Abstract
Secondary transporters use alternating-access mechanisms to couple uphill substrate movement to downhill ion flux. Most known transporters use a 'rocking bundle' motion, wherein the protein moves around an immobile substrate-binding site. However, the glutamate-transporter homolog GltPh translocates its substrate-binding site vertically across the membrane, through an 'elevator' mechanism. Here, we used the 'repeat swap' approach to computationally predict the outward-facing state of the Na+/succinate transporter VcINDY, from Vibrio cholerae. Our model predicts a substantial elevator-like movement of VcINDY's substrate-binding site, with a vertical translation of ~15 Å and a rotation of ~43°. Our observation that multiple disulfide cross-links completely inhibit transport provides experimental confirmation of the model and demonstrates that such movement is essential. In contrast, cross-links across the VcINDY dimer interface preserve transport, thus revealing an absence of large-scale coupling between protomers.
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Acknowledgements
We thank A. Banerjee for helpful discussions and M. Maduke, J. Faraldo-Gómez, G. Rudnick and M. Mayer for critical review of the manuscript. A.V.-J. is supported as a recipient of the L'Oreal Chile–United Nations Educational, Scientific and Cultural Organization (UNESCO) Women in Science Fellowship and the L'Oreal-UNESCO Rising Talent Award. This work was supported by the Division of Intramural Research of the US National Institutes of Health, National Institute of Neurological Disorders and Stroke.
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L.R.F. and J.A.M. conceived the project. C.F.-F., A.V.-J. and D.K. carried out computational modeling, and C.F.-F. and L.R.F. analyzed data and directed computational modeling efforts. J.A.M. and C.M. designed and planned experiments. C.M. performed experiments and supervised G.A.F., who performed dimer-interface experiments. Y.L. performed MS and interpreted MS data. C.M., C.F.-F., L.R.F. and J.A.M. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Sequence alignment for modeling.
(a) Refined sequence alignment between the template (i.e., reordered X-ray crystal structure), and the normal sequence of VcINDY, which corresponds to the repeat-swapped model. The percentage of identical residues is ~24% for the full-length alignment, including peripheral elements; within the repeats, ~20% of the residues are identical. The sequence range of each repeat in the model is indicated by rectangles under the target sequences, colored as in Fig. 1a. The secondary structure (helix) assignment obtained with DSSP for the X-ray structure is indicated by dark blue rectangles. Amino acids are colored as followed: red for acidic (asp and glu), dark blue for basic (his, lys, and arg), cyan for polar (ser, thr, asn, and gln), yellow for aromatic (tyr, trp, and phe), pink for helix-breaking (pro and gly), and pale yellow for cysteine and aliphatic (ala, val, ile, leu, and met). (b) Schematic indicating which structural repeat segments (colored as in (a)) are modeled on which segments of the crystal structure template. The peripheral helices (TM 1) in the model and template are shown in gray.
Supplementary Figure 2 Crystal structure of VcINDY compared with the repeat-swapped model.
(a) The X-ray crystal structure of a VcINDY protomer in an inward-facing conformation (left) is compared with the repeat-swapped protomer model in an outward-facing conformation (right). Structures are viewed along the plane of the membrane, with the extracellular side at the top. The Bendix plugin [Dahl A.C.E., Chavent M., Sansom M.S.P., Bendix: intuitive helix geometry analysis and abstraction. Bioinformatics 2012;28:2193–4] to the program VMD [Humphrey, W., Dalke, A. and Schulten, K., VMD - Visual Molecular Dynamics, J Molec Graphics 1996;14:33-38] was used to represent the helices. To quantify the conformational change of the protein, the rotation axis, rotation angle, and displacement of the helices were evaluated. The black line indicates the rotation axis, which lies approximately perpendicular to the symmetry axis. The displacement values suggest an elevator-like movement of the transport domain containing the substrates (spheres) across the membrane, and this displacement is associated with a rotation of 43°. (b) Displacement per residue was determined as the distance between Cα atoms in the crystal structure and the model (black line) after aligning the structures using the scaffold domain. Note that the model built prior to adding intradomain restraints, while using the same refined alignment (“Refined model”, red line) implies a similar conformational change in the transport domain relative to the scaffold as the model built using intradomain restraints (“Final model”, black line). By this analysis, the transport domain can be considered to comprise helices HP1-TM5-TM6 and HP2-TM10-TM11.
Supplementary Figure 3 Activity and stability of cysteine mutants.
(a) Initial rates of 3H-succinate transport by wild type VcINDY (VcINDYwt) and cysteine-free VcINDY (Cysless) after reconstitution into liposomes. Triplicate datasets were used and error bars represent S.E.M. (b) Size exclusion chromatography (SEC) traces showing the normalized absorbance at A280 for Cysless and the 4 double cysteine mutants; A120C V165C (red), A346C V364C (yellow), T154C V272C (brown), and L60C S381C (light blue). V0 is ~8 ml and the VcINDY elution peak is centered on 12.5 ml on the S200 SEC column.
Supplementary Figure 4 Inward-stabilizing cross-links abolish the transport activity of VcINDY.
