Insights into divalent cation regulation and G13-coupling of orphan receptor GPR35

<jats:title>Abstract</jats:title><jats:p>Endogenous ions play important roles in the function and pharmacology of G protein-coupled receptors (GPCRs) with limited atomic evidence. In addition, compared with G protein subtypes G<jats:sub>s</jats:sub>, G<jats:sub>i/o</jats:sub>, and G<jats:sub>q/11</jats:sub>, insufficient structural evidence is accessible to understand the coupling mechanism of G<jats:sub>12/13</jats:sub> protein by GPCRs. Orphan receptor GPR35, which is predominantly expressed in the gastrointestinal tract and is closely related to inflammatory bowel diseases (IBDs), stands out as a prototypical receptor for investigating ionic modulation and G<jats:sub>13</jats:sub> coupling. Here we report a cryo-electron microscopy structure of G<jats:sub>13</jats:sub>-coupled GPR35 bound to an anti-allergic drug, lodoxamide. This structure reveals a novel divalent cation coordination site and a unique ionic regulatory mode of GPR35 and also presents a highly positively charged binding pocket and the complementary electrostatic ligand recognition mode, which explain the promiscuity of acidic ligand binding by GPR35. Structural comparison of the GPR35–G<jats:sub>13</jats:sub> complex with other G protein subtypes-coupled GPCRs reveals a notable movement of the C-terminus of α5 helix of the Gα<jats:sub>13</jats:sub> subunit towards the receptor core and the least outward displacement of the cytoplasmic end of GPR35 TM6. A featured ‘methionine pocket’ contributes to the G<jats:sub>13</jats:sub> coupling by GPR35. Together, our findings provide a structural basis for divalent cation modulation, ligand recognition, and subsequent G<jats:sub>13</jats:sub> protein coupling of GPR35 and offer a new opportunity for designing GPR35-targeted drugs for the treatment of IBDs.</jats:p>