The Three-Dimensional Structure of 3′-Deoxycytidine

Abstract

Structural analysis of 3′-deoxycytidine and comparison with 2′-deoxynucleosides reveals no noticeable effect on the conformation of the molecule due to the lack of 3′-oxygen atom. There are two crystallographically independent molecules and both adopt the anti conformation with C3′-endo sugar puckering. A ‘head-to-tail’ packing of the molecules along the b axis results in a virtual ‘2′-5′ polycytidylic acid chain.     

Three-Dimensional cryoEM Reconstruction of Native LDL Particles to 16? Resolution at Physiological Body Temperature

Background

Low-density lipoprotein (LDL) particles, the major carriers of cholesterol in the human circulation, have a key role in cholesterol physiology and in the development of atherosclerosis. The most prominent structural components in LDL are the core-forming cholesteryl esters (CE) and the particle-encircling single copy of a huge, non-exchangeable protein, the apolipoprotein B-100 (apoB-100). The shape of native LDL particles and the conformation of native apoB-100 on the particles remain incompletely characterized at the physiological human body temperature (37°C).

Methodology/Principal Findings

To study native LDL particles, we applied cryo-electron microscopy to calculate 3D reconstructions of LDL particles in their hydrated state. Images of the particles vitrified at 6°C and 37°C resulted in reconstructions at ∼16 Å resolution at both temperatures. 3D variance map analysis revealed rigid and flexible domains of lipids and apoB-100 at both temperatures. The reconstructions showed less variability at 6°C than at 37°C, which reflected increased order of the core CE molecules, rather than decreased mobility of the apoB-100. Compact molecular packing of the core and order in a lipid-binding domain of apoB-100 were observed at 6°C, but not at 37°C. At 37°C we were able to highlight features in the LDL particles that are not clearly separable in 3D maps at 6°C. Segmentation of apoB-100 density, fitting of lipovitellin X-ray structure, and antibody mapping, jointly revealed the approximate locations of the individual domains of apoB-100 on the surface of native LDL particles.

Conclusions/Significance

Our study provides molecular background for further understanding of the link between structure and function of native LDL particles at physiological body temperature.     

A New Crystal Form of the Fab Fragment of a Monoclonal Antibody to Human Interleukin-2: The Three-Dimensional Structure at 2.7 Å Resolution

The three-dimensional structure of the antigen-binding fragment of a monoclonal antibody to human interleukin-2 in a new crystal form (space group P2₁2₁2₁; unit cell parameters: a = 42.82 Å, b = 90.68 Å, and c = 139.82 Å) was determined by the X-ray molecular replacement method at the resolution of 2.7 Å. The protein folding and the stereochemistry of its antigen-binding site were comparatively analyzed.     

The Refined Crystal Structure of Toxic Shock Syndrome Toxin-1 at 2.07 Å Resolution

The pyrogenic toxin toxic shock syndrome toxin-1 fromStaphylococcus aureusis a causative agent of the toxic shock syndrome disease. It belongs to a family of proteins known as superantigens that cross-link major histocompatibility class II molecules and T-cell receptors leading to the activation of a substantial number of T cells. The crystal structure of this protein has been refined to 2.07 Å with anR_crystvalue of 20.4% for 51,240 reflections. The final model contains three molecules in the asymmetric unit with good stereochemistry and a root-mean-square deviation of 0.009 Å and 1.63° from ideality for bond lengths and bond angles, respectively. The overall fold is considerably similar to that of other known microbial superantigens (staphylococcal enterotoxins). However, a detailed structural analysis shows that toxic shock syndrome toxin-1 lacks several structural features that affect its specificity for Vβ elements of the T-cell receptor and also its recognition by major histocompatibility class II molecules.     

