35 research outputs found
Structural basis of sterol recognition by human hedgehog receptor PTCH1
Hedgehog signaling is central in embryonic development and tissue regeneration. Disruption of the pathway is linked to genetic diseases and cancer. Binding of the secreted ligand, Sonic hedgehog (ShhN) to its receptor Patched (PTCH1) activates the signaling pathway. Here, we describe a 3.4-Ă… cryo-EM structure of the human PTCH1 bound to ShhNC24II, a modified hedgehog ligand mimicking its palmitoylated form. The membrane-embedded part of PTCH1 is surrounded by 10 sterol molecules at the inner and outer lipid bilayer portion of the protein. The annular sterols interact at multiple sites with both the sterol-sensing domain (SSD) and the SSD-like domain (SSDL), which are located on opposite sides of PTCH1. The structure reveals a possible route for sterol translocation across the lipid bilayer by PTCH1 and homologous transporters.ISSN:2375-254
Structure of the connexin-43 gap junction channel in a putative closed state
Gap junction channels (GJCs) mediate intercellular communication by connecting two neighbouring cells and enabling direct exchange of ions and small molecules. Cell coupling via connexin-43 (Cx43) GJCs is important in a wide range of cellular processes in health and disease (Churko and Laird, 2013; Liang et al., 2020; Poelzing and Rosenbaum, 2004), yet the structural basis of Cx43 function and regulation has not been determined until now. Here, we describe the structure of a human Cx43 GJC solved by cryo-EM and single particle analysis at 2.26 Ă… resolution. The pore region of Cx43 GJC features several lipid-like densities per Cx43 monomer, located close to a putative lateral access site at the monomer boundary. We found a previously undescribed conformation on the cytosolic side of the pore, formed by the N-terminal domain and the transmembrane helix 2 of Cx43 and stabilized by a small molecule. Structures of the Cx43 GJC and hemichannels (HCs) in nanodiscs reveal a similar gate arrangement. The features of the Cx43 GJC and HC cryo-EM maps and the channel properties revealed by molecular dynamics simulations suggest that the captured states of Cx43 are consistent with a closed state
Structural basis of adenylyl cyclase 9 activation
Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four conformations described here show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation
Structural basis of adenylyl cyclase 9 activation
Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four conformations described here show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation
Systematic identification of structure-specific protein–protein interactions
The physical interactome of a protein can be altered upon perturbation, modulating cell physiology and contributing to disease. Identifying interactome differences of normal and disease states of proteins could help understand disease mechanisms, but current methods do not pinpoint structure-specific PPIs and interaction interfaces proteome-wide. We used limited proteolysis–mass spectrometry (LiP–MS) to screen for structure-specific PPIs by probing for protease susceptibility changes of proteins in cellular extracts upon treatment with specific structural states of a protein. We first demonstrated that LiP–MS detects well-characterized PPIs, including antibody–target protein interactions and interactions with membrane proteins, and that it pinpoints interfaces, including epitopes. We then applied the approach to study conformation-specific interactors of the Parkinson’s disease hallmark protein alpha-synuclein (aSyn). We identified known interactors of aSyn monomer and amyloid fibrils and provide a resource of novel putative conformation-specific aSyn interactors for validation in further studies. We also used our approach on GDP- and GTP-bound forms of two Rab GTPases, showing detection of differential candidate interactors of conformationally similar proteins. This approach is applicable to screen for structure-specific interactomes of any protein, including posttranslationally modified and unmodified, or metabolite-bound and unbound protein states
Characterisation of the ligand binding sites in the translocator protein TSPO using the chimeric bacterial-mammalian constructs
The translocator protein TSPO is in an important diagnostic and therapeutic target in a range of pathologies, including neuroinflammation and cancer. Despite the availability of several structures of TSPO homologues, our understanding of the molecular determinants that govern high-affinity interactions of TSPO with its ligands is incomplete. Here, in order to decipher the key structural elements of TSPO responsible for interactions with its ligands, we designed a panel of chimeric proteins mimicking the mammalian substrate binding site grafted onto the backbone of the Rhodobacter sphaeroides TSPO homologue, RsTSPO. One of the designed chimeric constructs, RsMouse, could be heterologously expressed and displayed improved binding affinities for the known TSPO drugs diazepam, PK11195 and NBD-FGIN-1-27. Furthermore, the chimeric protein had improved interactions with NBD-cholesterol, a fluorescent analogue of the presumed natural substrate of TSPO. Partial modifications of the transmembrane helix bundle in the chimeric construct differentially affected binding of the TSPO drugs and the natural substrates of TSPO, consistent with the presence of multiple ligand binding sites in the protein. Based on the available structures of TSPO homologues, the substrate interactions may involve a lateral opening of the protein in the TM1-3, and stabilisation of TM4-5 is important for drug-like ligand binding. These observations are consistent with our experimental results, which show that the determinants of high-affinity ligand interactions of TSPO are distinct for different classes of ligands.ISSN:1046-5928ISSN:1096-027
Expression and purification of the mammalian translocator protein for structural studies
<div><p>The translocator protein (TSPO) is an 18 kDa polytopic membrane protein of the outer mitochondrial membrane, abundantly present in the steroid-synthesising cells. TSPO has been linked to a number of disorders, and it is recognised as a promising drug target with a range of potential medical applications. Structural and biochemical characterisation of a mammalian TSPO requires expression and purification of the protein of high quality in sufficiently large quantities. Here we describe detailed procedures for heterologous expression and purification of mammalian TSPO in HEK293 cells. We demonstrate that the established procedures can be used for untagged TSPO as well as for C-terminally fused TSPO constructs. Our protocol can be routinely used to generate high-quality TSPO preparations for biochemical and structural studies.</p></div
Optimisation of the TSPO-YFP fusion constructs.
<p>(A) Construct design for TSPO-YFP fusions. The linker between the proteins was shortened with two amino acid steps. (B): Sequence alignment of linker region for all five fusion constructs. (C) Expression level of the best clone of the TSPO-YFP fusion constructs after stable cell line generation analysed by FSEC.</p
Overview of the screening procedures performed for homologue selection.
<p>(A) Small-scale expression test: in-gel fluorescence showing the expression levels of C-terminal GFP-10xHis constructs expressed in HEK293T cells and solubilised in 1% DM (1 human, 2 mouse, 3 rat, 4 dog, 5 bovine, 6 pig, 7 sheep, 8 chicken, 9 <i>Xenopus laevis</i>, 10 <i>danio rerio</i>, 11 <i>danaus plexippus</i>, 12 <i>drosophila melanogaster</i>). (B) Radioligand binding assay with [<sup>3</sup>H]PK11195 performed with all 12 C-terminally GFP-tagged TSPO homologues. (C) Thermostability of nine eukaryotic TSPO homologues assessed by incubating the in DDM solubilised proteins for one hour at a range of temperatures and subsequent analysis by FSEC. Control sample was incubated at 4°C (D) Small-micelle detergent screen of dog, bovine, pig and sheep TSPO. DDM-solubilised TSPO was diluted 10-fold into a buffer containing a secondary detergent, analysed by FSEC and the peak height was compared to the DDM-solubilised control sample.</p
Stable HEK293 cell line expressing a cleavable TSPO.
<p>Comparison of the best clones of bovine and pig TSPO using FSEC. Normalised to total protein concentration with Bradford assay, determined using the cell lysates. Pig TSPO shows a 2-fold higher expression compared to bovine TSPO and was therefore chosen for subsequent large-scale expression.</p