Every voltage-gated ion channel (VGIC) has a pore domain (PD) made from four subunits, each comprising an antiparallel transmembrane helix pair bridged by a loop. The extent to which PD subunit structure requires quaternary interactions is unclear. Here, we present crystal structures of a set of bacterial voltage-gated sodium channel (BacNaV) ‘pore only’ proteins that reveal a surprising collection of non-canonical quaternary arrangements in which the PD tertiary structure is maintained. This context-independent structural robustness, supported by molecular dynamics simulations, indicates that VGIC-PD tertiary structure is independent of quaternary interactions. This fold occurs throughout the VGIC superfamily and in diverse transmembrane and soluble proteins. Strikingly, characterization of PD subunit-binding Fabs indicates that non-canonical quaternary PD conformations can occur in full-length VGICs. Together, our data demonstrate that the VGIC-PD is an autonomously folded unit. This property has implications for VGIC biogenesis, understanding functional states, de novo channel design, and VGIC structural origins.
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Coordinates and structures factors for NaVAb1p (DM), NaVAb1p (bicelles), CaVSp1p (bicelles), NaVAe1/Sp1CTDp (DDM), NaVAe1/Sp1CTDp–SAT09, and NaVAe1/Sp1CTDp–SAT09 are deposited in the RCSB under accession codes NaVAb1p (DM) (PDB: 7PGG), NaVAb1p (bicelles) (PDB: 7PGI), CaVSp1p (bicelles) (PDB: 7PGF), NaVAe1/Sp1CTDp (DDM) (PDB: 7PGH), NaVAe1/Sp1CTDp:SAT09 complex (PDB: 7PGB), and NaVAe1/Sp1CTDp:ANT05 complex (PDB: 7PG8). Source data are provided with this paper.
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We thank K. Brejc, L. Jan, and T. Kortemme for comments on the manuscript; S. Wong for expert technical assistance in protein preparation. This work was supported by grants NIH-NHLBI R01-HL080050, NIH-NIDCD R01-DC007664, and the Program for Breakthrough Biomedical Research, which is partially funded by the Sandler Foundation to D. L. M., NIH-NIGMS R35 GM122603 to W. F. D., NIH-NIGMS R21-GM100224 and R01-GM137109 to M. G., NIH-NIGMS GM117372 to A. A. K., and an AHA postdoctoral fellowships to C. A. and M. L. This work is based on research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (Grant P30 GM124165). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357.
The other authors declare no competing interests.
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a-c, 2Fo-Fc Electron density (1σ) for NaVAb1p (firebrick) a, side view, b, extracellular view, and c, Single subunit. d, Exemplar NaVAb1p Composite OMIT map density (1σ). e, Superposition of NaVAb1p structures determined in detergent (firebrick) and bicelles (cyan). f, Comparison of lipid bound to the P1 helix in NaVAb1p (firebrick) and NaVAe1p (PDB 5HK7)18. This lipid is modeled as phosphoenthanolamine and the 2Fo-Fo density (1.0σ, marine mesh) shows a well-defined acyl chain sitting between the P1 and S6 helices of NaVAb1p. g-i, 2Fo-Fc Electron density (1σ) for CaVSp1p (cyan) g, side view, h, extracellular view, and i, Single subunit. j, Exemplar CaVSp1p Composite OMIT map density (1σ). k, Close up of the CaVSp1p S6 (marine)-Neck (olive) junction showing two subunits. Select residues are indicated. l-q, 2Fo-Fc Electron density (1σ) for NaVAe1/Sp1CTDp (marine). l-n, inside-outB tetramer l, side view, m, extracellular view, and n, single subunit. o-q, inside-outC tetramer o, side view, p, extracellular view, and q, single subunit.
Extended Data Fig. 2 Structure comparison of selectivity filters from canonical and non-canonical quaternary assemblies.
a, NaVAb1p (firebrick), b, CaVSp1p (cyan), and c, NaVAe1/Sp1CTDp inside-outC form(marine) compared with the NaVAe1p canonical structure (orange) (PDB:5HK7)18. Selectivity filter TrpIn and TrpOut conformations are indicated. d, NaVAb1p, CaVSp1p, and NaVAe1p sequence comparison. Residue numbers and positions of unified numbering scheme for the selectivity filter (SF) (-1 to +3) as defined by Shaya et al.11 are shown.
a, Superposition showing the rigid body movements that connect canonical NaVAe1p (orange), inside-outA NaVAb1p (firebrick), inside-outB NaVAe1/Sp1CTDp (pale green), and inside-outC NaVAe1/Sp1CTDp (marine) conformations. C-tails of each monomer are superposed. The selectivity filter of each monomer is magenta. All inside-out forms are related to NaVAe1p by a 45° rotation round the hinge followed by varied degrees of translation indicated by the arrow. Rotation around the Hinge parallel to the membrane is indicated. b, Extracellular view of ‘a’. Location of central ion conducting pore is indicated by the open circle. NaVAe1p tetramer is shown with one orange and three white subunits. Arrow shows the relationships among the inside-out forms. c, Superposition of the CTDs from NaVAe1/Sp1CTDp inside-outB (pale green) and inside-outC (marine) conformations with CaVSp1p (cyan). d, Details of the central core from ‘c’.
a, Minimum distance between Trp199 and Phe218 Cα positions during simulation. Inset shows exemplar Trp199 (pink) Trpin and Trpout conformations at 6.00 µs and 6.73 μs, respectively. Pink sphere indicates Phe218 Cα. b, Ser225 and Ser226 hydrogen bond to the S6 helix backbone. C, Quantification of hydrogen bond persistence given as the proportion of time spent hydrogen bonded in each of three simulation replicates for the Phe211-Ser225 and Ile222-Ser226 pairs.
a, 2Fo-Fc (2σ) electron density for sFab SAT09:NaVAe1/Sp1CTDp. sFab SAT09 light (limon) and heavy (yellow orange) chains and NaVAe1/Sp1CTDp (magenta) are indicated. b, 2Fo-Fc (1σ) electron density for sFab ANT05:NaVAe1/Sp1CTDp. sFab ANT05 light (aquamarine) and heavy (yellow) chains and NaVAe1/Sp1CTDp (orange) are indicated. c, Composite OMIT map density for SAT09:NaVAe1/Sp1CTDp. Colors are as in ‘a’. d, Sequence comparisons of the light chain (LC) and heavy chain (HC) CDRs (blue and green, respectively) for sFabs SAT09 (black) and ANT05 (grey). CDR sequences are shown in italics.
a, Superposition of NaVAe1/Sp1CTDp from the sFab SAT09:NaVAe1/Sp1CTDp complex and NaVAe1p from the canonical structure (PDB:5HK7)18. Channel elements are colored as follows, S5 (bright orange), SF (red), P1 and P2 helices (teal), S6 (marine), neck (olive) coiled-coil (forest). b, Superposition of NaVAe1/Sp1CTDp (marine) and NaVAe1/Sp1CTDp from the SAT09 complex (magenta). c, sFabSAT09:NaVAe1/Sp1CTDp complex asymmetric unit. d, Extracellular view of a sFabSAT09:NaVAe1/Sp1CTDp pentameric complex (top). e, Extracellular view of a NaVAe1/Sp1CTDp inside-outD. Channel elements are colored as in ‘A’. f, Superposition showing the rigid body movements that connect conformations of NaVAe1p (orange) and NaVAe1/Sp1CTDp from the sFabSAT09:NaVAe1/Sp1CTDp complex (marine). C-tails of each monomer are superposed. The selectivity filter of each monomer is magenta. Hinge is indicated. g, Extracellular view of ‘f’. Location of central ion conducting pore in NaVAe1p is indicated by the open circle. Arrow shows the NaVAe1p-NaVAe1/Sp1CTDp relationship. h, sFabSAT09:NaVAe1/Sp1CTDp complex contact map. Cα- Cα distances for (black) diagonal subunits at 20 Å and (red) neighboring subunits at 12 Å. Channel structural elements are indicated. Extracellular views of the PDs having channel elements colored as in ‘a’ are shown. Arrows indicate the diagonal (black) and neighbor (red) distance relations of the contact plots.
a, SEC-MALS chromatograms of 15 µM NaVAe1Sp1CTD purified in DDM. b, SEC-MALS chromatograms of 15 µM NaVAe1Sp1CTD in complex with 2.5-fold excess of sFab SAT09. The red and purple lines represents respectively the total molar mass and protein molar mass fitting results. c, Superimposition of NaVAe1Sp1CTD and SAT09:NaVAe1Sp1CTD SEC-MALS chromatograms from ‘a’ and ‘b’. Chromatograms for ‘a’ and ‘b’ are available as source data.
a, Extracellular view of the sFabANT05:NaVAe1/Sp1CTDp complex. sFabANT05 (green) is shown in cartoon and surface rendering. NaVAe1/Sp1CTDp (marine) is shown in cartoon rendering. b, Comparison of the binding modes of sFabANT05 (green) and sFabSAT09 (cyan) to NaVAe1/Sp1CTDp. NaVAe1/Sp1CTDp from the sFabANT05 complex is marine. NaVAe1/Sp1CTDp from the sFabSAT09 complex is cyan.
a, Superposition of the sFabSAT09 complex (solid yellow, green SAT09 and marine cylinders NaVAe1/Sp1CTDp on the NaVAe1p (space filling, white) canonical structure (PDB:5HK7)18 showing three of the four subunits. Selectivity filter (SF) region of NaVAe1p is colored red. b, Superose 6 10/300 FSEC profiles of K2P2.1(TREK-1)cryst-GFP alone (black) and with SAT09 (green). DDM-solubilized fraction from K2P2.1(TREK-1)cryst-GFP expressing Pichia pastoris cells (100 µl) was incubated with 1 nmol of sFab SAT09. c, SEC-MALS chromatograms of 9 µM NaVSp1 reconstituted in amphipol A8-35. d, SEC-MALS chromatogram of 9 µM NaVSp1-SAT09 complex reconstituted in amphipol A8-35 and taken after purification of the complex on Superose 6. e, Superimposition of NaVSp1 (dashed line) and SAT09:NaVSp1 (black) SEC-MALS chromatograms from ‘c’ and ‘d’. Chromatograms for ‘b-e’ are available as source data.
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Arrigoni, C., Lolicato, M., Shaya, D. et al. Quaternary structure independent folding of voltage-gated ion channel pore domain subunits. Nat Struct Mol Biol 29, 537–548 (2022). https://doi.org/10.1038/s41594-022-00775-x