Employing NaChBac for cryo-EM analysis of action on voltage-gated Na+ channels in nanodisc

Shuai Gaoa, William C. Valinskyb, Nguyen Cam Ona, Patrick R. Houlihanb, Qian Quc, Lei Liuc, Xiaojing Pand, David E. Claphamb, and Nieng Yana,1

aDepartment of Molecular Biology, Princeton University, Princeton, NJ 08544; bJanelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147; cTsinghua-Peking Joint Center for Life Sciences, Department of Chemistry, Tsinghua University, Beijing 100084, China; and dBeijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China

Contributed by Nieng Yan, April 27, 2020 (sent for review December 31, 2019; reviewed by Jian Payandeh and Peng Zhang) + – NaChBac, the first bacterial voltage-gated Na (Nav) channel to be (11 20). Among these, human Nav1.7 is of particular interest to the characterized, has been the prokaryotic prototype for studying the pharmaceutical industry for the development of novel killers; structure–function relationship of Na channels. Discovered nearly v mutations in Nav1.7 are associated with various pain disorders, such two decades ago, the structure of NaChBac has not been deter- as indifference to pain or extreme pain syndrome (21–28). The mined. Here we present the single particle electron cryomicro- structures of human Na 1.7 bound to the tarantula gating modifier scopy (cryo-EM) analysis of NaChBac in both detergent micelles v (GMTs) in combination with pore blockers (Protoxin II with and nanodiscs. Under both conditions, the conformation of NaCh- Bac is nearly identical to that of the potentially inactivated Na Ab. ; Huwentoxin IV [HWTX-IV] with ), in the v β β Determining the structure of NaChBac in nanodiscs enabled us to presence of 1and 2 subunits, were determined at overall reso-

examine gating modifier toxins (GMTs) of Nav channels in lipid lutions of 3.2 Å (17). The GMTs, which associate with the pe- bilayers. To study GMTs in mammalian Nav channels, we gener- ripheral regions of VSDs, were only poorly resolved as blobs that ated a chimera in which the extracellular fragment of the S3 and prevented even rigid-body docking of the toxin structures. S4 segments in the second voltage-sensing domain from Nav1.7 The poor resolution of the toxins may be due to the loss of in- replaced the corresponding sequence in NaChBac. Cryo-EM struc- teraction with the membrane in detergent micelles; intact mem- tures of the nanodisc-embedded chimera alone and in complex branes were reported to facilitate the action of these toxins (29–32). with HuwenToxin IV (HWTX-IV) were determined to 3.5 and 3.2 Understanding the binding and modulatory mechanism of GMTs BIOPHYSICS AND COMPUTATIONAL BIOLOGY Å resolutions, respectively. Compared to the structure of HWTX- may require structures of the channel–toxin complex in a lipid en- IV–bound human Na 1.7, which was obtained at an overall reso- v vironment, such as nanodiscs. However, the low yield of Na 1.7 lution of 3.2 Å, the local resolution of the toxin has been improved v from ∼6to∼4 Å. This resolution enabled visualization of toxin recombinant protein has impeded our effort to reconstitute the docking. NaChBac can thus serve as a convenient surrogate for purified protein into nanodiscs. We therefore sought to establish a

structural studies of the interactions between GMTs and Nav chan- nels in a membrane environment. Significance

NaChBac | Nav channels | nanodisc | electron cryomicroscopy (cryo-EM) | NaChBac was the first bacterial Nav channel to be characterized gating modifier toxins and has been a prototype in the study of the structure–

