Ordered Solvent Molecules Research Articles

X-ray Crystallographic Structure of Recombinant Eosinophil-derived Neurotoxin at 1.83 Å Resolution

The X-ray crystallographic structure of recombinant eosinophil-derived neurotoxin (rEDN) has been determined by molecular replacement methods and refined at 1.83 Å resolution to a conventional R-factor (=Σ || F o| − | F c|| /Σ | F o| ) of 0.152 with excellent stereochemistry. The molecular model of rEDN contains all 1081 non-hydrogen protein atoms, two non-covalently bound sulfate anions and 121 ordered solvent molecules. The polypeptide fold of rEDN is related to those observed in the homologous structures of RNase A, Onconase and angiogenin. rEDN is one of the largest members of the pyrimidine-specific ribonuclease superfamily of vertebrates and has small insertions in four of its seven loop structures and a large insertion from Asp115 to Tyr123. The non-covalently bound SO 4(A) and SO 4(B) anions occupy phosphate-binding subsites of rEDN. The active site SO 4(A) anion makes contacts in rEDN that are similar to those in RNase A and involve the side-chain atoms of Gln14, His15 and His129, and the NH group of Leu130. The SO 4(B) anion makes contacts with the side-chain atoms of Arg36 and Asn39 and the main-chain atoms of Asn39 and Gln40. The equivalent residues of RNase A cannot make contacts similar to those observed in rEDN. The SO 4(B) binding site of rEDN likely corresponds to the P −1subsite and may be representative of how other homologous RNases bind the P −1phosphate.

Mimicking the Vancomycin Carboxylate Binding Site: Synthetic receptors for sulfonates, carboxylates, and N‐protected α‐amino acids in water

AbstractThe novel H2O‐soluble cyclophanes 1 and 2 incorporating different anion‐recognition sites were prepared in short synthetic routes (Schemes 1 and 2) as first‐generation mimics of the natural, D‐Ala‐D‐Ala binding antibiotic vancomycin. The X‐ray crystal structure of 1, a tris(hydrochloride)salt, revealed an open, preorganized cavity of sufficient size for the incorporation of small aliphatic residues (Fig. 3). In the crystal, molecules of 1 are arranged in parallel stacks, generating two types of channels, an ‘intra‐stack’ channel passing through the cyclophane cavities and an ‘inter‐stack’ channel located between cyclophane stacks (Fig. 4). The strongest intermolecular interactions between macrocycles in the crystal are CO…︁HN H‐bonds between the carboxamide residues of adjacent cyclophanes in neighboring stacks (Fig. 5). The ‘intra‐’ and ‘inter‐stack’ channels incorporate the three ordered Cl− counterions and several, partially ordered solvent molecules (4 MeOH, 1 H2O) (Fig. 6). Counterion Cl(2) is located within the ‘intra‐stack’ channel and interacts with a protonated piperazinium N‐atom and both ‘intra‐stack’ MeOH molecules. The two other counterions, Cl(1) and Cl(3), are located within the ‘inter‐stack’ channel. They are connected to two MeOH and one H2O molecules and also interact both with the NH group of the protonated spiropiperidinium ring in 1, forming an infinite, chain‐like H‐bonding network …︁Cl(1)…︁HOH…︁MeOH…︁Cl(3)…︁HNH…︁Cl(1′)…︁. Both ‘intra‐’ and ‘inter‐stack’ MeOH molecules undergo weak CH…︁π interactions with neighboring aromatic rings. Cyclophane 1 complexed aromatic sulfonates in 0.5M KCl/DCl buffer in D2O, whereas the tetrakis(quaternary ammonium) receptor 2 bound the sodium salts of aliphatic and aromatic carboxylates and sulfonates, of N‐acylated α‐amino acids as well as of N‐acetyl‐D‐alanyl‐D‐alanine (Ac‐D‐Ala‐D‐Ala), a substrate of vancomycin, in pure H2O. In all of these complexes, ion pairing between the cationic recognition site in the periphery of the cyclophane receptor and the anionic substrates represents the major driving force for host‐guest association. The 1H‐NMR analysis of complexation‐induced changes in chemical shift clearly demonstrated that, in solution, this ion pairing exclusively takes place outside the cavity. Nevertheless, the macrocyclic bridges are essential for the efficiency of the anion‐recognition sites in the two cyclophane receptors 1 and 2. Control compounds 3 and 4 possess nearly the same anion‐recognition sites than 1 and 2, but lack their macrocyclic preorganization; as a consequence, they do not form stable ion‐pairing complexes with mono‐anionic substrates in the considered concentration ranges ( < 50 mM) in D2O.

