Showing papers on "Structural biology published in 2011"

Structure and function of an irreversible agonist-β2 adrenoceptor complex

Abstract: G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signalling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs, the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human β(2) adrenergic receptor (β(2)AR) as a guide, we designed a β(2)AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent β(2)AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound β(2)AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method, and determined its structure at 3.5 A resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 μs) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.

The structural basis of agonist-induced activation in constitutively active rhodopsin

Abstract: Structural studies of active states of the visual pigment rhodopsin, a G protein-coupled receptor, have previously been limited to apoprotein or opsin forms that do not contain the agonist all-trans-retinal. Two groups now report structures that reveal more details of the transformations involved in rhodopsin activation. Choe et al. solve the X-ray crystal structure of the metarhodopsin II intermediate of the photoreceptor rhodopsin, and Standfuss et al. determine the structure of a constitutively active mutant of rhodopsin bound to a peptide derived from the C-terminus of the G protein transducin. This study solves the X-ray crystal structure of a constitutively active mutant of rhodopsin, a G-protein-coupled receptor, bound to a peptide derived from the C-terminus of the G protein transducin. Comparison of this structure with the structure of ground-state rhodopsin suggests how translocation of the retinal β-ionone ring leads to a rotational tilt of transmembrane helix 6, the critical conformational change that occurs upon activation. G-protein-coupled receptors (GPCRs) comprise the largest family of membrane proteins in the human genome and mediate cellular responses to an extensive array of hormones, neurotransmitters and sensory stimuli. Although some crystal structures have been determined for GPCRs, most are for modified forms, showing little basal activity, and are bound to inverse agonists or antagonists. Consequently, these structures correspond to receptors in their inactive states. The visual pigment rhodopsin is the only GPCR for which structures exist that are thought to be in the active state1,2. However, these structures are for the apoprotein, or opsin, form that does not contain the agonist all-trans retinal. Here we present a crystal structure at a resolution of 3 A for the constitutively active rhodopsin mutant Glu 113 Gln3,4,5 in complex with a peptide derived from the carboxy terminus of the α-subunit of the G protein transducin. The protein is in an active conformation that retains retinal in the binding pocket after photoactivation. Comparison with the structure of ground-state rhodopsin6 suggests how translocation of the retinal β-ionone ring leads to a rotation of transmembrane helix 6, which is the critical conformational change on activation7. A key feature of this conformational change is a reorganization of water-mediated hydrogen-bond networks between the retinal-binding pocket and three of the most conserved GPCR sequence motifs. We thus show how an agonist ligand can activate its GPCR.

Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching

Abstract: The transport of small molecules across the inner mitochondrial membrane is catalysed by a large family of membrane proteins called mitochondrial carriers. More than 40 different carriers have so far been identified to selectively translocate different substrates, but only one crystal structure is available — that of the bovine ADP/ATP carrier (ANT1). Now the structure of mitochondrial uncoupling protein 2 (UCP2), a member of the carrier family that translocates protons across the mitochondrial inner membrane, has been determined using a solution nuclear magnetic resonance (NMR) method. Its overall structure of resembles that of ANT1 — despite their low sequence identity — but the matrix side of the channel is substantially more open in UCP2. This method overcomes some of the challenges associated with using NMR spectroscopy to determine the structure of membrane proteins, so it seems likely that it will be possible to use the approach to solve the high-resolution NMR structures of other membrane proteins of comparable size. Mitochondrial uncoupling protein 2 (UCP2) is an integral membrane protein in the mitochondrial anion carrier protein family, the members of which facilitate the transport of small molecules across the mitochondrial inner membrane1,2. When the mitochondrial respiratory complex pumps protons from the mitochondrial matrix to the intermembrane space, it builds up an electrochemical potential2. A fraction of this electrochemical potential is dissipated as heat, in a process involving leakage of protons back to the matrix2. This leakage, or ‘uncoupling’ of the proton electrochemical potential, is mediated primarily by uncoupling proteins2. However, the mechanism of UCP-mediated proton translocation across the lipid bilayer is unknown. Here we describe a solution-NMR method for structural characterization of UCP2. The method, which overcomes some of the challenges associated with membrane-protein structure determination3, combines orientation restraints derived from NMR residual dipolar couplings (RDCs) and semiquantitative distance restraints from paramagnetic relaxation enhancement (PRE) measurements. The local and secondary structures of the protein were determined by piecing together molecular fragments from the Protein Data Bank that best fit experimental RDCs from samples weakly aligned in a DNA nanotube liquid crystal. The RDCs also determine the relative orientation of the secondary structural segments, and the PRE restraints provide their spatial arrangement in the tertiary fold. UCP2 closely resembles the bovine ADP/ATP carrier (the only carrier protein of known structure4), but the relative orientations of the helical segments are different, resulting in a wider opening on the matrix side of the inner membrane. Moreover, the nitroxide-labelled GDP binds inside the channel and seems to be closer to transmembrane helices 1–4. We believe that this biophysical approach can be applied to other membrane proteins and, in particular, to other mitochondrial carriers, not only for structure determination but also to characterize various conformational states of these proteins linked to substrate transport.