function relationship of Nav and Cav channels. Despite ad-

oltage-gated sodium (Nav) channels initiate and propagate vances in the structural biology of prokaryotic and eukaryotic + Vaction potentials by selectively permeating Na across cell Nav channels, the structure of NaChBac had not been de- membranes in response to changes in the transmembrane elec- termined. Here we present single particle electron cryomicro- tric field (the membrane potential) in excitable cells (1–3). There scopy (cryo-EM) structures of NaChBac in detergent micelles and in nanodiscs, which exhibit identical conformation. NaCh- are nine subtypes of Nav channels in humans, Nav1.1 to 1.9, whose central role in electrical signaling is reflected by the great diversity Bac can be engineered to be a surrogate to study the interac- tions between gating modifier toxins (GMTs) and targeted of disorders that are associated with aberrant functions of Nav channels. To date, more than 1,000 mutations have been identified segments on eukaryotic Nav channels. Our comparative struc- tural characterizations emphasize the significance of the in human Nav channels that are related to epilepsy, arrhythmia, pain syndromes, autism spectrum disorder, and other diseases (4–6). membrane environment in the structural study of Nav channels and GMTs. Elucidation of the structures and functional mechanisms of Nav channels will shed light on the understanding of fundamental bi- Author contributions: S.G., D.E.C., and N.Y. designed research; S.G., W.C.V., N.C.O., P.R.H., ology and facilitate potential clinical applications. and Q.Q. performed research; X.P. contributed new reagents/analytic tools; S.G., W.C.V., The eukaryotic Nav channels are composed of a pore-forming N.C.O., P.R.H., Q.Q., L.L., X.P., and N.Y. analyzed data; and S.G. and N.Y. wrote the paper. α subunit and auxiliary subunits (7). The α subunit is made of one Reviewers: J.P., Genentech, Inc.; and P.Z., Institute of Plant Physiology and Ecology, single polypeptide chain that folds to four repeated domains (I to Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. IV) of six transmembrane (S1 to S6) segments. The S5 and S6 The authors declare no competing interest. segments from each domain constitute the pore region of the Published under the PNAS license. channel, which are flanked by four voltage-sensing domains Data deposition: Atomic coordinates and electron microscopy maps have been deposited (VSDs) formed by S1 to S4. The VSD, a relatively independent in the Protein Data Bank (PDB; http://www.rcsb.org) and Electron Microscopy Data Bank structural entity, provides the molecular basis for voltage sensing (EMDB; https://www.ebi.ac.uk/pdbe/emdb), respectively. For NaChBac in GDN and in nanodiscs and the chimera alone and in complex with HWTX-VI, the PDB codes are in voltage-dependent channels (Nav,Kv, and Cav channels) and 6VX3, 6VWX, 6VXO, and 6W6O, respectively; EMDB codes are EMD-21428, EMD-21425, enzymes (3, 8–10). EMD-21446, and EMD-21560, respectively. Owing to the technological advancement of electron cryomicro- 1To whom correspondence may be addressed. Email: [email protected]. scopy (cryo-EM), we were able to resolve the atomic structures of This article contains supporting information online at https://www.pnas.org/lookup/suppl/ Cav and Nav channels from different species and in complex with doi:10.1073/pnas.1922903117/-/DCSupplemental. distinct auxiliary subunits, animal toxins, and FDA-approved drugs First published June 8, 2020.

