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Quaternary structure independent folding of voltage-gated ion channel pore domain subunits

Nature Structural & Molecular Biology volume 29, pages 537–548 (2022)Cite this article

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Abstract

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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Fig. 1: Structures of BacNaV PDs.
Fig. 2: Exemplar structural homologs of the VGIC-PD fold, identified using DALI30,31.
Fig. 3: BacNaV PD structural relationships and CTD comparisons.
Fig. 4: BacNaV PD contact maps.
Fig. 5: BacNaV PD monomer is stable in a bilayer.
Fig. 6: sFab SAT09 recognizes the BacNaV SF.
Fig. 7: Evidence for non-canonical PD quaternary structure in full-length BacNaVs.

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Data availability

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.

References

  1. Yu, F. H. & Catterall, W. A. The VGL-chanome: a protein superfamily specialized for electrical signaling and ionic homeostasis. Sci. STKE 2004, re15 (2004).

    Article  PubMed  Google Scholar 

  2. Isacoff, E. Y., Jan, L. Y. & Minor, D. L. Jr Conduits of life’s spark: a perspective on ion channel research since the birth of neuron. Neuron 80, 658–674 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Zhao, Y., McVeigh, B. M. & Moiseenkova-Bell, V. Y. Structural pharmacology of TRP channels. J. Mol. Biol. 433, 166914 (2021).

  4. Catterall, W. A., Wisedchaisri, G. & Zheng, N. The chemical basis for electrical signaling. Nat. Chem. Biol. 13, 455–463 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Jin, X. et al. Targeting two-pore channels: current progress and future challenges. Trends Pharmacol. Sci. 41, 582–594 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Natale, A. M., Deal, P. E. & Minor, D. L.Jr. Structural insights into the mechanisms and pharmacology of K2P potassium channels. J. Mol. Biol. 433, 166995 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Chakrapani, S., Sompornpisut, P., Intharathep, P., Roux, B. & Perozo, E. The activated state of a sodium channel voltage sensor in a membrane environment. Proc. Natl Acad. Sci. USA 107, 5435–5440 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Li, Q. et al. Structural mechanism of voltage-dependent gating in an isolated voltage-sensing domain. Nat. Struct. Mol. Biol. 21, 244–252 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Li, Q., Wanderling, S., Sompornpisut, P. & Perozo, E. Structural basis of lipid-driven conformational transitions in the KvAP voltage-sensing domain. Nat. Struct. Mol. Biol. 21, 160–166 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Shaya, D. et al. Voltage-gated sodium channel (NaV) protein dissection creates a set of functional pore-only proteins. Proc. Natl Acad. Sci. USA 108, 12313–12318 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Shaya, D. et al. Structure of a prokaryotic sodium channel pore reveals essential gating elements and an outer ion binding site common to eukaryotic channels. J. Mol. Biol. 426, 467–483 (2014).

    Article  CAS  PubMed  Google Scholar 

  12. McCusker, E. C. et al. Structure of a bacterial voltage-gated sodium channel pore reveals mechanisms of opening and closing. Nat. Commun. 3, 1102 (2012).

    Article  PubMed  CAS  Google Scholar 

  13. McCusker, E. C., D’Avanzo, N., Nichols, C. G. & Wallace, B. A. A simplified bacterial “pore” provides insight into the assembly, stability and structure of sodium channels. J. Biol. Chem. 286, 16386–16391 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Payandeh, J. & Minor, D. L. Jr Bacterial voltage-gated sodium channels (BacNas) from the soil, sea, and salt lakes enlighten molecular mechanisms of electrical signaling and pharmacology in the brain and heart. J. Mol. Biol. 427, 3–30 (2015).

