained from light scattering is shown at its corresponding position on the chromatogram in red. Panels show (a), SLC26Dg, and (b), the SLC26Dg-Nb5776 complex. (c) Glutaraldehyde (GA) crosslinking of reconstituted SLC26Dg. Samples were separated by SDS PAGE, the respective molecular weight of marker proteins is indicated. The band at around 130 kDa corresponds to a cross-linked SLC26Dg dimer, the band at around 55 kDa to monomeric SLC26Dg observed upon solubilization of proteoliposomes in SDS prior to glutaraldehyde addition. Left panel, SDS-PAGE gel of proteoliposomes reconstituted at a protein:lipid ratio of 1:50 (wt/wt); right panel, Western blot of proteoliposomes reconstituted at 1:250 and 1:500 indicating that crosslinking of SLC26Dg dimers also occurred at very low protein concentrations in the membrane. (d) Substrate selectivity. Competition of 14C-fumarate uptake by a 70-fold excess of other unlabeled dicarboxylates. (e) Concentration dependence of fumarate transport. The solid curve shows a fit to a Michaelis-Menten equation with a linear component due to the passive uptake of fumarate into liposomes. The dashed line shows the slope of the linear component. Specific activities were calculated assuming a complete incorporation of the protein into liposomes during reconstitution. (f) Membrane orientation of SLC26Dg (WT) and SLC26DgΔSTAS (TM) in proteoliposomes. Both proteins were expressed and reconstituted as N-terminal fusions to GFP. The membrane orientation was investigated by probing the accessibility of the HRV 3C protease cleavage site between the membrane transporter and GFP. In-gel fluorescence was measured after separation of the cleaved products on SDS PAGE. – refers to the untreated samples, p to samples treated with HRV 3C protease, and p/d to samples treated with HRV 3C protease and detergent. * indicates uncleaved protein. For both constructs, incubation with the protease decreased the GFP fluorescence of the uncleaved protein by approximately 50% indicating a similar random distribution of both orientations within the membrane. Molecular weight of markers (kDa) are indicated on the left. (g) Transport properties of SLC26DgΔSTAS. Time dependent uptake of 14C-fumarate into proteoliposomes containing SLC26DgΔSTAS (TM). ΔpH refers to an outside pH of 6.0 and a pH of 7.5 inside the liposomes, ΔNa+ to an external Na+ concentration of 50 mM and no Na+ inside the liposomes. Traces of WT and liposomes not containing any protein (with data presented in Fig. 1a) are shown for comparison. Proteoliposomes were prepared with equimolar amounts of protein. Equimolar protein amounts were also used for each data point. Data in d and g represents mean and s.e.m of 3 technical replicates, data in e represent mean and s.e.m of 6 technical replicates from two independent batches of proteoliposomes.

Supplementary Figure 3 Electron density of the SLC26Dg–Nb5776 complex.

Proteins are shown as sticks with carbons colored in green. 2Fo-Fc density (calculated at 3.2 Å, sharpened with b=90 and contoured at 1σ) calculated with phases obtained from the refined model is superimposed on the structure. Sections of different regions of the complex are shown: (a) Stereo view of the substrate binding site of the transmembrane domain with a bound DM molecule (with carbon atoms of the detergent colored in blue). The detergent binds to a similar location as a bound detergent in the UraA structure. (b) STAS domain viewed from the nanobody binding interface with α-helices 2 and 3 labeled. (c) View of the nanobody. The electron density for the nanobody is less well defined than the density of the transmembrane domain of SLC26Dg, which indicates a higher degree of disorder. The entire nanobody is shown. Supplementary Figs. 3, 4, 5, 6 were prepared with DINO (http://www.dino3d.org).

Supplementary Figure 4 STAS-domain structure.

(a) Ribbon representation of the STAS domain of SLC26Dg with secondary structure elements labeled. (b) Superposition of Cα-traces of the SLC26Dg structure (red) with the equivalent domain from the SLC26 transporter from Rhodobacter sphaeroides (SLC26Rs, blue, PDB code 3oiz), which shares 50% of identical residues. (c) Superposition of the SLC26Dg STAS domain on a structure of the equivalent domain of rat Prestin that lacks a long unstructured loop connecting α1 and β312 (SLC26A5, blue, PDB code 3llo). (d) Superposition of the SLC26Dg STAS domain on the STAS domain ACP complex of the E. coli transporter DauA11 (PDB code 3ny7). The DauA STAS domain is colored in blue, ACP in green. ACP binds to a region on the C-terminus of the STAS domain of DauA that is not defined in the electron density of SLC26Dg. The binding interface does not overlap with the nanobody-binding epitope on the STAS domain of SLC26Dg.

