The Hedgehog (Hh) pathway controls embryonic development and postnatal tissue maintenance and regeneration. Inhibition of Hh receptor Patched (Ptch) by the Hh ligands relieves suppression of signaling cascades. Here, we report the cryo-EM structure of tetrameric Ptch1 in complex with the palmitoylated N-terminal signaling domain of human Sonic hedgehog (ShhN p ) at a 4:2 stoichiometric ratio. The structure shows that four Ptch1 protomers are organized as a loose dimer of dimers. Each dimer binds to one ShhN p through two distinct inhibitory interfaces, one mainly through the N-terminal peptide and the palmitoyl moiety of ShhN p and the other through the Ca 2+ -mediated interface on ShhN p . Map comparison reveals that the cholesteryl moiety of native ShhN occupies a recently identified extracellular steroid binding pocket in Ptch1. Our structure elucidates the tetrameric assembly of Ptch1 and suggests an asymmetric mode of action of the Hh ligands for inhibiting the potential cholesterol transport activity of Ptch1.

Intracellular Ca signalling processes are fundamental to muscle contraction, neurotransmitter release, cell growth and apoptosis. Release of Ca from the intracellular stores is supported by a series of ion channels in sarcoplasmic or endoplasmic reticulum (SR/ER). Among them, two isoforms of the trimeric intracellular cation (TRIC) channel family, named TRIC-A and TRIC-B, modulate the release of Ca through the ryanodine receptor or inositol triphosphate receptor, and maintain the homeostasis of ions within SR/ER lumen. Genetic ablations or mutations of TRIC channels are associated with hypertension, heart disease, respiratory defects and brittle bone disease. Despite the pivotal function of TRIC channels in Ca signalling, their pore architectures and gating mechanisms remain unknown. Here we present the structures of TRIC-B1 and TRIC-B2 channels from Caenorhabditis elegans in complex with endogenous phosphatidylinositol-4,5-biphosphate (PtdIns(4,5)P, also known as PIP) lipid molecules. The TRIC-B1/B2 proteins and PIP assemble into a symmetrical homotrimeric complex. Each monomer contains an hourglass-shaped hydrophilic pore contained within a seven-transmembrane-helix domain. Structural and functional analyses unravel the central role of PIP in stabilizing the cytoplasmic gate of the ion permeation pathway and reveal a marked Ca-induced conformational change in a cytoplasmic loop above the gate. A mechanistic model has been proposed to account for the complex gating mechanism of TRIC channels.

Experiments with the transmembrane (TM) domains of the glycoprotein (GP) Ib-IX complex have indicated that the associations between the TM domains of these subunits play an important role in the proper assembly of the complex. As a first step toward understanding these associations, we previously found that the Ibb TM domain dimerized strongly in Escherichia coli cell membranes and led to Ibb TM-CYTO (cytoplasmic domain) dimerization in the SDS-PAGE assay, while neither Iba nor IX TM-CYTO was able to dimerize. In this study, we used the TOXCAT assay to probe the Ibb TM domain dimerization interface by Ala-and Leu-scanning mutagenesis. Our results show that this interface is based on a leucine zipper-like heptad repeat pattern of amino acids. Mutating either one of polar residues Gln129 or His139 to Leu or Ala disrupted Ibb TM dimerization dramatically, indicating that polar residues might form part of the leucine zipperbased dimerization interface. Furthermore, these specific mutational effects in the TOXCAT assay were confirmed in the thiol-disulfide exchange and SDS-PAGE assays. The computational modeling studies further revealed that the most likely leucine zipper interface involves hydrogen bonding of Gln129 and electrostatic interaction of the His139 side chain. Correlation of computer modeling results with experimental mutagenesis studies on the Ibb TM domain may provide insights for understanding the role of the association of TM domains on the assembly of GP Ib-IX complex.