    Article  CAS  PubMed  Google Scholar 

  15. Chatterjee, S. et al. The voltage-gated sodium channel pore exhibits conformational flexibility during slow inactivation. J. Gen. Physiol. 150, 1333–1347 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Saha, S. C. et al. Characterization of the prokaryotic sodium channel NavSp pore with a microfluidic bilayer platform. PLoS ONE 10, e0131286 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Bagneris, C. et al. Role of the C-terminal domain in the structure and function of tetrameric sodium channels. Nat. Commun. 4, 2465 (2013).

    Article  PubMed  CAS  Google Scholar 

  18. Arrigoni, C. et al. Unfolding of a temperature-sensitive domain controls voltage-gated channel activation. Cell 164, 922–936 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Naylor, C. E. et al. Molecular basis of ion permeability in a voltage-gated sodium channel. EMBO J. 35, 820–830 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Bagneris, C. et al. Prokaryotic NavMs channel as a structural and functional model for eukaryotic sodium channel antagonism. Proc. Natl Acad. Sci. USA 111, 8428–8433 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Deutsch, C. Potassium channel ontogeny. Annu Rev. Physiol. 64, 19–46 (2002).

    Article  CAS  PubMed  Google Scholar 

  22. Deutsch, C. The birth of a channel. Neuron 40, 265–276 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Gajewski, C., Dagcan, A., Roux, B. & Deutsch, C. Biogenesis of the pore architecture of a voltage-gated potassium channel. Proc. Natl Acad. Sci. USA 108, 3240–3245 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Delaney, E., Khanna, P., Tu, L., Robinson, J. M. & Deutsch, C. Determinants of pore folding in potassium channel biogenesis. Proc. Natl Acad. Sci. USA 111, 4620–4625 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. McDonald, S. K., Levitz, T. S. & Valiyaveetil, F. I. A shared mechanism for the folding of voltage-gated K+ channels. Biochemistry 58, 1660–1671 (2019).

    Article  CAS  PubMed  Google Scholar 

  26. Popot, J. L. & Engelman, D. M. Membrane protein folding and oligomerization: the two-stage model. Biochemistry 29, 4031–4037 (1990).

    Article  CAS  PubMed  Google Scholar 

  27. Popot, J. L., Gerchman, S. E. & Engelman, D. M. Refolding of bacteriorhodopsin in lipid bilayers. A thermodynamically controlled two-stage process. J. Mol. Biol. 198, 655–676 (1987).

    Article  CAS  PubMed  Google Scholar 

  28. Shaya, D. et al. Structure of a prokaryotic Sodium Channel Pore Reveals Essential Gating Elements and an Outer Ion Binding Site Common to Eukaryotic Channels. J. Mol. Biol. 426, 467–483 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Lupas, A. N. & Gruber, M. The structure of alpha-helical coiled coils. Adv. Protein Chem. 70, 37–78 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Holm, L. & Laakso, L. M. Dali server update. Nucleic Acids Res. 44, W351–W355 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Holm, L. & Rosenstrom, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Rushton, P. S. et al. Rice cellulose synthaseA8 plant-conserved region is a coiled-coil at the catalytic core entrance. Plant Physiol. 173, 482–494 (2017).

    Article  CAS  PubMed  Google Scholar 

  33. Zalewski, J. K. et al. Structure of the shroom-rho kinase complex reveals a binding interface with monomeric shroom that regulates cell morphology and stimulates kinase activity. J. Biol. Chem. 291, 25364–25374 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Grinzato, A. et al. High-Light versus low-light: effects on paired photosystem II supercomplex structural rearrangement in pea plants. Int. J. Mol. Sci. 21, 8643 (2020).

    Article  CAS  PubMed Central  Google Scholar 

  35. Payandeh, J., Scheuer, T., Zheng, N. & Catterall, W. A. The crystal structure of a voltage-gated sodium channel. Nature 475, 353–358 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Mravic, M. et al. Packing of apolar side chains enables accurate design of highly stable membrane proteins. Science 363, 1418–1423 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Engelman, D. M. & Steitz, T. A. The spontaneous insertion of proteins into and across membranes: the helical hairpin hypothesis. Cell 23, 411–422 (1981).

