Abstract
Allosteric modulators offer opportunities for pathway-selective GPCR signalling, but the structural mechanisms enabling biased allosteric modulation remain unclear. Here we identify a helix 8-proximate allosteric site in the immune-metabolic receptor GPR84 and define how it achieves Gi-biased signalling. Cryo-EM structures of the GPR84-Gi complexes bound to the orthosteric agonist OX04539 alone or in combination with the positive allosteric modulator (PAM) PSB-16671 reveal that PSB-16671 binds at the interface of TM1, TM7, and helix 8, a location distinct from previously characterized GPCR allosteric pockets. Molecular dynamics simulations and mutagenesis uncover a polar interaction network linking orthosteric and allosteric sites through conserved residues including Asp662.50, Asn1043.36, and Asn3627.45. Unexpectedly, disrupting this network enhances allosteric cooperativity, indicating that conformational flexibility within the network is essential for allosteric communication. PSB-16671 stabilizes a receptor conformation with pronounced TM6 displacement that favours Gi coupling while disfavouring β-arrestin recruitment. This Gi-biased profile sustains macrophage phagocytosis of cancer cells without the desensitization induced by balanced agonists. Sequence analysis suggests that helix 8-proximate allosteric sites may be broadly targetable across class A GPCRs, while receptor-specific contacts enable selective modulation. These findings establish structural and mechanistic principles for biased allosteric modulation applicable beyond GPR84.