www.pnas.org/cgi/doi/10.1073/pnas.1922903117 PNAS | June 23, 2020 | vol. 117 | no. 25 | 14187–14193 Downloaded by guest on October 1, 2021 surrogate system to facilitate understanding the modulation of Two spherical densities can be resolved in the EM map and Nav1.7 by GMTs. half maps in the selectivity filter (SF) of NaChBac-N in both C1 NaChBac from Bacillus halodurans was the first prokaryotic Nav and C4 symmetry, supporting the assignment of two ions (SI channel to be identified (33). More orthologs of this family were Appendix, Fig. S5). Although we cannot exclude the possibility of subsequently characterized (34). In contrast to eukaryotic counterparts, contaminating ions, we will still refer to the bound ions as Na+, bacterial Nav channels are homotetramers of 6-TM subunits. Bacterial which was present at 150 mM throughout purification. As ob- Nav channels, exemplified by NavAb and NavRh, have been exploited served in the structures of other homotetrameric Nav channels, as important model systems for structure–function relationship studies an outer Na+ coordination site is enclosed by the side groups of (35–38). Despite having been discovered two decades ago, the struc- Glu191 and Ser192 and an inner site is constituted by the eight ture of the prototypical NaChBac remains unknown. carbonyl oxygen groups from Thr189 and Leu190 (Fig. 1C and SI The characterized GMT binding sites are not conserved be- Appendix, Fig. S4B). tween prokaryotic and eukaryotic Nav channels. Bacterial Nav Calculation of the pore radii with the program HOLE (42) channel-based chimeras have been employed for structural anal- reveals that the intracellular gate, formed by Ile231 on S6, is ysis of the mechanisms of GMTs and small-molecule perturbation. closed (Fig. 1C). Note that Phe227, which is one helical turn In these chimeras, the extracellular halves of VSDs were replaced above Ile231, has up and down conformations that were resolved by the corresponding sequences from the eukaryotic counterparts. in the three-dimensional (3D) EM reconstruction (SI Appendix, These studies have afforded important insight into the function of Fig. S4C). In the down state, it is part of the closed gate, whereas GMTs at the VSD and have facilitated the structural elucidation in the up conformation it no longer contributes to the constric- of Nav channels in distinct conformations (39, 40). tion (Fig. 1C). These dual conformations of Phe at the in- The most commonly used scaffold protein is NavAb; however, tracellular gate are similar to those in the structures of human overexpression and purification of NavAb in insect cells is costly Nav1.7 and Nav1.5 (17, 20). and slow. Alternatively, NavRh cannot be recorded in any tested In each VSD, S4 exists as a 310 helix. Three gating charge heterologous system (36). Therefore, we focused on NaChBac. By residues on S4, Arg113/116/119, are above and one, Arg122, is overcoming technical challenges with recombinant NaChBac below the occluding Phe67 on S2 (Fig. 1D). The overall struc- suitable for cryo-EM analysis, we were able to solve the structures ture, which is similar to the first reported structure of NavAb of NaChBac in detergent micelles and in nanodiscs. A NaChBac (Protein Data Bank [PDB] code 3RVY), may represent a chimera that is able to bind to HWTX-IV was generated; its potentially inactivated conformation. structures alone and in complex with HWTX-IV in nanodiscs were determined to 3.5 and 3.2 Å resolutions, respectively. Structures of NaChBac-Nav1.7VSDII Chimera Alone and Bound to HWTX-IV. Inspired by the successful structural resolution of Results NaChBac in nanodiscs, we then attempted to generate chimeras Cryo-EM Analysis of NaChBac in Detergents and in Nanodiscs. Since to confer GMT binding activity to NaChBac. The linker between its discovery in 2001, each of our laboratories made various at- S3 and S4 on the second VSD (VSD )ofNa1.7 has been tempts to obtain structural information for NaChBac. Although