State-Targeting Stabilization of Adenosine A2A Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein

• Published in: International journal of molecular sciences Vol. 22; no. 23; p. 12906
• Main Authors: Mitsumoto, Masaya, Sugaya, Kanna, Kazama, Kazuki, Nakano, Ryosuke, Kosugi, Takahiro, Murata, Takeshi, Koga, Nobuyasu
• Format: Journal Article
• Language: English
• Published: Basel MDPI AG 29.11.2021; MDPI
• Subjects: Adenosine; adenosine A2A receptor; computational protein design; de novo designed protein; Design; Efficiency; fusion partner protein; G protein-coupled receptors; G-protein coupled receptor; Helices; Inverse agonists; Ligands; NMR; Nuclear magnetic resonance; Physiology; Protein G; protein stabilization; Proteins; Simulation; Spectrum analysis; Thermal stability
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms222312906

G-protein coupled receptors (GPCRs) are known for their low stability and large conformational changes upon transitions between multiple states. A widely used method for stabilizing these receptors is to make chimeric receptors by fusing soluble proteins (i.e., fusion partner proteins) into the intracellular loop 3 (ICL3) connecting the transmembrane helices 5 and 6 (TM5 and TM6). However, this fusion approach requires experimental trial and error to identify appropriate soluble proteins, residue positions, and linker lengths for making the fusion. Moreover, this approach has not provided state-targeting stabilization of GPCRs. Here, to rationally stabilize a class A GPCR, adenosine A2A receptor (A2AR) in a target state, we carried out the custom-made de novo design of α-helical fusion partner proteins, which can fix the conformation of TM5 and TM6 to that in an inactive state of A2AR through straight helical connections without any kinks or intervening loops. The chimeric A2AR fused with one of the designs (FiX1) exhibited increased thermal stability. Moreover, compared with the wild type, the binding affinity of the chimera against the agonist NECA was significantly decreased, whereas that against the inverse agonist ZM241385 was similar, indicating that the inactive state was selectively stabilized. Our strategy contributes to the rational state-targeting stabilization of GPCRs.