Molecular basis of enhanced GLP-1 signaling mediated by GLP-1(9-36) in conjunction with LSN3318839

Abstract Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) is essential for glucose metabolism and energy balance. It is primarily activated by the endogenous hormone GLP-1(7-36) and its weaker metabolite GLP-1(9-36). Although GLP-1(9-36) was previously considered biologically inactive, emerging evidence indicate that it exerts cardioprotective and insulinotropic actions. Here, we present cryo-electron microscopy structures showing that GLP-1(9-36) binds to an atypical site composed of the upper halves of transmembrane helices 1 (TM1) and 2 (TM2), extracellular loop 1, and the extracellular domain, distinct from the binding site of GLP-1(7-36). Meanwhile, LSN3318839, an orally effective positive allosteric modulator selective for GLP-1(9-36), anchors in the TM1-TM2 cleft and repositions GLP-1(9-36) to adopt a binding pose like GLP-1(7-36), thereby enhancing GLP-1R-mediated signal transduction. Mutagenesis and molecular dynamics simulations confirmed the crucial interactions and structural adjustments involved in this modulation. Our study uncovers a novel binding mode of GLP-1 metabolite and allosteric modulation that augments GLP-1R signaling, both are potentially insightful to unveil the physiological role of GLP-1(9-36) and design better small molecule therapeutics targeting GLP-1R.