Structural analysis reveals that water molecules mediate self-activation of GPR99

TL;DR

This study reveals how water molecules help activate the GPR99 receptor, offering insights for drug design.

Contribution

The study identifies a water-mediated polar network that stabilizes GPR99 self-activation via its ECL2 loop.

Findings

The second extracellular loop (ECL2) of GPR99 occupies the orthosteric binding pocket, promoting self-activation.

Structural water molecules form a polar network connecting ECL2 and the binding pocket, stabilizing receptor activity.

Abstract

GPR99 holds promise as a potential therapeutic target for inflammatory diseases. GPR99 exhibits marked basal activity when coupled with the Gq protein, its activation mechanism remains elusive. In this study, we determine the high-resolution structure of the human GPR99 in complex with the heterotrimeric miniGq in the ligand-free state using cryo-electron microscopy (cryo-EM). Our structural analysis and functional experiments reveal that the second extracellular loop (ECL2) of GPR99 occupies the orthosteric binding pocket, thereby promoting receptor self-activation. Moreover, we observe structural water molecules forming an extended polar network that connects ECL2 and the binding pocket, intricately linking these elements to the receptor’s functional activity. Structure-based mutagenesis experiments further validate the critical role of ECL2 in intracellular signal transduction of GPR99, offering a structural basis for exploring its function under physiological or pathological conditions. Additionally, these findings also provide a crucial theoretical framework for the design of drugs targeting GPR99.

GPR99 self-activation is promoted by the second extracellular loop (ECL2) occupying the orthosteric pocket, which is stabilized by a water-mediated polar network. These findings provide a structural basis for this receptor’s physiological roles and a theoretical framework for designing drugs targeting the receptor.

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