High Resolution Crystal Structures of Amphibian Red-Cell L Ferritin: Potential Roles for Structural Plasticity and Solvation in Function

Ferritin is a highly conserved multisubunit protein in animals, plants and microbes which assembles with cubic symmetry and transports hydrated iron ions and protons to and from a mineralized core in the protein interior. We report here the high resolution structures of recombinant amphibian red-cell L ferritin and two mutants solved under two sets of conditions. In one mutant, Glu56, 57, 58 and 60 were replaced with Ala, producing a lag phase in the kinetics of iron uptake. In the second mutant, His25 was replaced with Tyr with, at most, subtle effects on function. A molecule of betaine, used in the purification, is bound in all structures at the 2-fold axis near the recently identified heme binding site of bacterioferritin and horse spleen L ferritin. Comparisons of the five amphibian structures identify two regions of the molecule in which conformational flexibility may be related to function. The positions and interactions of a set of 10 to 18 side-chains, most of which are on the inner surface of the protein, are sensitive both to solution conditions and to the Glu→Ala mutation. A subset of these side-chains and a chain of ordered solvent molecules extends from the vicinity of Glu56 to 58 and Glu60 to the 3-fold channel in the wild type protein and may be involved in the transport of either iron or protons. The “spine of hydration” is disrupted in the Glu→Ala mutant. In contrast, H25Y mutation shifts the positions of backbone atoms between the site of the mutation and the 4-fold axis and side-chain positions throughout the structure; the largest changes in the position of backbone atoms are in the DE loop and E Helix, approximately 10 Å from the mutation site. In combination, these results indicate that solvation, structural plasticity and cooperative structural changes may play a role in ferritin function. Analogies with the structure and function of ion channel proteins such as annexins are noted.

Refined crystal structure of liver alcohol dehydrogenase-NADH complex at 1.8 A resolution.

The crystal structure of the ternary complex of horse liver alcohol dehydrogenase (LADH) with the coenzyme NADH and inhibitor dimethyl sulfoxide (DMSO) has been refined by simulated annealing with molecular dynamics and restrained positional refinement using the program X-PLOR. The two subunits of the enzyme were refined independently. The space group was P1 with cell dimensions a = 51.8, b = 44.5, c = 94.6 A, alpha = 104.8, beta = 102.3 and gamma = 70.6 degrees. The resulting crystallographic R factor is 17.3% for 62 440 unique reflections in the resolution range 10.0-1.8 A. A total of 472 ordered solvent molecules were localized in the structure. An analysis of secondary-structure elements, solvent content and NADH binding is presented.

Binding mode of azide to ferric Aplysia limacina myoglobin. Crystallographic analysis at 1.9 Å resolution

The binding mode of azide to the ferric form of Aplysia limacina myoglobin has been studied by X-ray crystallography. The three-dimensional structure of the complex has been refined at 1.9 A resolution to a crystallographic R-factor of 13.9%, including 126 ordered solvent molecules. Azide binds to the heme iron, at the sixth co-ordination position, and is oriented towards the outer part of the distal site crevice. This orientation is stabilized by an ionic interaction with the side-chain of Arg66 (E10) which, from an outer orientation in the 'aquo-met' ligand-free myoglobin, folds back towards the distal site in the presence of the anionic ligand. In the absence of a hydrogen bond donor residue at the distal E7 position in Aplysia limacina myoglobin, a different polar residue, Arg66 at the E10 topological position, has been selected by molecular evolution in order to grant ligand stabilization.

Expression of rat intestinal fatty acid binding protein in E. coli and its subsequent structural analysis: a model system for studying the molecular details of fatty acid-protein interaction

A prokaryotic expression vector containing the rec A promoter and a translational enhancer element from the gene 10 leader of bacteriophage T7 was used to direct efficient synthesis of rat intestinal fatty acid binding protein (I-FABP) in E. coli. Expression of I-FABP in E. coli has no apparent, deleterious effects on the organism. High levels of expression of I-FABP mRNA in supE+ strains of E. coli, such as JM101, is associated with suppression of termination at its UGA stop codon. This can be eliminated by using a supE-strain as MG1655 and by site-directed mutagenesis of the cDNA to create an in frame UAA stop codon. E. coli-derived rat I-FABP lacks its initiator Met residues. It has been crystallized with and without bound palmitate. High resolution x-ray crystallographic studies of the 131 residue apo- and holo-proteins have revealed the following. I-FABP contains 10 anti-parallel beta-strands organized into two orthogonally situated beta-sheets. The overall conformation of the protein resembles that of a clam--hence the term beta-clam. The bound ligand is located in the interior of the protein. Its carboxylate group forms part of a unique five member hydrogen bonding network consisting of two ordered solvent molecules as well as the side chains of Arg106 and Gln115. The hydrocarbon chain of the bound C16:0 fatty acid has a distinctive bent conformation with a slight left-handed helical twist. This conformation is maintained by interactions with the side chains of a number of hydrophobic and aromatic amino acids. Apo-I-FABP has a similar overall conformation to holo-I-FABP indicating that the beta-clam structure is stable even without bound ligand. The space occupied by bound ligand in the core of the holo-protein is occupied by additional ordered solvent molecules in the apo-protein. Differences in the side chain orientations of several residues located over a potential opening to the cores of the apo- and holo-proteins suggest that solvent may play an important role in the binding mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Refined apoprotein structure of rat intestinal fatty acid binding protein produced in Escherichia coli.

