Extracellular loop 2 of G protein-coupled olfactory receptors is critical for odorant recognition

Abstract

G protein-coupled olfactory receptors (ORs) enable us to detect innumerous odorants. They are also ectopically expressed, emerging as attractive drug targets. ORs can be promiscuous or highly specific, which is part of Nature′s strategy for odor discrimination. This work demonstrates that the extracellular loop 2 (ECL2) plays critical roles in OR promiscuity and specificity. Using site-directed mutagenesis and molecular modeling, we constructed 3D OR models in which ECL2 forms a lid of the orthosteric pocket. ECL2 controls the shape and the volume of the odorant-binding pocket, maintains the pocket hydrophobicity and acts as a gatekeeper of odorant binding. The interplay between the specific orthosteric pocket and the variable, less specific ECL2 controls OR specificity and promiscuity. The 3D models enabled virtual screening of new OR agonists and antagonists, exhibiting 78% hit rate in cell assays. This approach can be generalized to structure-based ligand screening for other GPCRs that lack high-resolution 3D structures.

Modeling the Orthosteric Binding Site of the G Protein-Coupled Odorant Receptor OR5K1

“…ECL2 is the largest and most structurally diverse extracellular loop of GPCRs, 63 and those of ORs are among the longest ECL2 in class A GPCR. 64 Loop modeling is highly challenging when sequence length reaches the size of the ECL2. [65][66][67] We remodeled this region using templates with higher similarities in terms of length and sequence composition (Figures S2 and S3).…”

“…Da with high-to-moderate hydrophobicity and their binding to ORs is driven by shape complementarity and mostly hydrophobic interactions. 64,75 L104 3.32 is conserved in 98% of orthologs investigated across 51 species, except for the receptor of the new world monkey Aotus nancymaae (XP_012332612.1), where a rather conservative amino acid exchange replaced the leucine at position 104 by an isoleucine (Figure S7, Table S5). Similarly, L255 6.51 of OR5K1 is conserved in 96% of all orthologs, except for the receptors of Aotus nancymaae, Loxodonta africana (African elephant, XP_003418985.1), and Urocitellus parryii (Arctic ground squirrel, XP_026258216.1).…”

“…Odorant molecules are typically small organic compounds of less than 300 Da with high-to-moderate hydrophobicity and their binding to ORs is driven by shape complementarity and mostly hydrophobic interactions. 64, 75…”

“…Odorant molecules are typically small organic compounds of less than 300 Da with high-tomoderate hydrophobicity and their binding to ORs is driven by shape complementarity and mostly hydrophobic interactions. 71,95 ORs are class A GPCRs for which we do not have experimental structures and that share very low sequence identity with non-sensory GPCRs.…”

“…The ECL2 is the largest and most structurally diverse extracellular loop of GPCRs, 70 and those of ORs are among the longest ECL2 in class A GPCRs. 71 Loop modeling is highly challenging when sequence length reaches the size of the OR ECL2. [72][73][74] A template selection based on sequence identity is rather difficult considering the high sequence and shape variability.…”