Probing Allosteric Binding Site Mapping in the Free Fatty Acid 2 receptor

Abstract

Understanding of orthosteric and allosteric communications in the G protein coupled receptors (GPCRs) at molecular level may lead to new strategies in rational design of selective and efficacious drugs. A recently de-orphanized GPCR, the free fatty acid receptor 2 (FFA2) with a potential role in the regulation of appetite and energy homeostasis is modulated by a selective ago-allosteric ligand, a phenylacetamide (S)-4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide (4-CMTB) and such, is a viable example to study these communications. In this work we have explored molecular basis of agonistic and allosteric properties of 4-CMTB and its analogues by employing computational modelling and pharmacological studies. Thus, we used available crystal structures of the ligand-activated GPCRs to model the binding site of 4-CMTB and examined computationally predicted residues of the transmembrane helices and loops by mutagenesis. Interestingly, although some single and double mutations of conserved and non-conserved residues affected the binding of a natural agonist, propionate, none of them changed the potency of 4-CMTB. The more substantial change, the swap of the second extracellar loop (EL2) between FFA2 and FFA3, abolished allosterism and not agonsim of 4-CMTB. Conformational analysis of EL2 in FFA2 revealed the possible mechanistic basis of allosteric communication. The results will be discussed in light of a general strategy to identify an allosteric binding site in GPCRs known to be more permissive with respect to the chemical structure bound to it compare to an orthosteric binding site. Importance of receptor conformational diversity and membrane environment in mapping of an allosteric binding site will be highlighted.