Structure of actinidin,after refinement at 1.7 Å resolution

The structure of the sulphydryl protease, actinidin, after refinement at 1.7 Å resolution, is described. The positions of most of the 1666 atoms have been determined with an accuracy better than 0.1 Å; only two residues (219 and 220) and the side-chain of a third (87) cannot be seen. In addition, the model contains 272 solvent molecules, all taken as water, except one which may be an ammonium ion. Atomic B values give a good indication of the mobility of different parts of the structure. Actinidin has a double domain structure, with one domain mostly helical in its secondary structure, and the other domain built around a twisted β-sheet. The geometry of hydrogen bonds in helices, β-structure and turns has been analysed. All are significantly non-linear, with the angle N-?…O ~160 °. Carbonyl groups are tilted outwards from the axis of each helix, the tilting apparently unaffected by whether or not additional hydrogen bonds are made (e.g. to water or side-chain atoms). Each domain is folded round a substantial core of non-polar side-chains, but the interface between domains is mostly polar. Interactions across this interface involve a network of eight buried water molecules, the buried carboxyl and amino groups of Glu35, Glu50, Lys181 and Lys17, other polar side-chains and a few hydrophobic groups. One other internal charged side-chain, that of Glu52, is adjacent to a buried solvent molecule, probably an ammonium ion. Other side-chain environments are described. One proline residue has a cis configuration. The sulphydryl group is oxidized, probably to SO₂^?, with one oxygen atom clearly visible but the other somewhat less certain. The active site geometry is otherwise compatible with the mechanism proposed by Drenth et al. (1975,1976) for papain. The positions of the 272 solvent molecules are described. The best-ordered water molecules are those that are internal (total of 17), in surface pockets, or in the intermolecular contact regions. These generally form three or four hydrogen bonds, two to proton acceptors and one or two to proton donors. Other water molecules make water bridges on the surface, sometimes covering the exposed edges of non-polar groups. Intermolecular contacts involve few protein atoms, but many water molecules.     

Microbial Hydroxylation of (−)-α-Santonin by Aspergillus niger

The microbiological transformation of a sesquiterpene lactone, (−)-α-santonin was carried out by using Aspergillus niger MIL 5024 and MIL 5025. Strain MIL 5024 brings about the transformation of (−)-α-santonin (400 mg) to 11-hydroxy-(−)-α-santonin (P1) (50.4 mg), 3,6,9-trihydroxy-9,10-seco-selina-1,3,5(10)-trien-12-oic acid-12,6-lactone (P2) (22.4 mg), and 3,6-dihydroxy-9,10-seco-selina-1,3,5(10)-trien-9,12-dioic acid-12,6-lactone (P3) (27.1 mg), which were isolated and characterized by UV, IR, mass and ¹H-NMR spectroanalyses. All of these products are described for the first time. Results similar to those with MIL 5024 were also obtained from the transformation of (−)-α-santonin by the other isolate, strain MIL 5025.     

Structure of bovine pancreatic phospholipase A2 at 1.7 Å resolution

The crystal structure of bovine pancreatic phospholipase A2 has been refined to 1.7 Å resolution. The starting model for this refinement was the previously published structure at a resolution of 2.4 Å (Dijkstra et al., 1978). This model was adjusted to the multiple isomorphous replacement map with Diamond's real space refinement program (Diamond, 1971,1974) and subsequently refined using Agarwal's least-squares method (Agarwal, 1978). The final crystallographic R-factor is 17.1% and the estimated root-mean-square error in the positional parameters is 0.12 Å. The refined model allowed a detailed survey of the hydrogen-bonding pattern in the molecule. The essential calcium ion is located in the active site and is stabilized by one carboxyl group as well as by a peptide loop with many residues unvaried in all known phospholipase A2 sequences. Five of the oxygen ligands octahedrally surround the ion. The sixth octahedral position is shared between one of the carboxylate oxygens of Asp49 and a water molecule. The entrance to the active site is surrounded by residues involved in the binding of micelle substrates. The N-terminal region plays an important role here. Its α-NH₊³ group is buried and interacts with Gln4, the carbonyl oxygen of Asn71 and a fully enclosed water molecule, which provides a link between the N terminus and several active site residues. A total of 106 water molecules was located in the final structure, most of them in a two-layer shell around the protein molecule. The mobility in the structure was derived from the individual atomic temperature factors. Minimum mobility is found for the main chain atoms in the central part of the two long α-helices. The active site is rather rigid.     

