Structural biology research center
Structural biology research center
Irannejad R.,University of California at San Francisco |
Tomshine J.C.,University of California at San Francisco |
Tomshine J.R.,University of California at San Francisco |
Chevalier M.,University of California at San Francisco |
And 8 more authors.
Nature | Year: 2013
A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited or no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute to the acute G-protein-mediated response. Evidence supporting these various latter hypotheses is indirect or subject to alternative interpretation, and it remains unknown if endosome-localized GPCRs are even present in an active form. Here we describe the application of conformation-specific single-domain antibodies (nanobodies) to directly probe activation of the β 2-adrenoceptor, a prototypical GPCR, and its cognate G protein, G s (ref. 12), in living mammalian cells. We show that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane, and suggest a versatile strategy for probing dynamic conformational change in vivo. © 2013 Macmillan Publishers Limited. All rights reserved.
Efremov R.G.,Max Planck Institute of Molecular Physiology |
Efremov R.G.,Structural Biology Research Center |
Efremov R.G.,Vrije Universiteit Brussel |
Leitner A.,ETH Zurich |
And 3 more authors.
Nature | Year: 2015
Muscle contraction is initiated by the release of calcium (Ca2+) from the sarcoplasmic reticulum into the cytoplasm of myocytes through ryanodine receptors (RyRs). RyRs are homotetrameric channels with a molecular mass of more than 2.2 megadaltons that are regulated by several factors, including ions, small molecules and proteins. Numerous mutations in RyRs have been associated with human diseases. The molecular mechanism underlying the complex regulation of RyRs is poorly understood. Using electron cryomicroscopy, here we determine the architecture of rabbit RyR1 at a resolutionof 6.1A° .We showthat the cytoplasmicmoiety ofRyR1containstwo largea-solenoiddomainsandseveralsmaller domains, with folds suggestive of participation in protein-protein interactions. The transmembrane domain represents a chimaera of voltage-gated sodiumand pH-activated ion channels.Weidentify the calcium-binding EF-hand domain and show that it functions as a conformational switch allosterically gating the channel. © 2015 Macmillan Publishers Limited.
De Meyer T.,Vlaams Institute for Biotechnology |
De Meyer T.,Ghent University |
Muyldermans S.,Structural Biology Research Center |
Muyldermans S.,Vrije Universiteit Brussel |
And 2 more authors.
Trends in Biotechnology | Year: 2014
Since the serendipitous discovery 20 years ago of bona fide camelid heavy-chain antibodies, their single-domain antigen-binding fragments, known as VHHs or nanobodies, have received a progressively growing interest. As a result of the beneficial properties of these stable recombinant entities, they are currently highly valued proteins for multiple applications, including fundamental research, diagnostics, and therapeutics. Today, with the original patents expiring, even more academic and industrial groups are expected to explore innovative VHH applications. Here, we provide a thorough overview of novel implementations of VHHs as research and diagnostic tools, and of the recently evaluated production platforms for several VHHs and VHH-derived antibody formats. © 2014 Elsevier Ltd.
Schueler-Furman O.,Hebrew University of Jerusalem |
Wodak S.J.,Structural Biology Research Center
Current Opinion in Structural Biology | Year: 2016
Allosteric regulation plays a key role in many biological processes, such as signal transduction, transcriptional regulation, and many more. It is rooted in fundamental thermodynamic and dynamic properties of macromolecular systems that are still poorly understood and are moreover modulated by the cellular context. Here we review the computational approaches used in the investigation of allosteric processes in protein systems. We outline how the models of allostery have evolved from their initial formulation in the sixties to the current views, which more fully account for the roles of the thermodynamic and dynamic properties of the system. We then describe the major classes of computational approaches employed to elucidate the mechanisms of allostery, the insights they have provided, as well as their limitations. We complement this analysis by highlighting the role of computational approaches in promising practical applications, such as the engineering of regulatory modules and identifying allosteric binding sites. © 2016
Ehrnstorfer I.A.,University of Zürich |
Geertsma E.R.,University of Zürich |
Geertsma E.R.,Goethe University Frankfurt |
Pardon E.,Structural biology research center |
And 4 more authors.
Nature Structural and Molecular Biology | Year: 2014
Members of the SLC11 (NRAMP) family transport iron and other transition-metal ions across cellular membranes. These membrane proteins are present in all kingdoms of life with a high degree of sequence conservation. To gain insight into the determinants of ion selectivity, we have determined the crystal structure of Staphylococcus capitis DMT (ScaDMT), a close prokaryotic homolog of the family. ScaDMT shows a familiar architecture that was previously identified in the amino acid permease LeuT. The protein adopts an inward-facing conformation with a substrate-binding site located in the center of the transporter. This site is composed of conserved residues, which coordinate Mn 2+, Fe 2+ and Cd 2+ but not Ca 2+. Mutations of interacting residues affect ion binding and transport in both ScaDMT and human DMT1. Our study thus reveals a conserved mechanism for transition-metal ion selectivity within the SLC11 family.
