Pseudo-threefold Symmetry Research Articles

Atomistic modeling of alternating access of a mitochondrial ADP/ATP membrane transporter with molecular simulations

The mitochondrial ADP/ATP carrier (AAC) is a membrane transporter that exchanges a cytosolic ADP for a matrix ATP. Atomic structures in an outward-facing (OF) form which binds an ADP from the intermembrane space have been solved by X-ray crystallography, and revealed their unique pseudo three-fold symmetry fold which is qualitatively different from pseudo two-fold symmetry of most transporters of which atomic structures have been solved. However, any atomic-level information on an inward-facing (IF) form, which binds an ATP from the matrix side and is fixed by binding of an inhibitor, bongkrekic acid (BA), is not available, and thus its alternating access mechanism for the transport process is unknown. Here, we report an atomic structure of the IF form predicted by atomic-level molecular dynamics (MD) simulations of the alternating access transition with a recently developed accelerating technique. We successfully obtained a significantly stable IF structure characterized by newly formed well-packed and -organized inter-domain interactions through the accelerated simulations of unprecedentedly large conformational changes of the alternating access without a prior knowledge of the target protein structure. The simulation also shed light on an atomistic mechanism of the strict transport selectivity of adenosine nucleotides over guanosine and inosine ones. Furthermore, the IF structure was shown to bind ATP and BA, and thus revealed their binding mechanisms. The present study proposes a qualitatively novel view of the alternating access of transporters having the unique three-fold symmetry in atomic details and opens the way for rational drug design targeting the transporter in the dynamic functional cycle.

Detection of Folding Sites of β-Trefoil Fold Proteins Based on Amino Acid Sequence Analyses and Structure-Based Sequence Alignment

The information on the 3D structure of a protein including its folding mechanism is encoded in its amino acid sequence. A β-trefoil protein is well known to have a remarkable 3D structure property, that is, the pseudo three-fold symmetry without clear hydrophobic packing. It is interesting to investigate how information on the folding mechanism to form such a topology is encoded in the amino acid sequence of a protein. In this study, analyses based on inter-residue average distance statistics and the conservation of hydrophobic residues are performed for sequences of 26 β-trefoil proteins to identify significant sites for the initial folding. Results are compared with the native 3D structures. The conserved hydrophobic residues are defined by multiple sequence alignment based on the 3D structures. It is confirmed that a conserved hydrophobic residue is always located in a β-strand. In particular, β-strands 5 and 6 are significant for the initial folding from the analyses based on the inter-residue average distance statistics. These results coincide well with the experimental data obtained so far for folding of some of the β-trefoil proteins. It is also confirmed that the conserved hydrophobic residues defined in this study contribute to form hydrophobic packing in β-trefoil proteins in general. Twelve conserved hydrophobic residue pairs are almost always observed to form packing in the 26 β-trefoil proteins from different superfamilies. We elucidate how the conserved hydrophobic residues in β-strands 5 and 6 contribute to the initial stage of folding of a β-trefoil protein. The common packing of the 12 conserved hydrophobic residue pairs are significant to form the whole β-trefoil fold structure.

Rational Organization of Lanthanide-Based SMM Dimers into Three-Dimensional Networks.

Optimization of the reaction of [Ln(hfac)3]·2H2O and pyridine-N-oxide (PyNO), which is known to afford double-bridged dimers, leads to triple-bridged dimers of formula [(Ln(hfac)3)2(PyNO)3] (Ln = Gd (1), Dy (2)) from which the Dy derivative (2) behaves as a single-molecule magnet (SMM). The pseudo threefold axis symmetry of this zero-dimensional building block makes possible its extension into a tridimensional network. By changing PyNO for 4,4'-bipyridine N,N'-dioxide (4,4'BipyNO) a tridimensional compound of formula {[Ln(hfac)3]2(4,4'BipyNO)2]} (Ln = Eu (3), Gd (4), and Dy (5)) is then rationally obtained. This covalent three-dimensional (3D) network has a remarkably high cell volume (V = 24 419 A(3)) and is an arrangement of interpenetrated 3D subnetworks whose triple-bridged dimers still behave as SMMs.