Crystal Structure of Bovine Heart Cytochrome c Oxidase at 2.8 Å Resolution

Thirteen different polypeptide subunits, each in one copy, five phosphatidyl ethanolamines and three phosphatidyl glycerols, two hemes A, three Cu ions, one Mg ion, and one Zn ion are detectable in the crystal structure of bovine heart cytochrome c oxidase in the fully oxidized form at 2.8 Å resolution. A propionate of hems a, a peptide unit (–CO–NH–), and an imidazole bound to Cu_A are hydrogen-bonded sequentially, giving a facile electron transfer path from Cu_A to heme a. The O₂ binding and reduction site, heme a ₃, is 4.7 Å apart from Cu_B. Two possible proton transfer paths from the matrix side to the cytosolic side are located in subunit I, including hydrogen bonds and internal cavities likely to contain randomly oriented water molecules. Neither path includes the O₂ reduction site. The O₂ reduction site has a proton transfer path from the matrix side possibly for protons for producing water. The coordination geometry of Cu_B and the location of Tyr²⁴⁴ in subunit I at the end of the scalar proton path suggests a hydroperoxo species as the two electron reduced intermediate in the O₂ reduction process.     

Crystal Structure of Monomeric Photosystem II from Thermosynechococcus elongatus at 3.6-? Resolution

The membrane-embedded photosystem II core complex (PSIIcc) uses light energy to oxidize water in photosynthesis. Information about the spatial structure of PSIIcc obtained from x-ray crystallography was so far derived from homodimeric PSIIcc of thermophilic cyanobacteria. Here, we report the first crystallization and structural analysis of the monomeric form of PSIIcc with high oxygen evolution capacity, isolated from Thermosynechococcus elongatus. The crystals belong to the space group C222₁, contain one monomer per asymmetric unit, and diffract to a resolution of 3.6 Å. The x-ray diffraction pattern of the PSIIcc-monomer crystals exhibit less anisotropy (dependence of resolution on crystal orientation) compared with crystals of dimeric PSIIcc, and the packing of the molecules within the unit cell is different. In the monomer, 19 protein subunits, 35 chlorophylls, two pheophytins, the non-heme iron, the primary plastoquinone Q_A, two heme groups, 11 β-carotenes, 22 lipids, seven detergent molecules, and the Mn₄Ca cluster of the water oxidizing complex could be assigned analogous to the dimer. Based on the new structural information, the roles of lipids and protein subunits in dimer formation of PSIIcc are discussed. Due to the lack of non-crystallographic symmetry and the orientation of the membrane normal of PSIIcc perpendicular (∼87°) to the crystallographic b-axis, further information about the structure of the Mn₄Ca cluster is expected to become available from orientation-dependent spectroscopy on this new crystal form.     

Bovine Carbonyl Lactoperoxidase Structure at 2.0? Resolution and Infrared Spectra as a Function of pH

Lactoperoxidase (LPO) is a hemeprotein catalyzing the oxidation of thiocyanate and I^? into antimicrobials and small aromatic organics after being itself oxidized by H₂O₂. LPO is excreted by the lungs, mammary glands, found in saliva and tears and protects mammals against bacterial, fungal and viral invasion. The Fe(II) form binds CO which inactivates LPO like many other hemeproteins. We present the 3-dimensional structure of CO?CLPO at 2.0? resolution and infrared (IR) spectra of the iron-bound CO stretch from pH?3 to 8.8?at 1 cm^?1 resolution. The observed Fe?CC?CO bond angle of 132° is more acute than the electronically related Fe(III), CN?CLPO with a Fe?CC?CN angle of 161°. The orientations of the two ligands are different with the oxygen of CO pointing towards the imidazole of distal His109 while the nitrogen of CN points away, the Fe(II) moves towards His109 while the Fe(III) moves away; both movements are consistent with a hydrogen bond between the distal His109 and CO, but not to the nitrogen of CN?CLPO. The IR spectra of CO?CLPO exhibit two major CO absorbances with pH dependent relative intensities. Both crystallographic and IR data suggest proton donation to the CO oxygen by His109 with a pK ?? 4; close to the pH of greatest enzyme turnover. The IR absorbance maxima are consistent with a first order correlation between frequency and Fe(III)/Fe(II) reduction potential at pH?7; both band widths at half-height correlate with electron density donation from Fe(II) to CO as gauged by the reduction potential.     