II v Escherichia coli characterized to be the primary binding site for HWTX-IV (43). the channel can be overexpressed in BL21(DE3) For one construct, we substituted the NaChBac sequence (resi- in mg/L LB culture and can be recorded in multiple heterologous dues 98 to 110) comprising the extracellular halves of S3 and S4 systems, it tends to aggregate during purification, preventing and their linker with the corresponding segment from the human crystallization trials or cryo-EM analysis. Na 1.7-VSD (residues 817 to 832); we refer to this as the After extensive trials for optimization of constructs, expres- v II chimera (Fig. 2A). sion, and purification conditions, the Yan laboratory determined Although both NaChBac and Na 1.7 can be recorded when the key to obtaining a monodispersed peak of NaChBac on size v expressed in HEK-293 cells, and the chimera maintained decent exclusion chromatography (SEC). At pH 10.0, NaChBac is protein yields from E. coli overexpression and biochemical be- compatible with several detergents, with glyco-diosgenin (GDN) havior in solution, currents could not be recorded from the being the best of those tested (SI Appendix, Fig. S1). We purified chimera (Fig. 2 B and C and SI Appendix, Fig. S6). This is not a NaChBac in the presence of 0.02% (wt/vol) GDN and solved its structure to 3.7 Å out of 91,616 selected particles using cryo-EM rare problem for Nav chimeras (39, 40, 44). Nevertheless, we (SI Appendix, Figs. S2 and S3 A, C, and D and Table S1). proceeded with cryo-EM analysis of the chimera alone and in the We then sought to reconstitute NaChBac into nanodiscs. presence of HWTX-IV. Preliminary trials revealed instability of the nanodisc-enclosed After some trials, the chimera alone was reconstituted into the NaChBac at pH 10.0. We modified the protocol and lowered brain polar lipid nanodiscs, and its complex with HWTX-IV was the pH to 8.0 when first incubating the protein with lipids. Ex- reconstituted in POPC nanodiscs. Their 3D EM reconstructions A were obtained at resolutions of 3.5 and 3.2 Å, respectively cellent samples were obtained after these procedures (Fig. 1 ), A B SI Appendix D and the structure of NaChBac in nanodiscs was obtained at 3.1 Å (Fig. 3 and and , Figs. S2 and S3 and Table S1). resolution out of 281,039 selected particles (SI Appendix, Figs. The overall structure of the chimera is nearly identical to S2–S4 and Table S1). For simplicity, we will refer to the struc- NaChBac except for the region containing the grafted segments. tures obtained in the presence of GDN and nanodiscs as In addition, four additional lipids are clearly resolved in both C SI NaChBac-G and NaChBac-N, respectively. chimera reconstructions, for a total of 12 lipids (Fig. 3 and Appendix, Fig. S7A). Structure of NaChBac in a Potentially Inactivated State. Despite The densities corresponding to HWTX-IV are observed on different pH conditions and distinct surrounding reagents, the the periphery of each VSD, with a local resolution of 4.2 to 4.6 Å structures of NaChBac remained nearly identical in GDN and in (Fig. 3B). The NMR structure of HWTX-IV (PDB ID 1MB6) nanodiscs, with a root-mean-square deviation of 0.54 Å over 759 (29) could be docked as a rigid body into these densities, al- Cα atoms for the tetrameric channel (Fig. 1B). For both struc- though the side chains were still indiscernible (SI Appendix, Fig. tures, application of a fourfold symmetry led to improved reso- S7 B and C). In the presence of the toxin, the local structure of lution, indicating identical conformations of the four protomers. the Nav1.7 fragment undergoes a slight outward shift (Fig. 3D). Because of the higher resolution, we will focus on NaChBac-N Because of the structural similarity between the chimera and for structural illustrations. In both reconstructions, two lipids are NaChBac-N, we will focus the structural analysis on the interface found to bind to each protomer (Fig. 1B). between the chimera and HWTX-IV.