    Article  CAS  PubMed  Google Scholar 

  38. Baker, E. N. & Hubbard, R. E. Hydrogen bonding in globular proteins. Prog. Biophys. Mol. Biol. 44, 97–179 (1984).

    Article  CAS  PubMed  Google Scholar 

  39. Uysal, S. et al. Crystal structure of full-length KcsA in its closed conformation. Proc. Natl Acad. Sci. USA 106, 6644–6649 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Dominik, P. K. & Kossiakoff, A. A. Phage display selections for affinity reagents to membrane proteins in nanodiscs. Methods Enzymol. 557, 219–245 (2015).

    Article  CAS  PubMed  Google Scholar 

  41. Folta-Stogniew, E. Oligomeric states of proteins determined by size-exclusion chromatography coupled with light scattering, absorbance, and refractive index detectors. Methods Mol. Biol. 328, 97–112 (2006).

    CAS  PubMed  Google Scholar 

  42. Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006).

    Article  CAS  PubMed  Google Scholar 

  43. Lolicato, M. et al. K2P2.1(TREK-1):activator complexes reveal a cryptic selectivity filter binding site. Nature 547, 364–368 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc. Natl Acad. Sci. USA 109, E690–E697 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Hesketh, S. J. et al. Styrene maleic-acid lipid particles (SMALPs) into detergent or amphipols: an exchange protocol for membrane protein characterisation. Biochim. Biophys. Acta Biomembr. 1862, 183192 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Cymer, F., von Heijne, G. & White, S. H. Mechanisms of integral membrane protein insertion and folding. J. Mol. Biol. 427, 999–1022 (2015).

    Article  CAS  PubMed  Google Scholar 

  47. Popot, J. L. & Engelman, D. M. Membranes do not tell proteins how to fold. Biochemistry 55, 5–18 (2016).

    Article  CAS  PubMed  Google Scholar 

  48. Ingolia, N. T., Lareau, L. F. & Weissman, J. S. Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147, 789–802 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Bostrom, K. et al. Pulse-chase studies of the synthesis and intracellular transport of apolipoprotein B-100 in Hep G2 cells. J. Biol. Chem. 261, 13800–13806 (1986).

    Article  CAS  PubMed  Google Scholar 

  50. Payandeh, J., Gamal El-Din, T. M., Scheuer, T., Zheng, N. & Catterall, W. A. Crystal structure of a voltage-gated sodium channel in two potentially inactivated states. Nature 486, 135–139 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Tang, L. et al. Structural basis for inhibition of a voltage-gated Ca2+ channel by Ca2+ antagonist drugs. Nature 537, 117–121 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Zhang, X. et al. Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel. Nature 486, 130–134 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Mackenzie, C. O. & Grigoryan, G. Protein structural motifs in prediction and design. Curr. Opin. Struct. Biol. 44, 161–167 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Alva, V. & Lupas, A. N. From ancestral peptides to designed proteins. Curr. Opin. Struct. Biol. 48, 103–109 (2018).

    Article  CAS  PubMed  Google Scholar 

  55. Hammon, J., Palanivelu, D. V., Chen, J., Patel, C. & Minor, D. L. Jr A green fluorescent protein screen for identification of well-expressed membrane proteins from a cohort of extremophilic organisms. Protein Sci. 18, 121–133 (2009).

    CAS  PubMed  Google Scholar 

  56. Dominik, P. K. et al. Conformational chaperones for structural studies of membrane proteins using antibody phage display with nanodiscs. Structure 24, 300–309 (2016).

    Article  CAS  PubMed  Google Scholar 

  57. Denisov, I. G., Grinkova, Y. V., Lazarides, A. A. & Sligar, S. G. Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size. J. Am. Chem. Soc. 126, 3477–3487 (2004).