Chapter Eight - Antiporters of the Mitochondrial Carrier Family

The eukaryotic transport protein family SLC25 consists of mitochondrial carriers (MCs) that are recognized on the sequence level by a threefold repeated and conserved signature motif. The majority of MCs characterized so far catalyzes strict exchanges of substrates across the mitochondrial inner membrane. The substrates are nucleotides, metabolic intermediates, and cofactors that are required in cytoplasmic and matrix metabolism. This review summarizes and discusses the current knowledge of the antiport mechanism(s) of MCs that has been deduced from determining transport characteristics and by analyzing structural, sequence, and mutagenesis data. The mode of transport varies among different MCs with respect to how the substrate translocation depends on the electrical and pH gradients across the mitochondrial inner membrane, for example, the ADP/ATP carrier is electrogenic (electrophoretic), the GTP/GDP carrier is dependent on the pH gradient, the aspartate/glutamate carrier is dependent on both, and the oxoglutarate/malate carrier is independent of them. The structure of the bovine ADP/ATP carrier consists of a six-transmembrane α-helix bundle with a pseudo-threefold symmetry and a closed matrix gate. By using this structure as a template in homology modeling, residues engaged in substrate binding and the formation of a cytoplasmic gate in MCs have been proposed. The functional importance of the residues of the binding site, the matrix, and the cytoplasmic gates is supported by transport activities of different MCs with single point mutations. Cumulative evidence has been used to postulate a general transport mechanism for MCs.

Creation and structure determination of an artificial protein with three complete sequence repeats

Symfoil-4P is a de novo protein exhibiting the threefold symmetrical β-trefoil fold designed based on the human acidic fibroblast growth factor. First three asparagine-glycine sequences of Symfoil-4P are replaced with glutamine-glycine (Symfoil-QG) or serine-glycine (Symfoil-SG) sequences protecting from deamidation, and His-Symfoil-II was prepared by introducing a protease digestion site into Symfoil-QG so that Symfoil-II has three complete repeats after removal of the N-terminal histidine tag. The Symfoil-QG and SG and His-Symfoil-II proteins were expressed in Eschericha coli as soluble protein, and purified by nickel affinity chromatography. Symfoil-II was further purified by anion-exchange chromatography after removing the HisTag by proteolysis. Both Symfoil-QG and Symfoil-II were crystallized in 0.1 M Tris-HCl buffer (pH 7.0) containing 1.8 M ammonium sulfate as precipitant at 293 K; several crystal forms were observed for Symfoil-QG and II. The maximum diffraction of Symfoil-QG and II crystals were 1.5 and 1.1 Å resolution, respectively. The Symfoil-II without histidine tag diffracted better than Symfoil-QG with N-terminal histidine tag. Although the crystal packing of Symfoil-II is slightly different from Symfoil-QG and other crystals of Symfoil derivatives having the N-terminal histidine tag, the refined crystal structure of Symfoil-II showed pseudo-threefold symmetry as expected from other Symfoils. Since the removal of the unstructured N-terminal histidine tag did not affect the threefold structure of Symfoil, the improvement of diffraction quality of Symfoil-II may be caused by molecular characteristics of Symfoil-II such as molecular stability.