Trimeric Structure of (+)-Pinoresinol-forming Dirigent Protein at 1.95 ? Resolution with Three Isolated Active Sites

Control over phenoxy radical-radical coupling reactions in vivo in vascular plants was enigmatic until our discovery of dirigent proteins (DPs, from the Latin dirigere, to guide or align). The first three-dimensional structure of a DP ((+)-pinoresinol-forming DP, 1.95 Å resolution, rhombohedral space group H32)) is reported herein. It has a tightly packed trimeric structure with an eight-stranded β-barrel topology for each DP monomer. Each putative substrate binding and orientation coupling site is located on the trimer surface but too far apart for intermolecular coupling between sites. It is proposed that each site enables stereoselective coupling (using either two coniferyl alcohol radicals or a radical and a monolignol). Interestingly, there are six differentially conserved residues in DPs affording either the (+)- or (−)-antipodes in the vicinity of the putative binding site and region known to control stereoselectivity. DPs are involved in lignan biosynthesis, whereas dirigent domains/sites have been implicated in lignin deposition.     

Crystal Structure of P13K SH3 Domain at 2.0 Å Resolution

The P13K SH3 domain, residues 1 to 85 of the P1 – 3 kinase p85 subunit, has been characterized by X-ray diffraction. Crystals belonging to space groupP4₃2₁2 diffract to 2.0 Å resolution and the structure was phased by single isomorphous replacement and anomalous scattering (SIRAS). As expected, the domain is a compact β barrel with an over-all conformation very similar to the independently determined NMR structures. The X-ray structure illuminates a discrepancy between the two NMR structures on the conformation of the loop region unique to P13K SH3. Furthermore, the ligand binding pockets of P13K SH3 domain are occupied by amino acid residues from symmetry-related P13K SH3 molecules: the C-terminal residues I(82) SPP of one and R18 of another. The interaction modes clearly resemble those observed for the P13K SH3 domain complexed with the synthetic peptide RLP1, a class 1 ligand, although there are significant differences. The solid-state interactions suggest a model of protein – protein aggregation that could be mediated by SH3 domains.     

Refinement and Analysis of the Mature Zika Virus Cryo-EM Structure at 3.1 Å Resolution

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Cryoelectron Microscopy Structure of Purified γ-Secretase at 12 Å Resolution

γ-Secretase, an integral membrane protein complex, catalyzes the intramembrane cleavage of the β-amyloid precursor protein (APP) during the neuronal production of the amyloid β-peptide. As such, the protease has emerged as a key target for developing agents to treat and prevent Alzheimer's disease. Existing biochemical studies conflict on the oligomeric assembly state of the protease complex, and its detailed structure is not known. Here, we report that purified active human γ-secretase in digitonin has a total molecular mass of ∼ 230 kDa when measured by scanning transmission electron microscopy. This result supports a complex that is monomeric for each of the four component proteins. We further report the three-dimensional structure of the γ-secretase complex at 12 Å resolution as obtained by cryoelectron microscopy and single-particle image reconstruction. The structure reveals several domains on the extracellular side, three solvent-accessible low-density cavities, and a potential substrate-binding surface groove in the transmembrane region of the complex.     