Tompa P.,Structural Biology Research Center |
Tompa P.,Vrije Universiteit Brussel |
Tompa P.,Hungarian Academy of Sciences
Chemical Society Reviews | Year: 2016
Signal transduction is the primary process by which cells respond to changes in their physical and chemical environments. Cellular response is initiated through a signaling protein (a receptor), which interacts with the "signal", most often a novel molecule outside or inside the cell. The mechanism of activation of the receptor is a conformational change and/or covalent modification, which then sets in motion a signaling pathway, i.e. a cascade of modification and binding events that relay and amplify the message to eventually alter the state of the cell. In reflection of this general perception, concepts such as the "second messenger" and the "phosphorylation cascade" dominate our views of signal transduction. The idea I advocate here is that the non-covalent change in protein conformation itself might serve as the initial or intermittent "signal" in the cascade, and it is often the primary event being recognized and interpreted by downstream receptor(s). This signaling principle is intertwined with many other cellular regulatory concepts, such as (pathway) allostery, conformational spread, induced folding/unfolding, conformational memory, the hierarchical assembly of complexes, and the action of regulatory chaperones and prions. By elaborating on many examples and also recent advances in experimental methodology, I show that conformational signaling, although thus far underappreciated, is a general and robust signaling principle that most of the time operates in close interplay with covalent signals in the cell. © 2016 The Royal Society of Chemistry.
Hubin E.,University of Twente |
Hubin E.,Vrije Universiteit Brussel |
Hubin E.,Structural Biology Research Center |
Van Nuland N.A.J.,Vrije Universiteit Brussel |
And 3 more authors.
Cellular and Molecular Life Sciences | Year: 2014
The aggregation and deposition of the amyloid-β peptide (Aβ) in the brain has been linked with neuronal death, which progresses in the diagnostic and pathological signs of Alzheimer's disease (AD). The transition of an unstructured monomeric peptide into self-assembled and more structured aggregates is the crucial conversion from what appears to be a harmless polypeptide into a malignant form that causes synaptotoxicity and neuronal cell death. Despite efforts to identify the toxic form of Aβ, the development of effective treatments for AD is still limited by the highly transient and dynamic nature of interconverting forms of Aβ. The variability within the in vivo "pool" of different Aβ peptides is another complicating factor. Here we review the dynamical interplay between various components that influence the heterogeneous Aβ system, from intramolecular Aβ flexibility to intermolecular dynamics between various Aβ alloforms and external factors. The complex dynamics of Aβ contributes to the causative role of Aβ in the pathogenesis of AD. © 2014 The Author(s).
Nagae T.,Nagoya University |
Kawamura T.,Nagoya University |
Chavas L.M.G.,Structural Biology Research Center |
Niwa K.,Nagoya University |
And 3 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2012
Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressure-denaturation process of IPMDH. © 2012 International Union of Crystallography Printed in Singapore - all rights reserved.
Versees W.,Vrije Universiteit Brussel |
Versees W.,Structural Biology Research Center
Structure | Year: 2015
In this issue of Structure, Glatt and colleagues report the structure of the Kti11/Kti13 heterodimer. This study reveals how dimerization and Fe2+ binding are required for modification of both tRNA and EF2, thus suggesting a mechanism for regulation of translation elongation via two different pathways. © 2015 Elsevier Ltd.
Sounier R.,Montpellier University |
Mas C.,Montpellier University |
Steyaert J.,Vrije Universiteit Brussel |
Steyaert J.,Structural Biology Research Center |
And 7 more authors.
Nature | Year: 2015
μ-Opioid receptors (μORs) are G-protein-coupled receptors that are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone. The recent structures of the μOR in inactive and agonist-induced active states (Huang et al., ref. 2) provide snapshots of the receptor at the beginning and end of a signalling event, but little is known about the dynamic sequence of events that span these two states. Here we use solution-state NMR to examine the process of μOR activation using a purified receptor (mouse sequence) preparation in an amphiphile membrane-like environment. We obtain spectra of the μOR in the absence of ligand, and in the presence of the high-affinity agonist BU72 alone, or with BU72 and a G protein mimetic nanobody. Our results show that conformational changes in transmembrane segments 5 and 6 (TM5 and TM6), which are required for the full engagement of a G protein, are almost completely dependent on the presence of both the agonist and the G protein mimetic nanobody, revealing a weak allosteric coupling between the agonist-binding pocket and the G-protein-coupling interface (TM5 and TM6), similar to that observed for the β2-adrenergic receptor. Unexpectedly, in the presence of agonist alone, we find larger spectral changes involving intracellular loop 1 and helix 8 compared to changes in TM5 and TM6. These results suggest that one or both of these domains may play a role in the initial interaction with the G protein, and that TM5 and TM6 are only engaged later in the process of complex formation. The initial interactions between the G protein and intracellular loop 1 and/or helix 8 may be involved in G-protein coupling specificity, as has been suggested for other family A G-protein-coupled receptors. © 2015 Macmillan Publishers Limited. All rights reserved.