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Fig. 1. Structural determination of NaChBac in GDN micelles and in nanodiscs. (A) Final purification step for NaChBac in MSP2N2-surrounded nanodiscs. Shown here is a representative SEC and Coomassie blue-stained sodium dodecyl (lauryl) sulfate–polyacrylamide gel electrophoresis. (B) Structures of NaChBac in GDN and nanodiscs are nearly identical. The structure of NaChBac-N (in a nanodisc) is colored orange for the pore domain and blue for VSDs, and that of NaChBac-G (in GDN) is colored gray. Two perpendicular views of the superimposed structures are shown. The bound ions and lipids are shown as purple spheres and black sticks, respectively. (C) Permeation path of NaChBac-N. (Left) The pore radii, calculated by HOLE (41), and (Right) the SF and the intracellular gate. (D) Structure of the VSD. Three gating charge residues are above the occluding Phe67 and one residue below. All structural figures were prepared in PyMol (https://pymol.org/2/).

HWTX-IV Is Half-Inserted in the Lipid Bilayer. Although the local Lys32 with alanine and examined their action on human Nav1.7 resolution of the toxin is insufficient for side chain analysis, its through electrophysiology (Fig. 4D). While R26A had a negligible backbone could be traced. Half of the toxin appears to be sub- effect on toxin activity, K27A exhibited slightly reduced inhibition. A merged in the densities corresponding to the nanodisc (Fig. 4 ). In contrast, HWTX-IV (K32A) no longer inhibited Nav1.7 activa- When the two reconstructions are overlaid (Fig. 4B), the toxin tion (Fig. 4 D and E). These characterizations support the place- bound to the chimera is positioned lower (toward the nanodisc) ment of basic residues at the HWTX-IV interface with the grafted than in the GDN-surrounded human Nav1.7 structure. In addi- Nav1.7 segment. tion, the S3–S4 loop of VSDII, which was not resolved in human Based on chemical principles and these experiments, we fine- Nav1.7 (17), is now visible in the chimera (SI Appendix, Fig. S7A). tuned the structural model. Lys32 of HWTX-IV interacts with These differences support the importance of the lipid bilayer in Glu822 and, along with the backbone amide of Tyr33, forms hy- positioning HWTX-IV and its binding segment. drogen bonds with the backbone groups of Leu825 and Ala826, Based on structural docking, a cluster of three hydrophobic respectively. This also leaves HWTX-IV’s Lys27 close to Glu822 residues, Ile5, Phe6, and Trp30, of HWTX-IV are completely in the Nav1.7 chimera, while Arg26 is beyond the range of effec- immersed in the lipid bilayer. These side chain assignments are tive electrostatic interaction (Fig. 4C). Even at excessive doses (2 supported by two observations. First, among the 35 residues in μM), HWTX-IV failed to attenuate wild-type NaChBac current HWTX-IV, six Cys residues form three pairs of central disulfide (SI Appendix, Fig. S6); this observation supports our conclusion bonds to stabilize the toxin structure. Second, the surface of the that the coordination of HWTX-IV is due to the grafted Nav1.7 toxin is amphiphilic, and the polar side of the toxin is unlikely to segment and not the surrogate NaChBac structure. be embedded in a hydrophobic environment. This residue as- signment placed several positively charged residues, including Discussion Arg26, Lys27, and Lys32, in the vicinity of the grafted Nav1.7 Here we report the long-sought structures of NaChBac, the segment for potential polar interactions (Fig. 4C). prototypical bacterial Nav channel that has been extensively Supported by the coordination observed in the toxin-bound biophysically characterized. The convenience of overexpressing structure, Glu822 and Glu829 in Nav1.7 have been reported to the recombinant protein in E. coli and reconstituting it into be critical for HWTX-IV sensitivity. Cross-linking experiments nanodiscs made NaChBac a suitable target for high-resolution suggested an important interaction between Nav1.7-Glu822 and cryo-EM analysis. We showed that NaChBac can be used as a Lys27 of HWTX-IV (47–49). Based on these structural impli- surrogate to study the specific interactions between modulators cations, we substituted the basic residues Arg26, Lys27, and and human Nav channels through chimera engineering. The shift

Gao et al. PNAS | June 23, 2020 | vol. 117 | no. 25 | 14189 Downloaded by guest on October 1, 2021 Fig. 2. Engineering of a NaChBac-Nav1.7VSDII chimera. (A) A diagram for the chimera design. Residues 98 to 110 of NaChBac are replaced by the corre- sponding sequence, colored pink, from human Nav1.7. (B) Current–voltage relationships of the chimera (orange; n = 7), Nav1.7 (black; n = 6), and NaChBac (purple; n = 7) elicited from a step protocol from −70 to +70 mV (in 10-mV increments). Macroscopic currents were not reliably detected for the chimera. (C) Final SEC purification of the chimera in the POPC nanodisc. (Right) Coomassie blue-stained SDS/PAGE for the indicated fractions.

of the binding position of the toxin in nanodiscs from that ob- Notwithstanding this progress, we note that the VSDs of the served in detergent micelles highlights the importance of a lipid chimera still remain in the same up conformation, as in the apo- environment for structural investigation of the function of GMTs chimera or in NaChBac; however, HWTX-IV was expected to (Fig. 4B). suppress activation of Nav channels (43). The lack of functional

A B

CD

Fig. 3. Cryo-EM structures of the chimera alone and in complex with HWTX-IV. (A and B) Resolution maps for the apo (A) and HWTX-IV-bound chimera (B). The resolution maps were calculated with Relion 3.0 (45) and prepared in Chimera (46). (C) Structural shift of the chimera upon HWTX-IV binding. (Left) Extracellular view of the superimposed NaChBac-N and chimera. (Right) Conformational deviation of the grafted region in the VSD. (D) HWTX-IV attaches to the periphery of VSD. The S3–S4 loop moves toward the cavity of the VSD upon HWTX-IV binding.