Effects of Size of Carbohydrate Chain on Protease Digestion of Aspergillus niger Endo-β-1,4-glucanase

Three different carbohydrate-depleted enzymes were prepared from an endo-β-l,4-glucanase of Aspergillus niger IF031125 by treatment with endo-β-N-acetylglucosaminidase or α-mannosidase. They were purified by Concanavalin A-Sepharose affinity and DEAE ion-exchange column chromatographies. The molecular sizes of these enzymes had been decreased from 40 kDa containing 9.0% carbohydrate to 39, 38, and 37kDa with carbohydrate at 4.5, 1.3, and 0.8% (wt/wt), respectively. The native and these carbohydrate-depleted enzymes were compared in their enzymatic properties, and it was found that they were identical in their catalytic activities and both thermal and pH stabilities. However, the 37-kDa enzyme was more susceptible to proteolysis by Savinase, proteinase K, and Pronase E. On the other hand, the specific protease trypsin showed no such effect on activity of all enzymes. These results suggested that the core structure of the asparagine-linked sugar chain, which consisted of three monosaccharide residues, contributed to the high stability of the endo-β-l,4-glucanase against protease digestion.     

Enhancing the activity and thermostability of thermostable β-glucosidase from a marine Aspergillus niger at high salinity

To evaluate the effect of salinity on the catalyzing ability of β-glucosidase in the marine fungus Aspergillus niger, the thermodynamic parameters of the β-glucosidase were investigated at different salinities. At the optimum salinity of 6% NaCl (w/v) solution, the optimum temperature and pH of the β-glucosidase activity was 66 °C and 5.0, respectively. Under these conditions, the β-glucosidase activity increased 1.46 fold. The half-life of denaturation in 6% NaCl (w/v) solution was approximately twice as long as that in NaCl free solution. The Gibb's free energy for denaturation, ΔG, was 2 kJ/mol higher in 6% NaCl (w/v) solution than in NaCl free solution. The melting point (68.51 °C) in 6% NaCl (w/v) solution was 1.71 °C higher than that (66.80 °C) in NaCl free solution. Similarly, the activity and thermostability of the pure β-glucosidase increased remarkably at high salinity. The thermostable β-glucosidase, of which the activity and the thermostability are remarkably enhanced at high salinity, is valuable for industrial hydrolyzation of cellulose in high salinity environments.     

Characterization of Recombinant Rhamnogalacturonan α-l-Rhamnopyranosyl-(1,4)-α-d-Galactopyranosyluronide Lyase from Aspergillus aculeatus : An Enzyme That Fragments Rhamnogalacturonan I Regions of Pectin

The four major oligomeric reaction products from saponified modified hairy regions (MHR-S) from apple, produced by recombinant rhamnogalacturonan (RG) α-l-rhamnopyranosyl-(1,4)-α-d-galactopyranosyluronide lyase (rRG-lyase) from Aspergillus aculeatus, were isolated and characterized by ¹H-nuclear magnetic resonance spectroscopy. They contain an alternating RG backbone with a degree of polymerization of 4, 6, 8, and 10 and with an α-Δ-(4,5)-unsaturated d-galactopyranosyluronic acid at the nonreducing end and an l-rhamnopyranose at the reducing end. l-Rhamnopyranose units are substituted at C-4 with β-galactose. The maximum reaction rate of rRG-lyase toward MHR-S at pH 6.0 and 31°C was 28 units mg⁻¹. rRG-lyase and RG-hydrolase cleave the same alternating RG I subunit in MHR. Both of these enzymes fragment MHR by a multiple attack mechanism. The catalytic efficiency of rRG-lyase for MHR increases with decreasing degree of acetylation. Removal of arabinose side chains improves the action of rRG-lyase toward MHR-S. In contrast, removal of galactose side chains decreased the catalytic efficiency of rRG-lyase. Native RG-lyase was purified from A. aculeatus, characterized, and found to be similar to the rRG-lyase expressed in Aspergillus oryzae.