[2P-2] Poster 2 (2P-38ー2P-88)

[2P-87*] State-selective stabilization of a G-protein coupled receptor by tailor-made design of fusion partner proteins

^○Masaya Mitsumoto¹, Kanna Sugaya², Kazuki Kazama², Ryosuke Nakano², Takahiro Kosugi³, Takeshi Murata², Nobuyasu Koga^1,3,4 (1.SOKENDAI, 2.Dept. of Sci., Chiba Univ., 3.NINS, ExCELLS, 4.IMS)

GPCRs transmit signals from the outside to the inside cell to drive physiological processes. The receptors are in equilibria between the active and inactive. The conformational displacement upon the state transition has been suggested for the transmembrane helices 5 and 6 (TM5 and TM6), connected by the intracellular loop 3 (ICL3). We hypothesized that the remodeling of the ICL3 region enables the state-selective stabilization of the receptors. Here, we computationally designed soluble proteins, and made the fusion with a prototypical GPCR, A_2AR, by replacing the ICL3 region with the designs so that the TM5 and TM6 are rigidly elongated without kinks or intervening loops. The designs were found to fold into monomeric unique structures with high thermal stabilities. The chimeric A_2ARs fused with the designs were found to be purified with higher yields than the wild type, and the melting temperatures were also ~4.3 °C higher. Moreover, for the chimera with a design, the binding ability was detected against an antagonist (ZM241385) but not an agonist (NECA), indicating that the success in the state-selectively stabilization at the inactive state. Our study suggests the importance of ICL3 for the state transition.