Triple-stranded ferric helices: a π–π interaction-driven structural hierarchy of Fe5, Fe7, and Fe17 clusters

Three polypyridine ligand-supported multinuclear iron complexes, [Fe5], [Fe7] and [Fe17], were synthesized and their physical properties were investigated. The complexes had triple-stranded helical structures with pseudo threefold symmetry, and were stabilized by varying degrees of intramolecular π-π stacking. The pentanuclear iron complex consisted of two Fe(II) and three Fe(III) ions, supported by three ligands, while the heptanuclear complex comprised four Fe(II) centres, three Fe(III) ions, and six ligands, and the heptadecanuclear complex contained seventeen Fe(III) ions and nine ligands. Electrochemical studies revealed that the pentanuclear and heptanuclear iron complexes showed pseudo-reversible three- and five-step redox behaviours, respectively. Magnetic measurements conducted on the pentanuclear and heptanuclear complexes revealed that antiferromagnetic interactions were operative between neighbouring iron ions through the oxo- and pyrazole-bridges.

Structural insights into the specific anti-HIV property of actinohivin: structure of its complex with the α(1-2)mannobiose moiety of gp120.

Actinohivin (AH) is an actinomycete lectin with a potent specific anti-HIV activity. In order to clarify the structural evidence for its specific binding to the α(1-2)mannobiose (MB) moiety of the D1 chains of high-mannose-type glycans (HMTGs) attached to HIV-1 gp120, the crystal structure of AH in complex with MB has been determined. The AH molecule is composed of three identical structural modules, each of which has a pocket in which an MB molecule is bound adopting a bracket-shaped conformation. This conformation is stabilized through two weak C-H...O hydrogen bonds facilitated by the α(1-2) linkage. The binding features in the three pockets are quite similar to each other, in accordance with the molecular pseudo-threefold symmetry generated from the three tandem repeats in the amino-acid sequence. The shape of the pocket can accept two neighbouring hydroxyl groups of the O(3) and O(4) atoms of the equatorial configuration of the second mannose residue. To recognize these atoms through hydrogen bonds, an Asp residue is located at the bottom of each pocket. Tyr and Leu residues seem to block the movement of the MB molecules. Furthermore, the O(1) atom of the axial configuration of the second mannose residue protrudes from each pocket into an open space surrounded by the conserved hydrophobic residues, suggesting an additional binding site for the third mannose residue of the branched D1 chain of HMTGs. These structural features provide strong evidence indicating that AH is only highly specific for MB and would facilitate the highly specific affinity of AH for any glycoprotein carrying many HMTGs, such as HIV-1 gp120.

Promiscuity of the euonymus carbohydrate-binding domain.

Plants synthesize small amounts of carbohydrate-binding proteins on exposure to stress. For example, on exposure to drought, high salt, wounding and by treatment with some plant hormones or by pathogen attack. In contrast to the ‘classical’ plant lectins that are mostly located in the vacuolar compartment, this new class of inducible lectins is present in the cytoplasm and in the nucleus. Taking into account that any physiological role of plant lectins most likely relies on their specific carbohydrate-binding activity and specificity, the discovery of these stress-related lectins provides strong evidence for the importance of protein-carbohydrate-interactions in plant cells. Hitherto, six families of such nucleocytoplasmic lectins have been identified in plants. This review will focus on the nucleocytoplasmic lectins with one or more Euonymus lectin (EUL) domain(s). The carbohydrate-binding specificity of EUL proteins from a monocot, a dicot and a lower plant has been compared. Furthermore, modeling of the different EUL domains revealed a similar ß-trefoil fold consisting of three bundles of ß-sheet organized around a pseudo three-fold symmetry axis. Despite the sequence similarity and the conserved amino acids in the binding site, glycan array analyses showed that the EUL domain has a promiscuous carbohydrate-binding site capable of accommodating high mannose N-glycans, blood group B related structures and galactosylated epitopes.

Tetraethylammonium tri-μ-phenolato-bis[tricarbonylmanganate(I)

The title compound, (C8H20N)[Mn2(C6H5O)3(CO)6], was synthesized from [Mn(CO)3(CH3CN)3]BF4 and (C8H20N)(OC6H5). The binuclear anion exhibits a pseudo-threefold symmetry and contains two six-coordinate Mn atoms. Each metal atom is coordinated by three facially oriented CO ligands and three doubly-bridging phenolate ligands. The average O—Mn—O bond angle is 74.9 (7)° in the Mn2O3 metal–phenolate dimeric core, yielding a distorted octa­hedron for each metal.

