Molecular architecture and the structural basis for anion interaction in prestin and SLC26 transporters

Abstract

Prestin (SLC26A5) is a member of the SLC26/SulP anion transporter family. Its unique quasi-piezoelectric mechanical activity generates fast cellular motility of cochlear outer hair cells, a key process underlying active amplification in the mammalian ear. Despite its established physiological role, it is essentially unknown how prestin can generate mechanical force, since structural information on SLC26/SulP proteins is lacking. Here we derive a structural model of prestin and related transporters by combining homology modelling, MD simulations and cysteine accessibility scanning. Prestin's transmembrane core region is organized in a 7+7 inverted repeat architecture. The model suggests a central cavity as the substrate-binding site located midway of the anion permeation pathway, which is supported by experimental solute accessibility and mutational analysis. Anion binding to this site also controls the electromotile activity of prestin. The combined structural and functional data provide a framework for understanding electromotility and anion transport by SLC26 transporters.

Figure 1. Structural model of prestin derived by homology modelling and MD simulations.

(a) Overall structure of the rPres TMD as derived from the MD simulation seen from the side (upper panel) and from the extracellular space (lower panel). Colours indicate the two inverted repeats (repeat TM1–TM7 in green, repeat TM8–TM14 in magenta). (b) Same prestin model, highlighting core (pink) and gate (blue-violet) domains, that were initially identified and defined for the UraA structure. (c) Localization of the protein regions NLC1 (red) and NLC2 (orange) that have been implicated in the generation of electromotility and anion selectivity. Although distant in the primary sequence (see also Fig. 2) both regions interact closely, with their intertwined TMs building the core of the molecule. Specifically, these domains comprise the symmetry-related TMs 3 and 10 that are unusual in featuring central antiparallel β-sheets that contribute to substrate binding in UraA.

Figure 2. Schematic representation of the topology and structural motifs of rPres.

Cylinders represent helices, arrows represent the two β-strands β3 and β10 in TM3 and TM10. Protein regions NLC1 and NLC2 are highlighted in red and orange, respectively. Topological localization of repeats I and II and of core and gate domains are indicated. Each repeat can be superimposed onto the other (inverted) repeat by a 180° rotation around a symmetry axis parallel to the membrane. Black numbers refer to amino-acid positions in the rPres sequence. Cytoplasmic C and N termini not drawn to scale.

Figure 3. Topology determination by substituted cysteine accessibility scanning.

(a) rPres_ΔCys(R236C) as an example for an amino-acid position accessible from the extracellular side. NLC was measured in the whole-cell patch-clamp configuration while MTSES or MTSET (1 mM) were applied to the extracellular side via an application capillary (left panel). Changes in V_1/2 during the application (middle panel) indicate covalent modification of cysteine 236. Representative NLC traces before (black) and after application of MTSES (red) or MTSET (blue) are shown in the right panels. (b) rPres_ΔCys(S465C) as an example for an amino-acid position accessible from the intracellular side. Extracellular application of either MTSES or MTSET was totally ineffective (upper panels). Application of MTSES or MTSET (1 mM) to the intracellular side via the patch pipette (lower panels) abolished NLC with a time course consistent with diffusion of the reagents into the cytoplasm. (c) Summary of MTS modification results for all cysteine-substituted mutants obtained as in a and b. Data obtained with MTSES and MTSET are shown in red or blue, respectively. Black bars indicate control experiments without application of MTS reagents. The upper panel shows the absolute values of V_1/2 shifts on application of MTSES or MTSET. For clarity, effects of extracellular application are plotted upwards and effects of intracellular application are plotted downwards. The lower panel shows relative (rel) changes in NLC amplitude on application of MTS reagents from the same set of experiments. As in the upper panel, amplitude changes in response to extracellular application are plotted upwards and effects of intracellular application are plotted downwards. Detailed data for each position are presented in Supplementary Fig. 4. Data are given as mean±s.e.m. with n≥5 experiments for each condition. (d,e) Schematic and structural representation of the results, showing the location of all examined amino-acid positions as predicted by the model. Positions accessible to MTS compounds from the extracellular side are coloured in green, those accessible from the intracellular side are coloured yellow.

Figure 4. The anion translocation pathway.