Structural Biology of the Toll-Like Receptor Family

Abstract: Innate immune receptors respond to common structural patterns in microbial molecules and are called pattern recognition receptors. Toll-like receptors (TLRs) play critical roles in the innate immune system by recognizing microbial lipids, carbohydrates, nucleic acids, and proteins. Precise definition of the ligand "pattern" of TLRs has been difficult to determine primarily owing to a lack of high-resolution structures. Recently, the structures of several TLR-ligand complexes and the intracellular signaling domains have been determined by X-ray crystallography. This new structural information, combined with extensive biochemical and immunological data accumulated over decades, sheds new light on ligand-recognition and -activation mechanisms. In this review, we summarize the TLR structures and discuss proposed ligand-recognition and -activation mechanisms.

Structural Analysis of Macromolecular Assemblies by Electron Microscopy

Abstract: 1.1. Light and Electron Microscopy and Their Impact in Biology To fully understand biological processes from the metabolism of a bacterium to the operation of a human brain, it is necessary to know the three-dimensional (3D) spatial arrangement and dynamics of the constituent molecules, how they assemble into complex molecular machines, and how they form functional organelles, cells, and tissues. The methods of X-ray crystallography and NMR spectroscopy can provide detailed information on molecular structure and dynamics. At the cellular level, optical microscopy reveals the spatial distribution and dynamics of molecules tagged with fluorophores. Electron microscopy (EM) overlaps with these approaches, covering a broad range from atomic to cellular structures. The development of cryogenic methods has enabled EM imaging to provide snapshots of biological molecules and cells trapped in a close to native, hydrated state.1,2 Because of the importance of macromolecular assemblies in the machinery of living cells and progress in the EM and image processing methods, EM has become a major tool for structural biology over the molecular to cellular size range. There have been tremendous advances in understanding the 3D spatial organization of macromolecules and their assemblies in cells and tissues, due to developments in both optical and electron microscopy. In light microscopy, super-resolution and single molecule methods have pushed the resolution of fluorescence images to ∼50 nm, using the power of molecular biology to fuse molecules of interest with fluorescent marker proteins.(3) X-ray cryo-tomography is developing as a method for 3D reconstruction of thicker (10 μm) hydrated samples, with resolution reaching the 15 nm resolution range.(4) In EM, major developments in instrumentation and methods have advanced the study of single particles (isolated macromolecular complexes) in vitrified solution as well as in 3D reconstruction by tomography of irregular objects such as cells or subcellular structures.1,5−7 Cryo-sectioning can be used to prepare vitrified sections of cells and tissues that would otherwise be too thick to image by transmission EM (TEM).8,9 In parallel, software improvements have facilitated 3D structure determination from the low contrast, low signal-to-noise ratio (SNR) images of projected densities provided by TEM of biological molecules.10−14 Alignment and classification of images in both 2D and 3D are key methods for improving SNR and detection and sorting of heterogeneity in EM data sets.(14) The resolution of single-particle reconstructions is steadily improving and has gone beyond 4 A for some icosahedral viruses and 5.5 A for asymmetric complexes such as ribosomes, giving a clear view of protein secondary structure elements and, in the best cases, resolving the protein or nucleic acid fold.15,16