RNA channelling by the eukaryotic exosome

The eukaryotic exosome is a key nuclease for the degradation, processing and quality control of a wide variety of RNAs. Here, we report electron microscopic reconstructions and pseudo-atomic models of the ten-subunit Saccharomyces cerevisiae exosome in the unbound and RNA-bound states. In the RNA-bound structures, extra density that is visible at the entry and exit sites of the exosome channel indicates that a substrate-threading mechanism is used by the eukaryotic exosome. This channelling mechanism seems to be conserved in exosome-like complexes from all domains of life, and might have been present in the most recent common ancestor.

Structural Convergence between Cryo-EM and NMR Reveals Intersubunit Interactions Critical for HIV-1 Capsid Function

Mature HIV-1 particles contain conical-shaped capsids that enclose the viral RNA genome and perform essential functions in the virus life cycle. Previous structural analysis of two- and three-dimensional arrays of the capsid protein (CA) hexamer revealed three interfaces. Here, we present a cryoEM study of a tubular assembly of CA and a high-resolution NMR structure of the CA C-terminal domain (CTD) dimer. In the solution dimer structure, the monomers exhibit different relative orientations compared to previous X-ray structures. The solution structure fits well into the EM density map, suggesting that the dimer interface is retained in the assembled CA. We also identified a CTD-CTD interface at the local three-fold axis in the cryoEM map and confirmed its functional importance by mutagenesis. In the tubular assembly, CA intermolecular interfaces vary slightly, accommodating the asymmetry present in tubes. This provides the necessary plasticity to allow for controlled virus capsid dis/assembly.

Phase transitions and H 2O motions in [Ca(H 2O) 4](ClO 4) 2 studied by infrared spectroscopy, inelastic/quasielastic incoherent neutron scattering and proton magnetic resonance. Part II

Fourier transform middle infrared spectroscopy, inelastic/quasielastic incoherent neutron scattering and proton magnetic resonance data for [Ca(H 2O) 4](ClO 4) 2 in the temperature range of 20–300 K and 110–300 K, respectively, are reported. Lack of infrared bands broadening with increasing temperature indicated that molecular reorientational correlation time is longer than 10 −12 s. The elastic peak of neutron inelastic scattering registered in the high temperature phase shows broadening, which can be well described by a model of 180° instantaneous stochastic jumps of protons around the two-fold axis formed by the Ca O bond, assuming that only two out of four H 2O ligands per [Ca(H 2O) 4] 2+ formula unit undergoes fast reorientation with reorientational correlation time τ R(H 2O) of an order of 10 −11 s. The analysis of the second moment M 2 of 1H NMR line in the low temperature phase of [Ca(H 2O) 4](ClO 4) 2 revealed that the reorientation via 180° flips (jumps) of water molecules causes a very small changes of M 2. It confirms that reorientation of H 2O is fast even at 110 K. The reason for the first plateau of M 2 ≈ 9.5 × 10 −8 T 2, which was observed at ca. 110–190 K range, is the anisotropic reorientation of whole [Ca(H 2O) 4] 2+ cations around the two-fold symmetry axis going through two calcium atoms of the neighbouring [Ca(H 2O) 4] 2+ cations. Above the phase transition at T C 4 h , the anisotropic reorientation of [Ca(H 2O) 4] 2+ (around the pseudo three-fold symmetry axis formed by the Ca…O direction between [Ca(H 2O) 4] 2+ cation and ClO 4 − anion, lying along the hydrogen bridge bond O H…O) is setting on with a frequency of the order of a few kHz, consequently above T C 1 h one can observe next plateau of M 2 ≈ 2.5 × 10 −8 T 2.