(a) Cartoon representation of the dimeric VcINDY inward-facing crystal structure (left) and the outward-facing model (right) viewed from the membrane plane. Substrate and Na+ are shown as yellow and pink spheres. Cα-atoms of the inward-stabilizing residues are shown as black spheres (left-hand side protomers). (b) SDS-PAGE band-shift assay showing the number of free cysteines present in L60C S381C with (+) and without (-) prior treatment with HgCl2 (left panel) or CuPhen (right panel). The following protein species seen in the gels are indicated by colored arrows; unmodified VcINDY (red arrow), dimeric VcINDY (orange), singly PEGylated VcINDY (blue), and doubly PEGylated VcINDY (magenta). (c) Normalized initial rates of [3H]-succinate transport in the presence of proteoliposomes containing Cysless and L60C S381C mutant after treatment with (+) and without (-) HgCl2 and DTT. Results from triplicate datasets are shown and error bars represent S.E.M. These assays were repeated twice with the same outcome.
Supplementary Figure 5 Chemical cross-linking with CuPhen protects outward-stabilizing cysteine pairs from mPEG5K labeling.
SDS-PAGE band-shift assay showing the number of free cysteines present in Cysless and the three double cysteine mutants; A120C V165C, T154C V272C, andA346C V364C, with (+) and without (-) prior treatment with CuPhen. The following protein species seen in the gels are indicated by colored arrows; unmodified VcINDY (red arrow), dimeric VcINDY (orange arrow), singly PEGylated VcINDY (blue arrow), and doubly PEGylated VcINDY (magenta arrow). This experiment was repeated twice with the same outcome.
Supplementary Figure 6 Effects of cross-linkers and reducing agent on the oligomeric state and transport activity of cysteineless VcINDY and cysteine mutants.
SDS-PAGE of reconstituted single and double cysteine mutant following treatment with (+) and without (-) HgCl2 (a) and CuPhen (b). Proteoliposomes were treated with the highest concentrations of both crosslinkers used in other assays; 50 μM HgCl2 and 0.5 mM 2:1 CuPhen. Prior to loading the samples on the non-reducing SDS-PAGE gels, the samples were treated with 2% (w/v) SDS and 500 mM MMTS to protect the single cysteines from crosslinking in the gel upon denaturation. The expected positions of monomeric and dimeric VcINDY are indicated by arrows. (c-d) Initial rates of 3H-succinate transport into proteoliposomes containing cysless VcINDY in the presence of increasing concentrations of DTT (c) and HgCl2 (d). Results from triplicate datasets are shown; error bars represent S.E.M. Each experiment was performed a single time.
Supplementary Figure 7 Comparison of the models generated with and without intradomain restraints with the VcINDY X-ray structure (PDB 4F35).
(a) Cartoon representation of the model obtained before applying distance restraints within the scaffold and transport domains (“refined model”) and the model obtained after applying restraints (“final model”) and the VcINDY structure used as template. Note that the alignment used to build both models is the same. The repeat-swapped models were built and refined as protomers, and then duplicated to construct the dimer model (see Methods). Therefore individual protomers are shown here, though the final model is a dimer. The coloring scheme is that used in the transmembrane definition and the substrate mimic (citrate) and one sodium ion are shown as green, red, and purple spheres. (b) The coloring scheme reflects the conservation level identified using ConSurf [Ashkenazy H, Erez, E., Martz, E., Pupko, T. and Ben-Tal, N., ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 2010;38:W529–W533].
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–7 and Supplementary Table 1 (PDF 1962 kb)
Supplementary Data Set 1
Raw gel images from all figures (PDF 285 kb)
Supplementary Data Set 2
Model of VcINDY in the outward-facing conformation (TXT 505 kb)
Predicted transport-associated conformational change in VcINDY
The conformational change predicted for VcINDY is modeled by morphing the VcINDY inward-facing X- ray structure (PDB ID: 4F35) (start) with the repeat-swapped model of the outwardfacing conformation (end point). The interpolation between structure and model was done using the Morph2 server (http://molmovdb.mbb.yale.edu/molmovdb/morph/). VcINDY is represented as cartoon helices and the coloring scheme reflects the division between scaffold (blues), oligomerization domain (dark blue) and transport domain (orange), as shown in Figure 1. The sodium ion and the citrate are shown as purple and yellow spheres, respectively. The protein is viewed from the plane of the membrane, first, from the perspective of the other protomer, and secondly, along the dimer axis. (MPG 91127 kb)
Cross-linking during the transport-associated conformational change in VcINDY
See legend to Supplementary Movie 1 for more detail. Here, the Cβ atom of each of the residue pairs that crosslink in the outwardfacing conformation is also shown as a sphere, color-coded by crosslinking pair as follows: Ala120 and Val165 in magenta, Thr154 and Val272 in green and Ala346 and Val364 in red. (MPG 28117 kb)
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Mulligan, C., Fenollar-Ferrer, C., Fitzgerald, G. et al. The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanism. Nat Struct Mol Biol 23, 256–263 (2016). https://doi.org/10.1038/nsmb.3166
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DOI: https://doi.org/10.1038/nsmb.3166
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