Structural basis for the subunit assembly of the anaphase-promoting complex

Abstract: The anaphase-promoting complex or cyclosome (APC/C) is an unusually large E3 ubiquitin ligase responsible for regulating defined cell cycle transitions. Information on how its 13 constituent proteins are assembled, and how they interact with co-activators, substrates and regulatory proteins is limited. Here, we describe a recombinant expression system that allows the reconstitution of holo APC/C and its sub-complexes that, when combined with electron microscopy, mass spectrometry and docking of crystallographic and homology-derived coordinates, provides a precise definition of the organization and structure of all essential APC/C subunits, resulting in a pseudo-atomic model for 70% of the APC/C. A lattice-like appearance of the APC/C is generated by multiple repeat motifs of most APC/C subunits. Three conserved tetratricopeptide repeat (TPR) subunits (Cdc16, Cdc23 and Cdc27) share related superhelical homo-dimeric architectures that assemble to generate a quasi-symmetrical structure. Our structure explains how this TPR sub-complex, together with additional scaffolding subunits (Apc1, Apc4 and Apc5), coordinate the juxtaposition of the catalytic and substrate recognition module (Apc2, Apc11 and Apc10 (also known as Doc1)), and TPR-phosphorylation sites, relative to co-activator, regulatory proteins and substrates.

The Mechanism of Membrane-Associated Steps in Tail-Anchored Protein Insertion.

Abstract: Tail-anchored (TA) membrane proteins destined for the endoplasmic reticulum are chaperoned by cytosolic targeting factors that deliver them to a membrane receptor for insertion Although a basic framework for TA protein recognition is now emerging, the decisive targeting and membrane insertion steps are not understood Here we reconstitute the TA protein insertion cycle with purified components, present crystal structures of key complexes between these components and perform mutational analyses based on the structures We show that a committed targeting complex, formed by a TA protein bound to the chaperone ATPase Get3, is initially recruited to the membrane through an interaction with Get2 Once the targeting complex has been recruited, Get1 interacts with Get3 to drive TA protein release in an ATPase-dependent reaction After releasing its TA protein cargo, the now-vacant Get3 recycles back to the cytosol concomitant with ATP binding This work provides a detailed structural and mechanistic framework for the minimal TA protein insertion cycle

Structure and mechanism of the hexameric MecA–ClpC molecular machine

Abstract: Regulated proteolysis by ATP-dependent proteases is universal in all living cells. Bacterial ClpC, a member of the Clp/Hsp100 family of AAA+ proteins (ATPases associated with diverse cellular activities) with two nucleotide-binding domains (D1 and D2), requires the adaptor protein MecA for activation and substrate targeting. The activated, hexameric MecA–ClpC molecular machine harnesses the energy of ATP binding and hydrolysis to unfold specific substrate proteins and translocate the unfolded polypeptide to the ClpP protease for degradation. Here we report three related crystal structures: a heterodimer between MecA and the amino domain of ClpC, a heterododecamer between MecA and D2-deleted ClpC, and a hexameric complex between MecA and full-length ClpC. In conjunction with biochemical analyses, these structures reveal the organizational principles behind the hexameric MecA–ClpC complex, explain the molecular mechanisms for MecA-mediated ClpC activation and provide mechanistic insights into the function of the MecA–ClpC molecular machine. These findings have implications for related Clp/Hsp100 molecular machines. ATP-dependent proteases are found in all living cells, where they are involved in protein quality control. Clp/Hsp100 protein-unfolding enzymes, members of the AAA+ superfamily of ATP-dependent chaperones, deliver proteins to the proteolytic chamber of protease complexes. The ClpC protein unfolding unit requires the adaptor protein MecA for activation and substrate targeting to the ClpCP protease complex. A structural and biochemical analysis of the MecA–ClpC complex now reveals the organizational principles driving this important molecular machine. Regulated proteolysis by ATP-dependent proteases have a crucial role in protein quality control in cells. The Clp/Hsp100 proteins of the AAA+ superfamily of ATP-dependent chaperones unfold and translocate proteins into the proteolytic chamber of protease complexes. ClpC requires the adaptor protein MecA for activation and substrate targetting to the ClpCP protease complex. Here, a structural and biochemical analysis is presented of the MecA–ClpC complex revealing organizational principles and providing mechanistic insights into this complex molecular machine.