(a) TM segments defining the central cavity. A uracil molecule is deliberately positioned corresponding to its location in the UraA crystal structure. (b) TMD cavities identified by analysis of solvent trajectories are shown in red (cavities accessible from cytoplasm, IC) and blue (accessible from extracellular solution, EC). The intracellular access pathway to the central cavity (yellow dashed line) was identified as a continuous solvated channel with a diameter compatible with passage of oxalate. (c) Intracellular view of the rPres model coloured according to TM segment distances from the anion pathway. Green helices are close to the pathway, distant helices are shown in grey. (d) Substituted cysteine accessibility confirms the model’s prediction of an inward-open anion access pathway for rPres. Shown is a lateral view onto the core domain summarizing the results of a SCAM scan along the entire TM10. Portions of the TM domain of rPres are not shown to enable view onto TM10. Experimental accessibility of TM10 residues to MTS reagents is indicated by colour code. Intra- and extracellularly accessible positions are highlighted in red and blue, respectively. Yellow positions were inaccessible from both sides. TM10 mutants at positions shown in grey were dysfunctional and could not be tested for accessibility. Original data are shown in Supplementary Fig. 6.

Figure 5. Identification of a candidate central binding site.

(a) Structural superposition of rPres (red) and UraA (blue). TM3 and TM10 are rendered in ribbon style, with antiparallel β-strands β3 and β10 as arrows. R399, S398 and Glu290 are highlighted, demonstrating the structural correspondence of Ser398 (rPres) to Glu290, which is involved in substrate binding in UraA. (b) Alignment of TM10 of UraA with various vertebrate prestins and invertebrate homologues (zPres=zebrafish prestin; ciSLC26, cePres and dmPres denote homologues from Ciona intestinalis (ciSLC26aα), Caenorhabditis elegans (Uniprot Acc. Q3LTR4) and Drosophila melanogaster (Q9VVM6), respectively). Sequence alignment with UraA is according to the structural superposition shown in a.

Figure 6. Mutations in the central cavity affect NLC and anion transport.

(a) Representative NLC recordings from the rPres mutants indicated. (b) Mean NLC amplitudes of rPres WT and R399 mutants. (c) Reduced oxalate (ox) sensitivity of NLC in the S398C mutant. Representative NLC traces from cells first recorded with standard pipette solutions (black) and subsequently repatched with a pipette solution containing additional 10 mM oxalate (green). (d) Mean fold change in NLC amplitude obtained as in c. (e) rPres R399S is insensitive to salicylate. Representative NLC recordings measured in control (contr) conditions (black) and from the same cells during application of 10 mM extracellular salicylate (blue). (f) Average fold changes in peak NLC amplitudes obtained from experiments as in e. (g) Representative transport currents from cPres and the mutants indicated in the absence of divalent substrates (black) and during subsequent application of extracellular 10 mM sulphate (red) or 10 mM oxalate (green). (h) Average transport currents of cPres-binding site mutant, normalized to cell size. Colour coding as in g. (i) Altered divalent selectivity of S404 mutants. Data were obtained from experiments as shown in g. The number of experiments (individual cells) is given in all bar graphs. Error bars indicate s.e.m. All described rPres and cPres mutants showed normal membrane localization, indicating intact folding and processing of the protein.

Figure 7. Solvent accessibility of an amino acid in the proposed central binding site.

(a) Upper panels, application of extracellular MTSES (0.1 mM; red) but not MTSET (1 mM; blue) to the extracellular side rapidly inhibits transport currents by cPres(S404C), indicating accessibility of this site. Lower panels, intracellular MTSET inhibits transport. Currents in the left panels are shown normalized to initial current (black). (b) Summary plot of remaining currents after application of MTS reagents obtained as in a. WT cPres was insensitive to both reagents (see Supplementary Fig. 10). Error bars denote s.e.m. and the number of experiments is indicated above bars. (c) Representative NLC traces from rPres_ΔCys(S398C) obtained before (black) and after application of 1 mM MTSES (red) or MTSET (blue) from the extracellular (upper panels) or intracellular (lower panels) side. (d) Summary plot of NLC changes after application of MTS compounds obtained as in c. Upper panel shows fold changes of peak NLC. Lower panel presents MTS-induced changes of V_1/2. Error bars denote s.e.m. (e) Central location of residue S398 (green sphere) according to the structural model. Core (pink) and gate domains (blue-violet) indicated by colours. (f) Working model for alternating access transport mechanism consistent with the dual accessibility of position S404 in cPres. Alternate accessibility of the substrate-binding site may result from rotational movement between gate and core domains as hypothesized for UraA. Shading of outward-facing conformation (E_o) symbolizes lack of extracellular accessibility of homologous position S398 in rPres, suggesting that outward-out conformation may not be reached.