Comparison of copper imine and amine podates: geometric consequences of podand size and donor type

The imine podands tris[(2-nitrobenzylidene)aminoethyl]amine and tris[(2-nitrobenzylidene)aminopropyl]amine both stabilize copper(I), forming {tris[(2-nitrobenzylidene)aminoethyl]amine-kappa4N}copper(I) perchlorate acetonitrile disolvate, [Cu(C27H27N7O6)]ClO4.2CH3CN, (II), and {tris[(2-nitrobenzylidene)aminopropyl]amine-kappa4N}copper(I) perchlorate, [Cu(C30H33N7O6)]ClO4, (VI), respectively. The larger propyl-based ligand is a poorer fit for the CuI ion. The reduced amine podand tris[(2-nitrobenzyl)aminoethyl]amine binds CuII and the resulting compound, chloro{tris[(2-nitrobenzyl)aminoethyl]amine-kappa4N}copper(II) chloride ethanol solvate, [Cu(C27H33N7O6)Cl]Cl.C2H5OH, (IV), shows both intra- and intermolecular hydrogen bonding, which gives rise to RRS or SSR conformations in the podand strands rather than the expected pseudo-threefold symmetry.

Crystal structure of conserved hypothetical protein Aq1575 from Aquifex aeolicus.

The crystal structure of a conserved hypothetical protein, Aq1575, from Aquifex aeolicus has been determined by using x-ray crystallography. The protein belongs to the domain of unknown function DUF28 in the Pfam and PALI databases for which there was no structural information available until now. A structural homology search with the DALI algorithm indicates that this protein has a new fold with no obvious similarity to those of other proteins of known three-dimensional structure. The protein reveals a monomer consisting of three domains arranged along a pseudo threefold symmetry axis. There is a large cleft with approximate dimensions of 10 A x 10 A x 20 A in the center of the three domains along the symmetry axis. Two possible active sites are suggested based on the structure and multiple sequence alignment. There are several highly conserved residues in these putative active sites. The structure based molecular properties and thermostability of the protein are discussed.

Complexation Properties of Heteroditopic Cryptands towards Cu2+, Zn2+, Cd2+, and Pb2+ in Aqueous Solution: Crystal Structures of [(H5L1)(ClO4)5]·4 H2O and [(NiL2Cl)Cl]·5.5 H2O·CH3OH

The protonation and metal ion (Cu2+, Zn2+, Cd2+, Pb2+) complexation abilities of three heteroditopic cryptands in solution are described and the relevant equilibrium constants are reported. The two distinct binding subunits of the ligands act cooperatively in the coordination of M(H2O)2+ (M = metal ion) species. The cryptand L1 binds five protons in very acidic solution and crystallizes as [(H5L1)(ClO4)5].4 H2O in the monoclinic space group P21ln with a = 14.281(4), b = 14.313(7), c = 24.082(8) A, β = 92.00(2)°, Z = 4, R = 0.079, and wR2 = 0.2010. The molecule shows an endo-endo conformation with a pseudo-threefold symmetry axis passing through the two bridgehead nitrogen atoms. All the cryptands (L1-L3) readily form inclusion complexes with Cu2+, Ni2+, Zn2+, Cd2+, and Pb2+ ions at the tren end of the cavity. The crystal structure of [(NiL2Cl)Cl].5.5 H2O.CH3OH is also reported [C2/c, a = 26.671(6), b = 16.784(4), c = 18.591(8) A, β = 101.006(14)°, Z = 8, R = 0.091, and wR2 = 0.1424]. The NiII ion resides in a trigonal-bipyramidal coordination environment with the equatorial sites occupied by the three secondary nitrogen atoms of the cryptand and the axial sites occupied by a bridgehead nitrogen atom and a Cl- ion.