Structural biology of Gram-positive bacterial adhesins.

Abstract: The structural biology of Gram-positive cell surface adhesins is an emerging field of research, whereas Gram-negative pilus assembly and anchoring have been extensively investigated and are well understood. Gram-positive surface proteins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) and individual proteins that assemble into long, hair-like organelles known as pili have similar features at the primary sequence level as well as at the tertiary structural level. Some of these conserved features are essential for their transportation from the cytoplasm and for cell wall anchoring. More importantly, the MSCRAMMs and the individual pilins are assembled with building blocks that are variants of structural modules used for human immunoglobulins. MSCRAMMs target the host's extracellular matrix proteins, such as collagen, fibrinogen, and fibronectin, and they have received considerable attention from structural biologists in the last decade, who have primarily been interested in understanding their interactions with host tissue. The recent focus is on the newly discovered pili of Gram-positive bacteria, and in this review, we highlight the advances in understanding of the individual pilus constituents and their associations and stress the similarities between the individual pilins and surface proteins.

DARS-RNP and QUASI-RNP: New statistical potentials for protein-RNA docking

Abstract: Background Protein-RNA interactions play fundamental roles in many biological processes. Understanding the molecular mechanism of protein-RNA recognition and formation of protein-RNA complexes is a major challenge in structural biology. Unfortunately, the experimental determination of protein-RNA complexes is tedious and difficult, both by X-ray crystallography and NMR. For many interacting proteins and RNAs the individual structures are available, enabling computational prediction of complex structures by computational docking. However, methods for protein-RNA docking remain scarce, in particular in comparison to the numerous methods for protein-protein docking.

Bound anions differentially stabilize multiprotein complexes in the absence of bulk solvent.

Abstract: The combination of ion mobility separation with mass spectrometry is an emergent and powerful structural biology tool, capable of simultaneously assessing the structure, topology, dynamics and composition of large protein assemblies within complex mixtures. An integral part of the ion mobility-mass spectrometry measurement is the ionization of intact multiprotein complexes and their removal from bulk solvent. This process, during which a substantial portion of protein structure and organization is likely to be preserved, imposes a foreign environment on proteins that may cause structural rearrangements to occur. Thus, a general means must be identified to stabilize protein structures in the absence of bulk solvent. Our approach to this problem involves the protection of protein complex structure through the addition of salts in solution prior to desorption/ionization. Anionic components of the added salts bind to the complex either in solution or during the electrospray process, and those that remain bound in the gas phase tend to have high gas phase acidities. The resulting ‘shell’ of counter-ions is able to carry away excess energy from the protein complex ion upon activation and can result in significant structural stabilization of the gas-phase protein assembly overall. By using ion mobility-mass spectrometry, we observe both the dissociation and unfolding transitions for four tetrameric protein complexes bound to populations of twelve different anions using collisional activation. The data presented here quantifies, for the first time, the influence of a large range of counter-ions on gas-phase protein structure and allows us to rank and classify counter-ions as structure stabilizers in the absence of bulk solvent. Our measurements indicate that tartrate, citrate, chloride and nitrate anions are amongst the strongest stabilizers of gas-phase protein structure identified in this screen. The rank order determined by our data is substantially different when compared to the known Hofmeister salt series in solution. While this is an expected outcome of our work, due to the diminished influence of anion and protein solvation by water, our data correlates well to expected anion binding in solution and highlights the fact that both hydration layer and anion-protein binding effects are critical for Hofmeister-type stabilization in solution. Finally, we present a detailed mechanism of action for counter-ion stabilization of proteins and their complexes in the gas-phase, which indicates that anions must bind with high-affinity, but must dissociate readily from the protein in order to be an effective stabilizer. Anion-resolved data acquired for smaller protein systems allows us to classify anions into three categories based on their ability to stabilize protein and protein complex structure in the absence of bulk solvent.