Supplementary Figure 5 SLC26Dg-Nb5776 interactions.

(a) Ribbon representation of the nanobody Nb5776 (blue) and the STAS domain of SLC26Dg (red). Both proteins interact via a parallel β-sheet formed by β5 of Nb5776 (containing the variable region CDR2) and β4 of the STAS domain and via side-chain interactions with a long loop of the nanobody connecting β-strands 8 and 9 (part of the variable region CDR3, *) that has changed its conformation upon binding to the transport protein. (b) Side-chain interactions between the Nb5776 and the STAS domain of SLC26Dg. (c) Ribbon representation of the Nb5776 structure. (d) Superposition of Cα-traces of the isolated Nb5776 (beige) and of its structure observed in the complex (blue). The difference in the conformation of the CDR3 region is apparent. (e) Stereo view of 2Fo-Fc density (calculated at 2.4 Å and contoured at 1σ) of a Nb5776 crystal superimposed on the structure. An asterisk indicates the region of CDR3 that has changed its conformation upon binding to SLC26Dg. (f) Interactions of the nanobody and the STAS domain between symmetry-related molecules in the crystals of the SLC26Dg-Nb5776 complex. Transmembrane and STAS domains of SLC26Dg are colored in green and red respectively, the nanobody is colored in blue. (g) SLC26Dg structure placed in a model of a lipid bilayer (obtained from http://www.lobos.nih.gov/mbs/coords.shtml). The molecular surface of the protein is shown with polar residues colored in green, acidic residues in red and basic residues in blue. The observed conformation of SLC26Dg would place the hydrophilic STAS domain (indicated by brackets) within the hydrophobic core of the lipid bilayer.

Supplementary Figure 6 TM-domain structure and functional behavior of a mutant.

(a) Stereo view of the SLC26Dg transmembrane domain. The protein is shown as ribbon with its N-terminal half colored in green and its C-terminal half in beige. (b) Stereo view of a superposition of the pseudo-symmetry related N- and C-terminal halves of the SLC26Dg transmembrane domain. The protein sub-domains are shown as Cα representation, the sidechains of the two pseudo-symmetry related glutamates facing the intracellular aqueous cavity as sticks. (c) Model of the SLC26Dg transmembrane domain in a lipid bilayer. The molecular surface of the protein is displayed with aromatic residues colored in beige and hydrophobic residues in yellow. Lipids are shown as sticks. (d) Arrangement of molecules in the unit cell of the SLC26DgΔSTAS crystal. The crystal is of space group P1 and contains 4 copies of the transport domain. (e) Stereo view of 2Fo-Fc electron density (calculated at 4.2 Å, sharpened with b=90 and contoured at 1σ) superimposed on a Cα-trace of the SLC26DgΔSTAS structure. The model was improved by refinement maintaining strong structural constraints. (f) Transport properties of the SLC26Dg double mutant E38A E241A. Time dependent uptake of 14C-fumarate (136 μM) into proteoliposomes. ΔpH refers to a pH of 6.0 outside and 7.5 inside the liposomes, pH 7.5 refers to symmetrical pH conditions. (g) Concentration dependence of fumarate transport in the mutant E38A E241A. Left panel: quantification of the protein reconstituted in the proteoliposomes. Proteoliposomes were solubilized with 1% DDM and samples before and after ultracentrifugation were taken to determine the fraction membrane-inserted proteins. Densitometric analysis of the samples after ultracentrifugation indicates a 7 times lower protein concentration in proteoliposomes containing the mutant E38A E241A. Right panel: the solid curve shows a fit to a Michaelis-Menten equation with a linear component due to the passive uptake of fumarate into liposomes. The dashed line shows the slope of the linear component. Specific activities were calculated in comparison to WT based on a quantification of the reconstituted protein in proteoliposomes (left panel). Data in f and g represent mean and s.e.m of 3 technical replicates.

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Geertsma, E., Chang, YN., Shaik, F. et al. Structure of a prokaryotic fumarate transporter reveals the architecture of the SLC26 family. Nat Struct Mol Biol 22, 803–808 (2015). https://doi.org/10.1038/nsmb.3091

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