Physical basis for the interaction between Drosophila ROS1 and the GPCR BOSS

Drosophila ROS1 (dROS1, Sevenless) is a receptor tyrosine kinase (RTK) essential for the differentiation of Drosophila R7 photoreceptor cells ^{1, 2} . Activation of dROS1 is mediated by binding to the extracellular region (ECR) of the GPCR (G protein coupled receptor) BOSS (Bride Of Sevenless) on adjacent cells ^{1, 3, 4} . Genetic evidence together with in vitro activity assays confirmed the activation of dROS1 by BOSS and identified subsequent downstream signaling pathways including SOS (Son of Sevenless) ^{1, 5} . However, the physical basis for how dROS1 interacts with the GPCR BOSS has long remained unknown. Here we provide the first structure, using Cryo-Electron Microscopy (CryoEM), of dROS1’s extracellular region, which mediates ligand binding. We show that the N-terminal region of dROS1 adopts a folded-over conformation harboring a novel structural domain. We further narrowed down the interacting binding epitopes on both dROS1 and BOSS. This includes a beta-strand in dROS1’s third Fibronectin type III (FNIII) domain and the C-terminal portion of BOSS’ ECR. Our mutagenesis studies, coupled with AlphaFold complex predictions, support a binding interaction mediated by a hydrophobic interaction and beta-strand augmentation between these regions. Our findings provide a fundamental understanding of the regulatory function of dROS1 and further provide mechanistic insight into the human ortholog and oncogene ROS1.