Assignment of PolyProline II Conformation and Analysis of Sequence – Structure Relationship

Abstract: Background Secondary structures are elements of great importance in structural biology, biochemistry and bioinformatics. They are broadly composed of two repetitive structures namely α-helices and β-sheets, apart from turns, and the rest is associated to coil. These repetitive secondary structures have specific and conserved biophysical and geometric properties. PolyProline II (PPII) helix is yet another interesting repetitive structure which is less frequent and not usually associated with stabilizing interactions. Recent studies have shown that PPII frequency is higher than expected, and they could have an important role in protein – protein interactions.

Structural Biology of TRP Channels

Abstract: Structural studies on TRP channels, while limited, are poised for a quickened pace and rapid expansion. As of yet, no high-resolution structure of a full length TRP channel exists, but low-resolution electron cryomicroscopy structures have been obtained for 4 TRP channels, and high-resolution NMR and X-ray crystal structures have been obtained for the cytoplasmic domains, including an atypical protein kinase domain, ankyrin repeats, coiled coil domains and a Ca2+-binding domain, of 6 TRP channels. These structures enhance our understanding of TRP channel assembly and regulation. Continued technical advances in structural approaches promise a bright outlook for TRP channel structural biology.

An introduction to membrane proteins.

Abstract: α-Helical membrane proteins are important for many biological functions. Due to physicochemical constraints, the structures of membrane proteins differ from the structure of soluble proteins. Historically, membrane protein structures were assumed to be more or less two-dimensional, consisting of long, straight, membrane-spanning parallel helices packed against each other. However, during the past decade, a number of the new membrane protein structures cast doubt on this notion. Today, it is evident that the structures of many membrane proteins are equally complex as for many soluble proteins. Here, we review this development and discuss the consequences for our understanding of membrane protein biogenesis, folding, evolution, and bioinformatics.

The Age of Protein Kinases

Abstract: Major progress has been made in unravelling of regulatory mechanisms in eukaryotic cells. Modification of target protein properties by reversible phosphorylation events has been found to be one of the most prominent cellular control processes in all organisms. The phospho-status of a protein is dynamically controlled by protein kinases and counteracting phosphatases. Therefore, monitoring of kinase and phosphatase activities, identification of specific phosphorylation sites, and assessment of their functional significance are of crucial importance to understand development and homeostasis. Recent advances in the area of molecular biology and biochemistry, for instance, mass spectrometry-based phosphoproteomics or fluorescence spectroscopical methods, open new possibilities to reach an unprecidented depth and a proteome-wide understanding of phosphorylation processes in plants and other species. In addition, the growing number of model species allows now deepening evolutionary insights into signal transduction cascades and the use of kinase/phosphatase systems. Thus, this is the age where we move from an understanding of the structure and function of individual protein modules to insights how these proteins are organized into pathways and networks. In this introductory chapter, we briefly review general definitions, methodology, and current concepts of the molecular mechanisms of protein kinase function as a foundation for this methods book. We briefly review biochemistry and structural biology of kinases and provide selected examples for the role of kinases in biological systems.

