Cryo-EM Study of Patched in Lipid Nanodisc Suggests a Structural Basis for Its Clustering in Caveolae

Abstract

The 12-transmembrane protein Patched (Ptc1) acts as a negative modulator for Hedgehog (Hh) signaling by depleting sterols in the inner leaflet of the plasma membrane that are required for the activation of downstream regulators in this pathway. The inhibition is alleviated by its proteinaceous ligand Hh. Ptc1 and its co-receptors are locally concentrated in invaginated microdomains known as caveolae for efficient signal transduction. Here, we reconstitute the mouse Ptc1 into lipid nanodiscs and determine its structure in this native-like membrane environment using single-particle cryo-electron microscopy (cryo-EM). The structure is overall similar to the mouse and human Ptc1 structures in amphipol and detergent environment but displays various conformational differences in the transmembrane region. Although most particles show a Ptc1 monomer in the artificial membrane, we observe Ptc1 dimers with distinct interaction patterns and different membrane curvatures. Analyzing the bent membranes reveals that the membrane curvatures are close to that reported in caveolae. We find that a “hand-shake” region rich in hydrophobic and aromatic residues in the ectodomain mediates inter-Ptc1 interactions under different membrane curvatures. We speculate that the plasticity of the hydrophobic interactions is important to accommodate membrane bending. Altogether, our data provide a plausible framework for Ptc1 clustering in the highly curved caveolae membrane.