The bioactive lipid sphingosine 1-phosphate (S1P) binds to five known G protein-coupled receptors, S1P1–5, and acts as a second messenger during cell signaling. Among them FTY720 targeting S1P1 is used for immunosuppressive agent for the treatment of autoimmune disease. However, FTY720 acts through multiple S1P receptors, the mechanism of action through one or more of these receptors may account for its side effects. In 2011, the X-ray crystal structure of antagonist-bound inactive state S1P1 was solved, but FTY720 is an agonist. Solving the structure of agonist-bound active state S1P1 is expected not only to elucidate the mechanism of S1P1, but to design of a more selective and effective drug.
First, we attempted purification of S1P1R and G protein complex for structural analysis. However, the expression level of wild-type S1P1 is very low. To improve this problem, co-expressing dominant negative Gi and Gβγ with S1P1 increased the yield and enhance the stability of S1P1-G protein heterotrimer complexes. Negative stain electron microscope (EM) and 2D class averages revealed uniformity and stable complex particles suitable for cryo-EM.