    Article  CAS  PubMed  Google Scholar 

  58. Miller, K. R. et al. T cell receptor-like recognition of tumor in vivo by synthetic antibody fragment. PLoS ONE 7, e43746 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Borowska, M. T., Dominik, P. K., Anghel, S. A., Kossiakoff, A. A. & Keenan, R. J. A YidC-like protein in the archaeal plasma membrane. Structure 23, 1715–1724 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Bailey, L. J. et al. Locking the elbow: improved antibody fab fragments as chaperones for structure determination. J. Mol. Biol. 430, 337–347 (2018).

    Article  CAS  PubMed  Google Scholar 

  61. Ritchie, T. K. et al. Chapter 11 — reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol. 464, 211–231 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Doublie, S. Preparation of selenomethionyl proteins for phase determination. Method Enzymol. 276, 523–530 (1997).

    Article  CAS  Google Scholar 

  63. Ujwal, R. & Bowie, J. U. Crystallizing membrane proteins using lipidic bicelles. Methods 55, 337–341 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Battye, T. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D Biol. Crystallogr 67, 271–281 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Evans, P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr D Biol. Crystallogr 67, 282–292 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008).

    Article  CAS  PubMed  Google Scholar 

  67. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol. Crystallogr 60, 2126–2132 (2004).

    Article  PubMed  CAS  Google Scholar 

  68. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol. Crystallogr. 67, 355–367 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Kabsch, W. XDS. Acta Crystallogr. D. Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr D Biol. Crystallogr. 69, 1204–1214 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Nocula-Lugowska, M., Lugowski, M., Salgia, R. & Kossiakoff, A. A. Engineering synthetic antibody inhibitors specific for LD2 or LD4 motifs of paxillin. J. Mol. Biol. 427, 2532–2547 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol. Crystallogr 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Skubak, P. & Pannu, N. S. Automatic protein structure solution from weak X-ray data. Nat. Commun. 4, 2777 (2013).

  74. Brunger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D. Biol. Crystallogr. 54, 905–921 (1998).

    Article  CAS  PubMed  Google Scholar 

  75. Brunger, A. T. Version 1.2 of the crystallography and NMR system. Nat. Protoc. 2, 2728–2733 (2007).

    Article  CAS  PubMed  Google Scholar 

  76. Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).

    Article  CAS  PubMed  Google Scholar 

  77. Jo, S., Kim, T. & Im, W. Automated builder and database of protein/membrane complexes for molecular dynamics simulations. PLoS ONE 2, e880 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  78. Case, D. A. et al. AMBER 2016. https://doi.org/10.13140/RG.2.2.27958.70729 (2016).

  79. Maier, J. A. et al. ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 11, 3696–3713 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Dickson, C. J. et al. Lipid14: the amber lipid force field. J. Chem. Theory Comput. 10, 865–879 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983).

    Article  CAS  Google Scholar 

  82. Salomon-Ferrer, R., Gotz, A. W., Poole, D., Le Grand, S. & Walker, R. C. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 2. Explicit solvent particle mesh ewald. J. Chem. Theory Comput. 9, 3878–3888 (2013).

    Article  CAS  PubMed  Google Scholar 

  83. Aqvist, J., Wennerstrom, P., Nervall, M., Bjelic, S. & Brandsdal, B. O. Molecular dynamics simulations of water and biomolecules with a Monte Carlo constant pressure algorithm. Chem. Phys. Lett. 384, 288–294 (2004).

    Article  CAS  Google Scholar 

  84. Miyamoto, S. & Kollman, P. A. Settle — an analytical version of the shake and rattle algorithm for rigid water models. J. Comput. Chem. 13, 952–962 (1992).

    Article  CAS  Google Scholar 

  85. Gomez, Y. K. et al. Taking the Monte-Carlo gamble: how not to buckle under the pressure! J. Comput. Chem. https://doi.org/10.1002/jcc.26798 (2021).

  86. Roe, D. R. & Cheatham, T. E. PTRAJ and CPPTRAJ: software for processing and analysis of molecular dynamics trajectory data. J. Chem. Theory Comput. 9, 3084–3095 (2013).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

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.

