Cryo-EM as a tool to characterize & exploit allostery in GPCRs
Allosteric modulation of G-protein coupled receptor (GPCR) activity represents a promising therapeutic avenue for next-generation drugs, yet this type of modulation is complex and understudied relative to more conventional orthosteric signaling. We combined structural data obtained by cryoEM with biophysical approaches such as HDX-MS and smFRET to obtain a more holistic picture of the mechanism of receptor activity modifying protein (RAMP)-induced negative allosteric modulation of a Family B GPCR, the glucagon receptor. We find that RAMP2-binding causes extensive conformational dynamics in the receptor extracellular domain, resulting in loss of agonist affinity and diminished activation at the intracellular face of the receptor. Further, we took advantage of our ability to select for particular receptor ensembles of interest and designed a screening strategy to select for small molecule positive and negative allosteric modulators (PAMs & NAMs) of the µOR. Using cryoEM, we identified our modulator binding sites as novel areas on the receptor responsible for allosteric effects on receptor function. The first identified potent, specific µOR NAM enters the brain and has clear effects on morphine-induced analgesia and side effects, though these effects remain counterintuitive and further study is required.