1,4,7,10,13,16,21,24-Octaazabicyclo[8.8.8]hexacosa-4,6,13,15,21,23-hexaene pentahydrate

The title compound, C 18 H 30 N 8 .5H 2 O, is a hexaimine cryptand which crystallizes with pseudo-threefold symmetry, each strand having the anti conformation. Water molecules are hydrogen bonded to five of the six imine N atoms.

New folds of plant lectins

Among the crystal structures of lectins determined recently, three — snowdrop lectin, jacalin and amaranthin — represent new lectin families. Their polypeptide folds share remarkably similar features and consist exclusively of β structure. Autonomously folded β-sheet subdomains, inter-related by a pseudothreefold symmetry, assembly to form β-prism or β-barrel structures which are stabilized by a hydrophobic core.

Three-dimensional reconstruction of Macrobdella decora (leech) hemoglobin by cryoelectron microscopy

Macrobdella decora hemoglobin was observed in vitreous ice by cryoelectron microscopy and subjected to three-dimensional reconstruction by the method of random conical tilt series. The refined volume has a resolution of 40 A and a D6 point-group symmetry. Its architecture, with its hexagonal bilayer appearance, resembles those of Lumbricus terrestris (oligochaete) and Eudistylia vancouverii (polychaete). When the reconstruction volume is viewed along its sixfold axis, the vertices of the upper hexagonal layer are rotated 16 degrees clockwise compared to those of the lower layer. In agreement with the "bracelet" model of Vinogradov et al., a central linker complex is decorated by 12 hollow globular substructures. The linker complex is made up of a central hexagonal toroid linked by 12 c5 connections to two bracelets of c3 connections, which are themselves linked via six c4 connections. The portion of the hollow globular substructure corresponding to the dodecamer of globin chains has a local pseudo threefold symmetry and is composed of three elongated structures visible when the volume is displayed at high threshold. The main difference between Macrobdella, Lumbricus, and Eudistylia hemoglobins is the presence in Macrobdella of a central hexagonal toroid instead of a compact flat hexagonal structure.

タンパク質結晶学と構造生物学 新しいタンパク質立体構造 ニワトリ卵黄膜由来の蛋白質ＶＭＯ‐Ｉの結晶構造解析

The crystal structure of vitelline membrane outer layer protein I (VMO-I), which was isolated from the outer layer of the vitelline membrane of hen's eggs, has been determined by X-ray analysis. VMO-I is composed of three homologous structures, each containing a β-sheet forming Greek key motif, which are in accordance with the three repeats in the sequence. These three homologous structures are related by a pseudo three-fold symmetry. This new folding motif, recently designated as the β-prism, has also been observed in the second domain of δ-endotoxin. Thus, VMO-I and δ-endotoxin constitute a new family with a novel folding motif. The VMO-I molecule has a groove-like cavity. This region contains invariant acidic residues in the three repeats. VMO-I is assumed to be an oligosaccharide binding protein like lysozyme, although details of its catalytic function remain to be solved. A docking model of an oligosaccharide to VMO-I has been constructed. This model shows that the cavity has a sufficient space to bind roughly pentamer saccharides. The structural features strongly suggest that the negatively-charged cavity in the top region of the protein may be involved in an unknown catalytic reaction.

Crystal structure of vitelline membrane outer layer protein I (VMO-I): a folding motif with homologous Greek key structures related by an internal three-fold symmetry.

The crystal structure of vitelline membrane outer layer protein I (VMO-I), which is isolated from the vitelline membrane outer layer of hen's eggs, has been determined by the multiple isomorphous replacement method and refined to an R-factor of 18.8% at 2.2 A resolution. The main chain folds into an unusual structure that consists of three beta-sheets forming Greek key motifs, which are related by an internal pseudo three-fold symmetry. The internal portion surrounded by these three beta-sheets is filled with hydrophobic side chains. This conformational feature coincides with three internal repeats in the sequence. Although a similar fold exists in the second domain of delta-endotoxin, there are significant structural differences between the two proteins, with the three-fold symmetry being most regular in VMO-I.