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Fig. 4. HWTX-IV inserts into the nanodisc. (A) HWTX-IV is half-submerged in the nanodisc. The map was low pass-filtered to show the nanodisc contour. Densities that likely belong to the extended S6 segments (labeled as CTD) are discernible at low resolution. (B) HWTX-IV resides lower in the nanodisc-

embedded chimera than that in the detergent-surrounded human Nav1.7. The 3D EM maps of HWTX-IV–bound Nav1.7 (EMD-9781) and the chimera, both low-pass filtered to ∼8 Å, are overlaid relative to the pore domain in the chimera. (C) Potential interactions between HWTX-IV and the chimera. Note that the local resolution does not support reliable side chain assignment of HWTX-IV. Please refer to SI Appendix, Fig. S6 for the densities of the toxin and the adjacent

channel segment. Shown here are the likely interactions based on the chemical properties of the residues. (D) Lys32 of HWTX-IV is critical for Nav1.7 sup- pression. Shown here are representative recordings of Nav1.7 current block by the indicated HWTX-IV variants, each applied at 200 nM, elicited from a single step protocol from −100 mV (holding) to −20 mV (maximal activation; 50 ms). (E) Percentage current block (mean ± SEM) by HWTX-IV variants. Control (no toxin; n = 4), WT (n = 4), R26A (n = 4), K27A (n = 5), and K32A (n = 5).

expression of chimeric channels also represents a drawback for Nanodisc Reconstitution. Lipid (POPC or BPL; Avanti) in chloroform was dried the surrogate system in the establishment of these structure– under a nitrogen stream and resuspended in reconstitution buffer containing 25 mM glycine, pH 10.0, 150 mM NaCl, and 0.7% DDM. Approximately 250 μg function relationships. Further optimization of NaChBac chi- μ mera is required to make it a more useful surrogate system. purified protein was mixed with 1 mg MSP2N2 and 750 g lipid (BPL for NaChBac, POPC for chimera), resulting in a protein:MSP:lipid molar ratio of μ Materials and Methods 1:12:520. Approximately 100 g TEV protease was added to the mixture to remove the His tag from MSP2N2. The mixture was incubated at 4 °C for 5 h Expression and Purification of NaChBac and the Nav1.7-NaChBac Chimera. The with gentle rotation. Bio-beads (0.3 g) were then added to remove deter- cDNA for full-length NaChBac was cloned into pET15b with an amino ter- gents from the system and facilitate nanodisc formation. After incubation at minal His6 tag. For the Nav1.7-NaChBac chimera, residues 98 to 110 of 4 °C overnight, Bio-beads were removed through filtration, and protein- NaChBac were replaced by residues 817 to 832 from Nav1.7 through stan- containing nanodiscs were collected for cryo-EM analysis after final purifi- dard two-step PCR. Overexpression in E. coli BL21(DE3) cells was induced cation by Ni-NTA resin and SEC in running buffer containing 25 mM Tris, pH with 0.2 mM IPTG (final concentration) at 22 °C when the OD600 reached 1.0 8.0, and 150 mM NaCl (Superose 6 Increase 10/300 GL; GE Healthcare). to 1.2. Cells were harvested after 16-h incubation at 22 °C, and cell pellets were resuspended in the buffer containing 25 mM Tris, pH 8.0, and 150 mM Chemical Synthesis of HWTX-IV and Mutants. HWTX-IV and its mutations were NaCl. Cells were disrupted by sonication (1.5 min/L), and insoluble fractions synthesized on 500 mg Rink Amide-AM resin (Tianjin Nankai HECHENG) (0.5 were removed by centrifugation at 27,000 × g for 10 min. The supernatant mmol/g resin) using the microwave peptide synthesizer (CEM Corporation) to was applied to ultracentrifugation at 250,000 × g for 1 h. The membrane- generate the C-terminal peptide amide. The resin was swelled in dime- containing pellets were resuspended in the extraction buffer containing thylformamide (DMF; J&K Scientific) for 10 min. The 9-fluorenylmethyloxycarbonyl 25 mM glycine, pH 10.0, 150 mM NaCl, and 2% (wt/vol) DDM, incubated at (Fmoc) protecting groups were removed by treatment with piperidine 4 °C for 2 h, and subsequently ultracentrifuged at 250,000 × g for 30 min. (10% vol/vol) and 0.1 M ethyl cyanoglyoxylate-2-oxime (Oxyma; Adamas-beta) The supernatant was applied to Ni-NTA resin, washed with 10 column vol- in DMF at 90 °C for 1.5 min. The coupling reaction was carried out using a DMF umes of wash buffer containing 25 mM glycine, pH 10.0, 150 mM NaCl, solution of Fmoc- (4 equivalent [equiv]; GL Biochem), Oxyma (4 equiv), 25 mM imidazole, and 0.02% GDN. Target proteins were eluted with four and N,N′-diisopropylcarbodiimide (DIC, 8 equiv; Adamas-beta) at 90 °C. Each column volumes of elution buffer containing 25 mM glycine, pH 10.0, coupling step required 3 min, and then the resin was washed ×3byDMF.The 150 mM NaCl, 250 mM imidazole, and 0.02% GDN. After concentration, peptide chain elongation proceeded until N-terminal coupling. proteins were further purified with SEC (Superdex 200 Increase 10/300 GL; Finally, a cleavage mixture, which contains trifluoroacetic acid (TFA; J&K Scien-

