We expressed full-length sp9C1 in mammalian cells as an eGFP fusion construct. Purification of the protein in digitonin micelles yielded biochemically monodisperse protein which was used for single particle cryoEM imaging (Extended Fig. 1a-c). We will first describe the overall architecture of sp9C1 obtained at pH 8 with K+ as the dominant cation which yielded a final reconstruction map at GS-FSC resolution of 3.8 Å (Extended Fig. 1d, e)
The EDs of the two subunits pack against each other (Figs. 1a-c, Supplementary Video 1) forming the core of the protein. Each ED comprises 13 transmembrane helices (TM1 through TM13), organized in a manner similar to other members of the SLC9 family (Extended Fig. 2a, b). Seven of the thirteen transmembrane helices (TMs 1–3 and 7–10) form the ‘dimerization domain’ and the remaining six (TMs 4–6 and 11–13) constitute the ‘core domain’ (Extended Fig. 2c). A wide, cytoplasmically-accessible funnel, lined with negatively charged residues, is observed between the dimerization and core domains of the ED (Extended Fig. 2c and d). The tip of the funnel features conserved residues poised to bind a Na+ ion21. Thus, in our structure the ED is in an “inward open” state. Despite the structural similarity at an individual subunit level, the dimeric organization of the two EDs in sp9C1 is markedly different from other SLC9s (Extended Fig. 2e). TM10 of the two subunits of sp9C1, which line the inter-subunit cavity, tilt further away from each other by ~ 12º causing their extracellular ends to splay apart by an additional ~ 12Å, thus widening the crevice at the ED-dimer interface. In all our reconstructions, we were able to reliably identify densities for three lipid and four detergent molecules in this extracellularly accessible cavity (Fig. 1c). Lipid occupancy of this cavity might determine the stability of the sp9C1 dimers and act as clamps, holding the dimerization domain in place as the core helices move during the gating cycle of the ED15,18,22,23.
The VSDs (each comprising helices S1 through S4) are structurally estranged from the EDs, appearing as “floating buoys” on either side of the ED-dimer interface. At the closest, the VSD (extracellular end of S1) is ~ 22Å away from the ED (extracellular end of TM7) of the same subunit (Fig. 1c). This arrangement is in striking contrast to VGICs where the VSDs form intimate contacts with the ion translocating pore domain and the shared interfaces profoundly impact voltage-dependent channel opening24. The lack of any direct contact between the VSD and the ED in sp9C1 indicates that the allosteric mechanism underlying its voltage-regulation is different from that in VGICs. It is noteworthy that while the local resolution of our density map (FSC = 0.5) reaches ~ 3.2Å in the core of the protein, it drops to 4–6Å in the peripheral regions, including the VSD (Extended Fig. 1e). This is probably due to its lose packing with the protein core which leads to its higher structural flexibility. Nevertheless we were able to reliably trace the backbone of most of the VSD helices (Extended Fig. 3a, b). The S4 helix of sp9C1-VSD, which is critical for its voltage-sensitivity, has six regularly spaced positively charged residues. The intracellular end of S4 forms a 9-residue-long 310-helix harboring three positive charges (R809, R812, K815) which are highly conserved in different sp9C1 orthologs (Extended Fig. 3c). The remaining three positive charges (K800, R803 and R806) are arranged on the extracellular, α-helical end of S4. These three residues are relatively less conserved, particularly in the mammalian variants of SLC9C1, which could contribute to functional divergence in their voltage-sensitivities. The limited resolution of the VSD precludes unambiguous deduction of its conformational state. However, given the strong hyperpolarizing voltages necessary to drive the sp9C1-VSD into its resting (or down) state19, it is likely that in our structure (at 0mV) it is in the activated (or up) conformation.
In each sp9C1 subunit, the CNBD hangs ~ 30Å below the inner leaflet of the membrane. It has an evolutionarily conserved fold comprising an 8-stranded β-jelly roll (βR1) flanked on the N-terminus by the αA helix and on the C-terminus by αB and αC helices. Unexpectedly, the CNBD is C-terminally linked to a second 8-stranded β-jelly roll (βR2) via an α-helical linker (αD helix) (Extended Fig. 3d). Extensive hydrophobic contacts and a possible cation-π interaction, mediated by conserved residues (W1091 and R1185), are observed at the interface between βR2 and αC helix (Extended Fig. 3d,e). The βR2s of the two subunits are structurally apposed against each other, and together with the CNBDs, form a “cytoplasmic cuff”. CNBDs of cyclic nucleotide modulated ion channels do not feature a comparable βR2 domain25–27. While the specific functional role of βR2 is unclear, we will presently discuss a possible role of the βR2 in guiding cyclic nucleotide dependent conformational changes in sp9C1.
A distinct scaffolding domain, which we refer to as the ‘allosteric ring domain’ (ARD), directs the three dimensional arrangement of the ED, VSD and CNBD in the context of the sp9C1 dimers (Fig. 1d). The ARD fills the gap between the cytoplasmic surface of the transmembrane domains and the CNBDs. It is formed by the association of the polypeptide segments connecting the ED and VSD (the Exchanger-Voltage-sensor Linker or EVL) and the VSD and CNBD (the Voltage-sensor-CNBD linker or VCL). Each of the linkers are shaped like two paddles, annotated as PN< X> or PC< X> (where N and C designate the N-terminal or C-terminal paddle and X is either EVL or VCL) connected by a flexible hinge. Inter-subunit interactions between PNEVL and PCVCL and intra-subunit contacts between PCEVL and PNVCL drive the assembly of the ARD. In addition, the ARD interacts intimately with the βR1 of CNBDs – PNEVL is nestled in a groove formed between PCVCL and βR1 of the neighboring subunit while PNVCL rests atop of the βR1 of the same subunit (Fig. 1d and Supplementary Video 1). The overall arrangement of the cytoplasmic domains and ARD of sp9C1 is similar to the SOS1 transporter17, which is a plant ortholog of SLC9s, although the latter does not feature a VSD and is not regulated by cAMP.
The characteristic arrangement of the ARD suggests that it may play an important role in the assembly of the sp9C1 dimers. To test this, we compared the stabilities of full length (FL) sp9C1 with 3 truncation mutants (named 950, 657 and 494 referring to the amino acid positions of the truncation sites) using Fluorescence Size Exclusion Chromatography (FSEC)28,29. All 4 proteins, expressed as eGFP fusion constructs and affinity purified in digitonin micelles, were largely monodisperse with retention volumes consistent with a dimeric assembly (Fig. 1e). However, when exchanged into mildly destabilizing conditions, a second peak, corresponding to a monomer, was observed. Constructs truncated at 657 and 494 were drastically more unstable than FL-sp9C1 as reflected by a much faster and greater extent of disassembly over a 10hr period. In both these constructs the ARD is partly or entirely deleted. Thus the intra- and inter-subunit interactions at the level of the ARD are critical for stability of the sp9C1 dimers. The 950 construct, in which the CNBD, βR2 and the C-terminal tail was deleted, also exhibited elevated breakdown relative to the full-length protein. Thus the closed cuff arrangement of the cytoplasmic domains also contributes to dimeric stability, either via direct inter-subunit interactions between the βR2 domains or by facilitating a stable arrangement of the ARD via the ARD-CNBD interfaces.