MAPPING THE SEQUENCE FITNESS LANDSCAPE OF CLASS C GPCRS FOR DETERMING CRITICAL REGIONS FOR ACTIVE AGONIST BINDING
Jeremiah Heredia, Erik Procko.
University of Illinois at Urbana Champaign, Urbana, IL.
G‐protein coupled receptors (GPCRs) are the largest class of membrane proteins and are major drug targets, yet most GPCR structures remain unsolved. One unusual subgroup is the class C GPCR proteins, which includes members for the recognition of neurotransmitters and sweet and savory tasting substances. Class-C GPCRs are dimeric and characterized by an extracellular hydrophilic ligand binding domain (LBD), an extracellular cysteine‐rich domain, and a C‐terminal 7 transmembrane domain. Receptor activation requires ligand interactions with the LBD, but it is unknown how ligand binding stabilizes a conformational change many angstroms away in the membrane‐spanning region. Our lab is researching how small molecule ligands are recognized and how ligand binding induces downstream signaling in class-C GPCRs. We have developed a method for comprehensively mapping the sequence‐fitness landscape of a GPCR. All possible single amino acid substitutions are combined in a library that is sorted for expression and high affinity ligand interactions. Deep sequencing pre‐ and post‐sort allows calculation of the phenotypic fitness of all sequence variants in a single experiment, and residues that are conserved for function are revealed. Our initial efforts have focused on simple GPCRs that recognize chemokines, and we hypothesize that planned sequence‐fitness landscape mapping of multidomain class-C GPCRs will determine regions critical for adopting their active, agonist‐bound conformation. The development of these saturation mutagenesis libraries could also be applied for the characterization of other receptor families and for engineering transmembrane proteins with new or improved properties.