Bondarenko V et al. (2022), Structures of highly flexible intracellular dom...

XB-ART-58976

Nat Commun 2022 Feb 10;131:793. doi: 10.1038/s41467-022-28400-x.

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Structures of highly flexible intracellular domain of human α7 nicotinic acetylcholine receptor.

Bondarenko V , Wells MM , Chen Q , Tillman TS , Singewald K , Lawless MJ , Caporoso J , Brandon N , Coleman JA , Saxena S , Lindahl E , Xu Y , Tang P .

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The intracellular domain (ICD) of Cys-loop receptors mediates diverse functions. To date, no structure of a full-length ICD is available due to challenges stemming from its dynamic nature. Here, combining nuclear magnetic resonance (NMR) and electron spin resonance experiments with Rosetta computations, we determine full-length ICD structures of the human α7 nicotinic acetylcholine receptor in a resting state. We show that ~57% of the ICD residues are in highly flexible regions, primarily in a large loop (loop L) with the most mobile segment spanning ~50 Å from the central channel axis. Loop L is anchored onto the MA helix and virtually forms two smaller loops, thereby increasing its stability. Previously known motifs for cytoplasmic binding, regulation, and signaling are found in both the helices and disordered flexible regions, supporting the essential role of the ICD conformational plasticity in orchestrating a broad range of biological processes.

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	Fig. 1. Functional α7nAChR TMD + ICD construct and membrane mimetic for structural studies.a Current traces and b concentration-dependent potentiation of Xenopus oocytes injected with purified TMD + ICD, showing channel activation by ivermectin (30 μΜ) and potentiation by PNU-120596. Data shown in black open circles (n = 5 sets) were fit to the Hill equation, EC50 = 10 ± 1 μM. Source data are provided as a Source Data file. c Overlay of 1H-15N TROSY-HSQC NMR spectra of α7nAChR TMD + ICD in LDAO micelles (red) and in asolectin nanodiscs (blue) collected at an 18.8-tesla NMR spectrometer at 45 °C. Many more residues are observable in micelle samples than in nanodiscs, a trend which is also true for spectra collected at other temperatures (Supplementary Fig. 3).
	Fig. 2. Structures of α7nAChR TMD + ICD.a Side view of a pentameric structure with marked residues (Cα atom shown in VDW) for measuring ICD dimensions. b A single subunit color-coded with secondary structures (cyan for α helix, blue for 310-helix, orange for turn, and silver for coil). c A bottom view of the pentameric structure. d Intensity (peak height) distribution of TMD + ICD residues obtained from a HNCOCA NMR spectrum. Missing bars are due to overlapping residues or prolines. Note that the distinctly high and low intensities for residues in loop regions and TMD or long ICD helices are indication of faster and slower motions, respectively. Source data are provided as a Source Data file.
	Fig. 3. Major interactions stabilizing ICD structures.a Uneven distribution of charge residues (blue: ARG and LYS; red: GLU and ASP) in the ICD. b–d Representative electrostatic interactions that stabilize tertiary structure (yellow), and quaternary structure (yellow–orange). e Representative hydrogen bonding in disordered loop regions. f Representative hydrogen bonding between adjacent subunits. g Representative hydrophobic interactions in the ICD. All dash lines in (c) and (d) < 3.2 Å.
	Fig. 4. Functional relevant motifs in ICD structures.(1) K307-L314, (2) L314-M323, (3) R322-R325, and (4) R340-S350 are represented by CA atom spheres for clarity, with selected residues highlighted with sticks. Residues are colored based on residue type: white-hydrophobic, green-hydrophilic, blue-basic, red-acidic. (5) L362-F367, represented by helix h3 in cartoon. (6) A GXXXG motif: G382 and G386 forms a hydrogen bond with a distance < 2.1 Å. (7) Proline-rich motifs: proline residues are presented in sticks and colored in black.
	Fig. 5. Ion permeation pathways.a Side view of the ion permeation pathway in the α7nAChR TMD + ICD. For clarity, only two subunits are shown. The pore lumen is represented with blue dots. Side chains of pore-lining residues are labeled and shown in sticks with color codes: red or blue for negative and positive charged residues and green or white for hydrophilic and hydrophobic residues, respectively. Source data are provided as a Source Data file. b Expanded view of a putative lateral portal formed mostly by charged and polar residues in adjacent MA helices.

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Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed