Structures of the R-type human Cav2.3 channel reveal conformational crosstalk of the intracellular segments.

The R-type voltage-gated Ca2+ (Cav) channels Cav2.3, widely expressed in neuronal and neuroendocrine cells, represent potential drug targets for pain, seizures, epilepsy, and Parkinson's disease. Despite their physiological importance, there have lacked selective small-molecule inhibitors targeting these channels. High-resolution structures may aid rational drug design. Here, we report the cryo-EM structure of human Cav2.3 in complex with α2δ-1 and ß3 subunits at an overall resolution of 3.1 Å. The structure is nearly identical to that of Cav2.2, with VSDII in the down state and the other three VSDs up. A phosphatidylinositol 4,5-bisphosphate (PIP2) molecule binds to the interface of VSDII and the tightly closed pore domain. We also determined the cryo-EM structure of a Cav2.3 mutant in which a Cav2-unique cytosolic helix in repeat II (designated the CH2II helix) is deleted. This mutant, named ΔCH2, still reserves a down VSDII, but PIP2 is invisible and the juxtamembrane region on the cytosolic side is barely discernible. Our structural and electrophysiological characterizations of the wild type and ΔCH2 Cav2.3 show that the CH2II helix stabilizes the inactivated conformation of the channel by tightening the cytosolic juxtamembrane segments, while CH2II helix is not necessary for locking the down state of VSDII.

Extracellular domain of PepT1 interacts with TM1 to facilitate substrate transport.

Mammalian peptide transporters, PepT1 and PepT2, mediate uptake of small peptides and are essential for their absorption. PepT also mediates absorption of many drugs and prodrugs to enhance their bioavailability. PepT has twelve transmembrane (TM) helices that fold into an N-terminal domain (NTD, TM1-6) and a C-terminal domain (CTD, TM7-12) and has a large extracellular domain (ECD) between TM9-10. It is well recognized that peptide transport requires movements of the NTD and CTD, but the role of the ECD in PepT1 remains unclear. Here we report the structure of horse PepT1 encircled in lipid nanodiscs and captured in the inward-open apo conformation. The structure shows that the ECD bridges the NTD and CTD by interacting with TM1. Deletion of ECD or mutations to the ECD-TM1 interface impairs the transport activity. These results demonstrate an important role of ECD in PepT1 and enhance our understanding of the transport mechanism in PepT1.

Inhibition of tetrameric Patched1 by Sonic Hedgehog through an asymmetric paradigm.

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 (ShhNp) 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 ShhNp through two distinct inhibitory interfaces, one mainly through the N-terminal peptide and the palmitoyl moiety of ShhNp and the other through the Ca2+-mediated interface on ShhNp. 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.

Pore architecture of TRIC channels and insights into their gating mechanism.

Intracellular Ca2+ signalling processes are fundamental to muscle contraction, neurotransmitter release, cell growth and apoptosis. Release of Ca2+ 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 Ca2+ 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 Ca2+ 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)P2, also known as PIP2) lipid molecules. The TRIC-B1/B2 proteins and PIP2 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 PIP2 in stabilizing the cytoplasmic gate of the ion permeation pathway and reveal a marked Ca2+-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.

The dimerization interface of the glycoprotein Ibß transmembrane domain corresponds to polar residues within a leucine zipper motif.

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 Ibß TM domain dimerized strongly in Escherichia coli cell membranes and led to Ibß TM-CYTO (cytoplasmic domain) dimerization in the SDS-PAGE assay, while neither Ibα nor IX TM-CYTO was able to dimerize. In this study, we used the TOXCAT assay to probe the Ibß 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 Ibß TM dimerization dramatically, indicating that polar residues might form part of the leucine zipper-based 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 Ibß TM domain may provide insights for understanding the role of the association of TM domains on the assembly of GP Ib-IX complex.