GE Healthcare) in running buffer containing 25 mM glycine, pH 10.0, tific), H2O, thioanisole (J&K Scientific), and 1,2-ethanedithiol (EDT; J&K Scientific) 150 mM NaCl, and 0.02% GDN. at a volume ratio of 87.5:5:5:2.5, was used to remove the protecting groups of

Gao et al. PNAS | June 23, 2020 | vol. 117 | no. 25 | 14191 Downloaded by guest on October 1, 2021 side chains and cleave the peptide from resin via a 3-h incubation. In the next (PDB ID 5EK0), was manually docked into the 3.7 Å EM map in Chimera (46)

step, the cleavage solution was collected and concentrated by pure N2 and manually adjusted in COOT (58), followed by refinement against the superfusion. After precipitation in cold diethyl ether and centrifugation, the corresponding maps by the phenix.real_space_refine program in PHENIX crude peptides were obtained. The linear peptide was analyzed and purified (59) with secondary structure and geometry restraints. by reverse-phase high-performance liquid chromatography (RP-HPLC). A definitive binding pose for the HWTX-IV could be determined, although The lyophilized linear peptide (4.1 mg, 1 μmol) was dissolved in 100 mL the density for many side chains did not allow unambiguous assignment. μ refolding buffer containing 100 mol reduced glutathione (GSH; Sigma), The NMR structure (PDB ID 1MB6) was manually docked into the EM map in μ · 10 mol oxidized glutathione (GSSG; J&K Scientific), and 10 mM Tris HCl, pH Chimera and adjusted in COOT. 8.0, at 25 °C overnight. The folded product was monitored and purified by RP-HPLC. Electrophysiological Characterizations. Plasmids carrying cDNAs were cotrans- fected with EGFP (9:1 ratio) into HEK293 cells for 24 h. Cells were selected for μ Cryo-EM Data Acquisition. Aliquots of 3.5 L concentrated samples were patch-clamp based on green fluorescence. loaded onto glow-discharged holey carbon grids (Quantifoil Cu R1.2/1.3, 300 Cells were bathed in a solution containing (mM) 140 Na-Gluconate, 4 KCl, mesh). Grids were blotted for 5 s and plunge-frozen in liquid ethane cooled 2 CaCl , 1 MgCl , 10 glucose, and 10 HEPES, pH 7.4. Thick-walled borosilicate by liquid nitrogen using a Vitrobot MarK IV (Thermo Fisher) at 8 °C with 2 2 patch pipettes of 2 to 5 MΩ contained (mM) 130 CsCH SO , 8 NaCl, 2 MgCl , 100% humidity. Grids were transferred to a Titan Krios electron microscope 3 3 2 2 MgATP, 10 EGTA, and 20 HEPES, pH 7.2. Solutions were 290 ± 5 mOsm. (Thermo Fisher) operating at 300 kV and equipped with a Gatan Gif Whole-cell currents were recorded at room temperature using an Axo- Quantum energy filter (slit width 20 eV) and spherical aberration (Cs) image corrector. Micrographs were recorded using a K2 Summit counting camera patch 200B patch-clamp amplifier controlled via a Digidata 1440A (Molec- (Gatan Company) in superresolution mode with a nominal magnification of ular Devices) and pClamp 10.3 software. Data were digitized at 25 kHz and 105,000×, resulting in a calibrated pixel size of 0.557 Å. Each stack of 32 low-pass-filtered at 5 kHz. frames was exposed for 5.6 s, with an exposure time of 0.175 s per frame. Cells were held at −100 mV and stepped from −70 to +70 mV in +10-mV − The total dose for each stack was ∼53 e /Å2. SerialEM was used for fully increments (1,000-ms step duration for NaChBac/chimera; 50-ms step dura- − automated data collection. All 32 frames in each stack were aligned, sum- tion for Nav1.7). After each step, cells were briefly stepped to 120 mV (50 med, and dose weighted (50) using MotionCorr2 (51) and twofold binned to ms) before returning to the holding potential (−100 mV). The intersweep a pixel size of 1.114 Å/pixel. The defocus values were set from −1.9 to −2.1 duration was 6.2 s for Nav1.7 and 15 s for NaChBac. Before recordings, μm and were estimated by Gctf (52). currents were corrected for pipette (fast) capacitance, whole-cell capaci- tance, and series resistance compensated to 80%. Leak currents and capac- Image Processing. Totals of 2,068, 1,729, 1,590, and 2,423 cryo-EM micro- itive transients were minimized using online P/4 leak subtraction. graphs were collected and 949,848, 923,618, 798,929, and 1,490,375 particles For HWTX-IV recordings, cells were held at −100 mV and stepped to −20 mV

