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

Abstract

NaChBac, the first bacterial voltage-gated Na+(Nav) channel to be characterized, has been the prokaryotic prototype for studying the structure–function relationship of Navchannels. Discovered nearly two decades ago, the structure of NaChBac has not been determined. Here we present the single particle electron cryomicroscopy (cryo-EM) analysis of NaChBac in both detergent micelles and nanodiscs. Under both conditions, the conformation of NaChBac is nearly identical to that of the potentially inactivated NavAb. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Navchannels in lipid bilayers. To study GMTs in mammalian Navchannels, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Nav1.7 replaced the corresponding sequence in NaChBac. Cryo-EM structures of the nanodisc-embedded chimera alone and in complex with HuwenToxin IV (HWTX-IV) were determined to 3.5 and 3.2 Å resolutions, respectively. Compared to the structure of HWTX-IV–bound human Nav1.7, which was obtained at an overall resolution of 3.2 Å, the local resolution of the toxin has been improved from ∼6 to ∼4 Å. This resolution enabled visualization of toxin docking. NaChBac can thus serve as a convenient surrogate for structural studies of the interactions between GMTs and Navchannels in a membrane environment.

Structural Basis of the Modulation of the Voltage‐Gated Calcium Ion Channel Cav1.1 by Dihydropyridine Compounds**

“…We had some concerns with potential structural perturbation of these highly dynamic molecular machines by detergents. The structural similarity of rCa v 1.1 in nanodiscs and in detergent micelles, which was also observed in our recently published NaChBac, [11] alleviates this concern and consolidates the structural findings obtained from the detergent‐embedded channels. More satisfyingly, the coordination of the backbones of all resolved DHP antagonists, nifedipine, [10c] amlodipine, and RBK, is conserved regardless of the surrounding milieu of detergents or nanodiscs.…”

“…The endogenous rCa v 1.1 complex isolated from the rabbit skeletal muscle was purified following our published protocols [10a, 11] . Please refer to the Supporting Information for details of nanodisc reconstitution with the membrane scaffold protein 2N2 (MSP2N2) and 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC) (Figure 1 B and Supporting Information, Figure S1).…”

Selective Targeting of Nav1.7 with Engineered Spider Venom-Based Peptides

“…Although the resolution was insufficient to map side chain orientation, it allowed the docking of HwTX-IV’s NMR structure onto the channel via backbone alignment. This confirmed the general orientation of HwTx-IV in the Neff and Tzakoniati models, and identified I5, F6, and W30 as residues that are immersed in the membrane [ 60 ] ( Figure 1 ).

Figure 1.

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“…Recently, Guo et al [ 60 ] attempted to produce a cryo-EM structure of HwTX-IV bound to a nanodisc-embedded bacterial Nav channel containing the extracellular halves of the human S3-S4 sequence. Although the resolution was insufficient to map side chain orientation, it allowed the docking of HwTX-IV’s NMR structure onto the channel via backbone alignment.…”

The propofol binding sites of prokaryotic voltage-gated sodium channels

“…We threaded the sequence of NaChBac through the three PDB structures using the SWISS-MODEL software [Waterhouse, et al, 2018). The accuracy of the NaChBac model in the inactivated state was confirmed by comparing it against the recently published cryoEM structure of potentially inactivated NaChBac ( Figure S8 ) (Gao et al, 2020).…”