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  1. Cristina Arrigoni & Marco Lolicato

    Present address: Department of Molecular Medicine, University of Pavia, Pavia, Italy

Authors and Affiliations

  1. Cardiovascular Research Institute, University of California, San Francisco, CA, USA

    Cristina Arrigoni, Marco Lolicato, David Shaya, Ahmed Rohaim, Felix Findeisen, Lam-Kiu Fong, Claire M. Colleran, William F. DeGrado, Michael Grabe & Daniel L. Minor Jr.

  2. Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA

    Lam-Kiu Fong, William F. DeGrado & Michael Grabe

  3. Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA

    Pawel Dominik, Sangwoo S. Kim & Anthony A. Kossiakoff

  4. Northeastern Collaborative Access Team, Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA

    Jonathan P. Schuermann

  5. Departments of Biochemistry and Biophysics, and Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA

    Daniel L. Minor Jr.

  6. California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA, USA

    Daniel L. Minor Jr.

  7. Kavli Institute for Fundamental Neuroscience, University of California, San Francisco, CA, USA

    Daniel L. Minor Jr.

  8. Molecular Biophysics and Integrated Bio-imaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Daniel L. Minor Jr.

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Contributions

C. A. and D. L. M. conceived the study and designed the experiments. D. S. purified and crystallized the initial structures of CaVSp1p and NaVAb1p in detergent. A. R. purified, crystallized, and determined the structure of NaVAb1p in bicelles. M. L. determined the structures of the NaVAe1Sp1CTDp and the SAT09 and ANT05 complexes, and refined all of the structures. F. F. determined structures of CaVSp1p and NaVAb1p in detergent. C. M. C. and C. A. expressed and purified the proteins and sFab complexes. C. A. crystallized NaVAe1/Sp1CTDp and the SAT09 and ANT05 complexes and performed the biochemical characterization. P. D. and A. A. K. provided the platform for the development of sFabs. P. D. and S. S. K. selected the sFabs. J. P. S. contributed to the ANT05 complex data collection and structure determination. L.-K. F. performed the simulations. L.-K. F., W. F. D. and M. G. analyzed the simulations. D. L. M. analyzed data and provided guidance and support. C. A., M. L., L.-K. F., W. F. D., M. G. and D. L. M. wrote the paper.

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Correspondence to Daniel L. Minor Jr..

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Nature Structural and Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Editor recognition statement Primary Handling editor: Florian Ullrich, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 BacNaV PDs exemplar electron density and structural details.

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.

Extended Data Fig. 3 Structure comparison of inside-out and canonical quaternary assemblies.

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’.

Extended Data Fig. 4 BacNaV pore loop and S6 hydrophilic residue dynamics.

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.

Extended Data Fig. 5 sFab:NaVAe1/Sp1CTDp complexes.

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.

Extended Data Fig. 6 Structure of the sFabSAT09:NaVAe1/Sp1CTDp complex.

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.

Extended Data Fig. 7 SEC-MALS analysis of NaVAe1Sp1CTD and SAT09:NaVAe1Sp1CTD.

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.

Source data

Extended Data Fig. 8 Structure of the sFabANT05:NaVAe1/Sp1CTDp complex.

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.

Extended Data Fig. 9 Characterization of sFab SAT09 binding.

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.

Source data

Extended Data Fig. 10 EPR mobility changes.

a, Side and b, Extracellular views of the CaVSp1p structure showing residues having changed mobility relative to the full length channel. Increased (orange), decreased (blue). Selectivity filter is colored red. EPR data are from 15.

Supplementary information

Source data

Source Data Fig. 2

DALI search results.

Source Data Fig. 7

Chromatograms for Fig. 7.

Source Data Extended Data Fig. 7

Chromatograms for Extended Data Fig. 7 (Note: Data in 7c are from 7a and 7b).

Source Data Extended Data Fig. 9

Chromatograms for Extended Data Fig. 9

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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

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