Control of dna movement in a nanopore at one nucleotide precision by a processive enzyme

Abstract: The invention herein disclosed provides for devices and methods that can detect and control an individual polymer in a mixture is acted upon by another compound, for example, an enzyme, in a nanopore. Of particular note is the stability of the system in a saline medium and to detect individual nucleotide bases in a polynucleotide in real time and which may be used to sequence DNA for many hours without change of reagents. The invention is of particular use in the fields of forensic biology, molecular biology, structural biology, cell biology, molecular switches, molecular circuits, and molecular computational devices, and the manufacture thereof.

The structural biology of ryanodine receptors.

Abstract: Ryanodine receptors are ion channels that allow for the release of Ca 2+ from the endoplasmic or sarcoplasmic reticulum. They are expressed in many different cell types but are best known for their predominance in skeletal and cardiac myocytes, where they are directly involved in excitation-contraction coupling. With molecular weights exceeding 2 MDa, Ryanodine Receptors are the largest ion channels known to date and present major challenges for structural biology. Since their discovery in the 1980s, significant progress has been made in understanding their behaviour through multiple structural methods. Cryo-electron microscopy reconstructions of intact channels depict a mushroom-shaped structure with a large cytoplasmic region that presents many binding sites for regulatory molecules. This region undergoes significant motions during opening and closing of the channel, demonstrating that the Ryanodine Receptor is a bona fide allosteric protein. High-resolution structures through X-ray crystallography and NMR currently cover ~11% of the entire protein. The combination of high- and low-resolution methods allows us to build pseudo-atomic models. Here we present an overview of the electron microscopy, NMR, and crystallographic analyses of this membrane protein giant.

Drug discovery toward antagonists of methyl-lysine binding proteins.

Abstract: The recognition of methyl-lysine and -arginine residues on both histone and other proteins by specific “reader” elements is important for chromatin regulation, gene expression, and control of cell-cycle progression. Recently the crucial role of these reader proteins in cancer development and dedifferentiation has emerged, owing to the increased interest among the scientific community. The methyl-lysine and -arginine readers are a large and very diverse set of effector proteins and targeting them with small molecule probes in drug discovery will inevitably require a detailed understanding of their structural biology and mechanism of binding. In the following review, the critical elements of methyl-lysine and -arginine recognition will be summarized with respect to each protein family and initial results in assay development, probe design, and drug discovery will be highlighted.

Overview: Structural Biology of Integrins

Abstract: Integrins are cell adhesion molecules that play important roles in many biological processes including hemostasis, immune responses, development, and cancer. Their adhesiveness is dynamically regulated through a process termed inside-out signaling. In addition, ligand binding transduces outside-in signals from the extracellular domain to the cytoplasm. Advances in the past several years have shed light on structural basis for integrin regulation and signaling, especially how the large-scale reorientations of the ectodomain are related to the inter-domain and intra-domain shape shifting that changes ligand-binding affinity. Experiments have also shown how the conformational changes of the ectodomain are linked to changes in the α- and β-subunit transmembrane and cytoplasmic domains.

Determinants of Gas-Phase Disassembly Behavior in Homodimeric Protein Complexes with Related Yet Divergent Structures

Abstract: The overall structure of a protein–protein complex reflects an intricate arrangement of noncovalent interactions. Whereas intramolecular interactions confer secondary and tertiary structure to individual subunits, intermolecular interactions lead to quaternary structure—the ordered aggregation of separate polypeptide chains into multisubunit assemblies. The specific ensemble of noncovalent contacts dictates the stability of subunit folds, enforces protein–protein binding specificity, and determines multimer stability. Consequently, noncovalent architecture is likely to play a role in the gas-phase dissociation of these assemblies during tandem mass spectrometry (MS/MS). To further advance the applicability of MS/MS to analytical problems in structural biology, a better understanding of the interplay between the structures and fragmentation behaviors of noncovalent protein complexes is essential. The present work constitutes a systematic study of model protein homodimers (bacteriophage N15 Cro, bacteriopha...