were autopicked by RELION-3.0 (45, 53, 54) for NaChBac in GDN, nanodiscs, (maximal activation) for 50 ms (NaV1.7) or 1,000 ms (NaChBac). After each step, chimera, and chimera-HWTX-IV in nanodiscs, respectively. Particle picking cells were stepped to −120 mV as described above. The intersweep duration, was performed by RELION-3.0 (55). All subsequent two-dimensional (2D) and capacitance correction, series resistance compensation, and P/4 leak sub- 3D classifications and refinements were performed using RELION-3.0. traction were identical to the I-V protocol described above. To study HWTX-IV, Reference-free 2D classification using RELION-3.0 was performed to remove the toxin was diluted to the working concentration in extracellular solution ice spots, contaminants, and aggregates, yielding 718,086, 569,435, 552,252, and bath-perfused at a rate of 1 to 2 mL for up to 15 min. Where block was and 713,753 particles. The particles were processed with a global search K = observed, maximum block typically occurred between 10 and 15 min of per- 1 or global multireference research K = 5 procedure using RELION-3.0 to fusion. Prior to HWTX-IV perfusion, cells were recorded for 5 min to establish determine the initial orientation alignment parameters using bin2 particles. peak current. A published PDB structure of human Nav1.7-NavAb chimera was used as the initial reference. After 40 iterations, the datasets from the last 6 iterations Data Availability. Atomic coordinates and EM maps have been deposited in (or last iteration) were subject to local search 3D classifications using four the PDB (http://www.rcsb.org) and Electron Microscopy Data Bank (EMDB; classes. Particles from good classes were then combined and reextracted https://www.ebi.ac.uk/pdbe/emdb), respectively. For NaChBac in GDN and in with a box size of 240 and binned pixel size of 1.114 Å for further re- nanodiscs and the chimera alone and in complex with HWTX-VI, the PDB finement and 3D classification, resulting in 450,478, 302,656, 87,482, and codes are 6VX3, 6VWX, 6VXO, and 6W6O, respectively; EMDB codes are 391,361 particles, which were subjected to autorefinement. The 3D recon- EMD-21428, EMD-21425, EMD-21446, and EMD-21560, respectively. structions were obtained at resolutions of 3.9, 3.1, 3.9, and 3.7 Å. Skip- alignment 3D classification using bin1 particles yielded datasets containing ACKNOWLEDGMENTS. We thank the cryo-EM facility at Princeton Imaging 91,616, 281,039, 31,147, and 49,149 particles and resulted in respective re- and Analysis Center, which is partially supported by the Princeton Center for constructions at 3.7, 3.1, 3.5, and 3.2 Å by using a core mask (56). Complex Materials, a NSF-Materials Research Science and Engineering Centers program (DMR-1420541). The work was supported by grant from Model Building and Structure Refinement. The initial model of NaChBac, which NIH (5R01GM130762). N.Y. is supported by the Shirley M. Tilghman endowed was built in SWISS-MODEL (57) based on the structure of Nav1.7-VSD4-NavAb professorship from Princeton University.

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