Integrative Structure Determination Of Alpha 7nachr Intracellular Domain

The intracellular domain (ICD) of eukaryotic pentameric ligand-gated ion channels (pLGICs) is involved in receptor assembly and trafficking, channel conductance and desensitization, and cytoplasmic signaling. Although the number of high-resolution eukaryotic pLGIC structures has grown rapidly in recent years, no structure obtained thus far contains a complete ICD. The dynamic nature of the ICD is a major obstacle for traditional structure determination using X-ray crystallography or cryo-EM. The α7 nicotinic acetylcholine receptor (α7nAChR) is an important pLGIC that plays a central role in nicotine addiction, Alzheimer's disease, schizophrenia, neuroinflammation, and other neurological processes. To determine α7nAChR ICD structures, we combined NMR and ESR experiments with computations in Rosetta. A suite of NMR experiments, including NOESY and paramagnetic relaxation enhancement (PRE) experiments, was used to determine secondary and tertiary structural restraints; double electron-electron resonance ESR and 19F PRE NMR provided long distance restraints to define quaternary structures; and Rosetta software provided an effective platform to integrate all these diverse types of experimental information into de novo structure calculations. Iterative Rosetta calculations yielded structures with low energy and high convergence, providing the first atomic view of the full-length ICD. This integrated approach can also be applied to solve ICD structures of other pLGICs. This work was supported by grants from the NIH.