The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanism

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.

Figure 1. Repeat-swap modeling of VcINDY

(a) Schematic representation of the topology of VcINDY colored according to the structural repeats. The blue and cyan helices comprise repeat unit 1 (RU1), while repeat unit 2 (RU2) is composed of the red and orange helices. (b) Cartoon representation of the X-ray crystal structure of a VcINDY protomer showing that RU1 is related to RU2 by two-fold pseudo-symmetry, with the symmetry axis parallel to the membrane. The black arrow represents the symmetry axis within the structure of the VcINDY protomer, viewed from within the plane of the membrane (left) and from the extracellular side of the protein (right). (c) A cartoon representation showing a structural alignment, built with TM-Align, of the repeats with the helices colored according to the topology in (a). The initial sequence alignment used to build a swapped-repeat model was generated based on this structural alignment.

Figure 2. Identification of VcINDY transporter domains

(a) Topology diagram of a VcINDY protomer colored according to the helices forming the scaffold (light blue), oligomerization (dark blue) and transport (orange) domains. Gray triangles indicate RU1 (down triangle) and RU2 (up triangle), respectively. (b) Cartoon representation of the dimeric VcINDY X-ray crystal structure in an inward-facing conformation (left) and the model in an outward-facing conformation (right) viewed from the membrane plane. Substrate and Na⁺ are shown asspheres, with their pathways indicated by gray arrows. Cα-atoms of the residues studied by crosslinking are shown as spheres colored in pairs (left-hand side protomers). (c) Surface representation of the VcINDY X-ray crystal structure (left) and model (right) viewed from the extracellular side. The substrate (yellow spheres, indicated by arrows) is visible from the extracellular side in the model (right), but not in the inward-facing crystal structure (left).

Figure 3. Chemical crosslinking of outward-stabilizing cysteine pairs

SDS-PAGE band-shift assay showing the number of free cysteines present in Cysless and the three double cysteine mutants; A120C V165C, T154C V272C, and A346C V364C, with (+) and without (−)prior treatment with HgCl₂. Relative positions of the cysteine pairs are shown in the cartoon representation of a VcINDY protomer (top). 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). Non-reducing SDS-PAGE gels were used and protein was visualized using Coomassie dye. The assay was performed on at least two separate occasions with the same outcome.

Figure 4. Mass spectrometric identification of crosslinked peptides

Representative LC-MS/MS spectra of disulfide-linked peptides detected from the digests of CuPhen-treated A120C V165C (a) and T154C V272C (b). Collision-induced dissociation (CID) spectrum of the disulfide linked peptide(inset) from the proteolytic digests (left), and the associated extracted ion chromatogram (XIC, right) for protein treated with crosslinking reagent (black line) or maintained in reducing conditions (red line). This experiment was repeated twice with separately prepared and treated protein. The annotation “i:j represents fragments from internal cleavage. For example y_2:5 represents the peptide fragment from the 2^nd to 5^th residue; “*” represents fragments with a neutral loss of water.

Figure 5. Stabilizing VcINDY in the outward-facing state abolishes transport

Normalized initial rates of [³H]-succinate transport in the presence of proteoliposomes containing Cysless and the three double cysteine mutants compatible with the outward-facing state after treatment with(+) and without (−) HgCl₂ and DTT. Relative positions of cysteine pairs are shown as in Fig. 3. Results from triplicate datasets are shown and error bars represent S.E.M. This experiment was repeated twice with fresh preparations of proteoliposomes.

Figure 6. Constraining the dimer interface has minimal effects on transport

(a) Surface representation of a VcINDY dimer viewed from the plane of the membrane (left) and a VcINDY protomer viewed from the dimer interface (right), if the VcINDY dimer is opened like a book. Cylinders represent the interfacial α-helices that make the intra-protomer contacts across the dimer interface. The colored spheres represent the positions of cysteine residues introduced to staple the VcINDY protomers together. (b) Coomassie-stained SDS-PAGE gels of purified cysteine mutants in detergent solution (‘D’)and His-tag Western blot analysis of VcINDY-containing proteoliposomes (‘PL’) with (+) and without (−)treatment with CuPhen. In the absence of CuPhen treatment (−), disulfides were prevented by addition of 1 mM DTT. The positions of monomeric and dimeric VcINDY are indicated. (c) Normalized initial transport rates of Cysless and indicated cysteine mutants with (+) and without (−) treatment with CuPhen (concentration of CuPhen used is indicated in the figure). Results are from at least triplicate datasets and error bars represent S.E.M. Western blots were performed three separate times and transport assays were repeated at least twice with the same outcome.

Figure 7. Proposed elevator-type transport mechanism in the VcINDY dimer

Cartoon representation of the transport mechanism inferred from the inward-facing crystal structure and outward-facing model of VcINDY. Blue shapes represent the scaffold and oligomerization domains and the orange shape is the transport domain. Substrates are represented by yellow spheres (succinate) and pink spheres (Na⁺ ions). In our scheme, the substrates bind the outward-facing state of VcINDY (left, model) at which point the transport domain undergoes a ~15 Å translocation and ~43° rotation into the inward-facing state (right, crystal structure), where substrate can be released into the cytoplasm. Empty transporter must then recycle back to the outward-facing state to restart the cycle.