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Abstract
The M2 muscarinic acetylcholine receptor (M2R) is a prototypical G protein-coupled receptor (GPCR) that responds to acetylcholine (ACh) and mediates various cellular responses in the nervous system. We recently established Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy for ligand binding to M2R reconstituted in lipid membranes, paving the way to understand the mechanism in atomic detail. However, the obtained difference FTIR spectra upon ligand binding contained ligand, protein, lipid, and water signals, so a vibrational assignment was needed for a thorough understanding. In the present study, we compared difference FTIR spectra between unlabeled and 2-13C labeled ACh, and assigned the bands at 1741 and 1246 cm−1 as the C Created by potrace 1.16, written by Peter Selinger 2001-2019 ]]> O and C–O stretches of ACh, respectively. The CO stretch of ACh in M2R is close to that in aqueous solution (1736 cm−1), and much lower in frequency than the free CO stretch (1778–1794 cm−1), indicating a strong hydrogen bond, which probably formed with N4046.52. We propose that a water molecule bridges ACh and N4046.52. The other ACh terminal is positively charged, and it interacts with negatively charged D1033.32. The present study revealed that D1033.32 is deprotonated (negatively charged) in both ACh-bound and free states, a suggested mechanism to stabilize the negative charge of D1033.32 in the free M2R.
Highlights
Kohei Suzuki,a Kota Katayama,ab Yuji Sumii,a Tomoya Nakagita,d Ryoji Suno,c Hirokazu Tsujimoto,d So Iwata,d Takuya Kobayashi,ce Norio Shibata a and Hideki Kandori *ab
Gprotein-coupled receptor (GPCR) signaling utilizes a coupling mechanism between the extracellular ligand-binding pocket and the cytoplasmic domain of the receptor that selectively interacts with a signaling transducer.[1,2]
What is the protonation state of D1033.32 in the ligand-free form? And how do N4046.52 and tyrosines alter their structures before and a er ACh binding? A similar question is applicable to ACh itself, namely how is the structure altered upon binding to M2R?
Summary
Kohei Suzuki,a Kota Katayama,ab Yuji Sumii,a Tomoya Nakagita,d Ryoji Suno,c Hirokazu Tsujimoto,d So Iwata,d Takuya Kobayashi,ce Norio Shibata a and Hideki Kandori *ab. It is important to understand the molecular mechanism behind selective ligandinduced changes in receptor conformation and speci c transducer-recognition for the development of GPCR-targeting drugs Both X-ray crystallography and cryo-electron microscopy techniques have played important roles in determining structures of inactive and active GPCR states bound to orthosteric and allosteric ligands.[3,4,5] In addition, spectroscopic techniques such as NMR and double electron–electron resonance (DEER) of multiple GPCR states have provided valuable information on their dynamic nature.[1,6,7,8,9] More recently, solution NMR was used to structurally link ligand-binding to M2 muscarinic acetylcholine receptor (M2R) with its G-protein coupling interface.[10]. Stimulus-induced difference Fourier-transform infrared (FTIR) spectroscopy is a powerful, sensitive and informative tool to investigate protein structural changes that accompany
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