Structural basis of human α7 nicotinic acetylcholine receptor activation

Dear Editor, Nicotinic acetylcholine receptors (nAChRs) are a class of pentameric ligand-gated ion channels (pLGICs) widely expressed in nervous system. nAChRs function as neurotransmitter receptors that respond to endogenous acetylcholine and choline, modulat- ing neuronal excitability and synaptic communication. The homomeric α 7 nAChR is among the most abundant subtypes of nAChR in the brain. Dysfunction of α 7 is found to be associated with several neuropsychiatric and neurologic disorders, including schizophrenia and Alzheimer ’ s disease. 1,2 Stimulation of α 7 has been reported to improve attention, cognitive performance, and neuronal resistance to injury. Therefore, agonists and positive allosteric modulators (PAMs) of α 7 have become hot candidates in the drug development for the treatment of α 7-related diseases. 3,4 EVP-6124 (abbreviated as EVP) is a high-af ﬁ nity α 7-selective agonist. 5 PNU-120596 (abbreviated as PNU) is the ﬁ rst reported α 7-selective PAM that could increase the peak current of the receptor evoked by agonists and delay channel desensitization. 6 Both EVP and PNU are in clinical trials for the treatment of Alzheimer ’ s disease, schizophrenia, and cognitive impairment. Despite the signi ﬁ cance of α 7 in physiology and pharmacology, the mechanisms underlying the activation of α 7 upon agonist and/or PAM binding remain elusive. Little is known about the structural basis of the higher selectivity of EVP and PNU for α 7,

incubation at 4 °C for 2 h, the insoluble fraction was removed by ultra-centrifugation at 180,000× g for 45 min at 4 °C and the supernatant was incubated with anti-Flag M2 affinity resin (Sigma) at 4 °C. The resin was then collected and washed with wash (W) buffer (150 mM NaCl, 0.06% GDN, 20 mM Tris, pH 8.0). The proteins were eluted with W buffer supplemented with 200 μg/mL FLAG peptide. After elution, the proteins were concentrated and further purified on a Superose 6 increase column (GE healthcare) equilibrated with 150 mM NaCl, 0.02% GDN, and 20 mM Tris, pH 8.0. Peak fractions of α7 protein were collected and concentrated to ~4 mg/mL for cryo-EM experiments.
For the sample preparation of different α7 complexes, a final concentration of 100 μM 2 EVP-6124, or 100 μM EVP-6124 and 100 μM PNU-120596 were added into the protein, respectively, and incubated for 1 h before cryo-EM sample verification.

Cryo-EM sample preparation and data acquisition
A total of 3 μL of protein sample at ~4 mg/mL was applied to freshly plasma-cleaned (H2/O2, 10 s) holey carbon grids (Quantifoil, R1.2/1.3, 300 mesh, Au). The grids were blotted for 5 s at 100% humidity and 4 °C with a Vitrobot Mark IV (Thermo Fisher Scientific) and plunge-frozen into liquid ethane cooled by liquid nitrogen. The blotted grids were stored in liquid nitrogen until imaging.
The data sets were collected on a Titan Krios cryo-electron microscope (FEI) operated at 300 kV equipped with a Gatan K2 Summit direct detection camera using the automated image acquisition software SerialEM 2 in counting mode. For apo form, movies were recorded with SA29, 000× magnification yielding a pixel size of 1.01 Å at University of Science and Technology of China. The total dose of 56 e -/Å 2 was fractionated to 32 frames with 0.18 s per frame. Nominal defocus values ranged from -1.0 to -1.4 μm. For EVP-bound form and EVP/PNU-bound form, movies were recorded with SA29, 000× magnification yielding a pixel size of 1.014 Å at Zhejiang University.
The total dose of 62 e -/Å 2 was fractionated to 40 frames with 0.2 s per frame. Nominal defocus values ranged from -1.1 to -1.3 μm. Data sets of the apo form, EVP-bound form and EVP/PNU-bound form included 2,047, 2,800 and 2,948 movies, respectively.

Cryo-EM data processing
All data sets were processed similarly in relion3.1 3 and cryoSPARC2 4 . Dosefractionated image stacks were subjected to beam-induced motion correction and doseweighting using UCSF MotionCor2 5 . Contrast transfer function parameters were estimated with Gctf 6 . For particle picking, around 2,000 particles were picked manually to generate templates for auto-picking in relion3.1. Auto-picked particles were extracted in relion3.1, then imported into cryoSPARC2 for subsequent 2D classification.
Particles from well-defined 2D averages were selected and combined for 3D 3 classification. An ab initio 3D reconstruction from the 2D average particles was generated in cryoSPARC2. The initial model was then used as a reference for 3D classification by heterogenous refinement without symmetry imposed. A selected class with continuous density for all transmembrane helices was used to perform further 3D classification by heterogenous refinement with C5 symmetry imposed in cryoCPARC2.
Particles in good class were subjected in non-uniform refinement and produced the final 3D reconstruction. The overall resolutions were estimated by applying a soft mask excluding detergent micelle and the gold-standard Fourier shell correlation (FSC) using the 0.143 criterion. Local resolution was determined using cryoCPARC2.

Model building, refinement and validation
Atomic models were built in the software Coot 7 . For the model of apo-α7, the ECD was built using the crystal structure of alpha7-AChBP chimera (PDB code: 3SQ9) as a reference, while the TMD and ICD were built de novo. Every residue was manually examined. The initial model was subjected to iterative manual rebuilding in Coot and real-space refinement in PHENIX 8 . The final model was validated using the module "comprehensive validation (cryo-EM)" in PHENIX 9 . The residues from the N-terminal signal peptide  and the linker between helices MX and MA (347-431) were not built due to the lack of corresponding densities. N-acetylglucosamine moieties were built to linking to Asn46 and Asn133 sites, respectively, based on the corresponding densities. A cholesterol mimic was assigned according to the extra density nestled the M3, M4 and MX helices in each subunit. To build the models of α7/EVP and α7/EVP/PNU complex, the model of apo form was docked into the individual EM density maps and further subjected to manual rebuilding, real-space refinement and validation. All the figures were prepared using UCSF Chimera 10 .

Electrophysiology experiments
Chinese hamster ovary (CHO) cells were cultured in DMEM/F12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL 4 streptomycin at 37 °C in a 5% CO2 incubator. For each transfection of a 24-well-plate well, 0.6 μg of plasmid encoding α7EM and 0.6 μg of plasmid encoding NACHO, or 0.6 μg of plasmid encoding α7, 0.6 μg of plasmid encoding NACHO and 0.2 μg of plasmid encoding eGFP were diluted and mixed with lipofectamine 3000 (Invitrogen), then added to the cells. After incubation for 5 h, the cells were transferred to poly-L-lysinecoated slides to culture for another 16-24 h in fresh medium. They were then used for electrophysiological recording.   Distances between the adjacent α-carbon of 16'Leu, 13'Val, 9'Leu and -2'Gly are shown in angstroms. c Plots of pore radius for receptors along the pore axis. The αcarbon position of 0'-Lys (Lys261) is set to zero. Channel pore radius was calculated using the HOLE program.