Rat intestinal fatty acid binding protein (I-FABP) is a member of a family of cytoplasmic hydrophobic ligand-binding proteins. To gain insights about the contribution of bound fatty acid to I-FABP's conformation and mechanism of ligand binding, we have determined the structure of Escherichia coli-derived rat apo-I-FABP to 1.96-A resolution and compared it to the recently refined structure of I-FABP with bound palmitate. Both apo- and holo-I-FABP are composed primarily of anti-parallel beta-strands which form two nearly orthogonal beta-sheets ("beta-clam"). The overall structures of the apo- and holo-I-FABP are nearly identical, with a root mean square (rms) difference of 0.37 A between C alpha atoms, 0.38 A between all main-chain atoms, and 0.94 A between all side-chain atoms. However, rms differences of greater than 1.3 A were noted for the side chains of Ile-23, Lys-27, Arg-56, Leu-72, Ala-73, and Asp-74. The space occupied by bound ligand in the core of the holoprotein is occupied in the apo-protein by ordered solvent molecules. This results in an increase in the total number of internal ordered solvent molecules from 7 in the holoprotein to 13 in apo-I-FABP. This finding, together with observed differences in the side-chain orientations of two residues (Arg-56 and Lys-27) situated over a potential opening to the cores of the apo- and holoproteins, suggests that solvent molecules play a critical role in ligand binding. Moreover, the data indicate that the beta-clam structure is stable even in the absence of bound ligand.

Refined structure of spinach glycolate oxidase at 2 Å resolution

The amino acid sequence of glycolate oxidase from spinach has been fitted to an electron density map of 2.0 Å nominal resolution and the structure has been refined using the restrained parameter least-squares refinement of Hendrickson and Konnert. A final crystallographic R-factor of 18.9% was obtained for 32,888 independent reflections from 5.5 to 2 Å resolution. The geometry of the model, consisting of 350 amino acid residues, the cofactor flavin mononucleotide and 298 solvent molecules, is close to ideal with root-means-quare deviations of 0.015 Å in bond lengths and 2.6 ° in bond angles. The expected trimodal distribution with preference for staggered conformation is obtained for the side-chain χ 1-angles. The core of the subunit is built up from the eight β-strands in the β α -barrel. This core consists of two hydrophobic layers. One in the center is made up of residues pointing in from the β-strands towards the barrel axis and the second, consisting of two segments of residues, pointing out from the β-strands towards the eight α-helices of the barrel and pointing from the helices towards the strands. The hydrogen bond pattern for the β-strands in the β α -barrel is described. There are a number of residues with 3 10-helix conformation, in particular there is one left-handed helix. The ordered solvent molecules are organized mainly in clusters. The average isotropic temperature factor is quite high, 27.1 Å 2, perhaps a reflection of the high solvent content in the crystal. The octameric glycolate oxidase molecule, which has 422 symmetry, makes strong interactions around the 4-fold axis forming a tight tetramer, but only weak interactions between the two tetramers forming the octamer.

Orthorhombic crystals and three-dimensional structure of the potent toxin II from the scorpion Androctonus australis Hector.

Orthorhombic crystals (space group P212121, a = 45.94 A, b = 40.68 A, c = 29.93 A) of the potent scorpion alpha-toxin II from Androctonus australis Hector were grown using sterile techniques. The structure was solved by a combination of heavy-atom and model phasing. Subsequently, it was refined at 1.8 A resolution by a fast-Fourier restrained least-squares procedure. The crystallographic R factor is 0.152 for data with 7.0 A greater than d greater than 1.8 A and F greater than 2.5 sigma (F) and 0.177 when all data are considered. Eighty-nine solvent molecules have been incorporated into the model. The dense core formed by the alpha-helical and antiparallel beta-sheet moieties and three of the four disulfide bridges is similar in variant 3, a toxin purified from the North American scorpion Centruroides sculpturatus, and in toxin II. However, the two molecules differ markedly in the orientation of loops protruding from the core. Toxin II seems to contain several highly ordered solvent molecules. Eight of them occupy a cavity consisting of the C-terminal region and a loop found only in scorpion alpha-toxins. The highly reactive and pharmacologically important Lys-58 is found at one of the extremes of this cavity, where it establishes a series of hydrogen bonds with protein and solvent atoms. The reactivities of the five lysine residues of toxin II are highly correlated with the formation of hydrogen bonds, hydrophobic interactions, and salt links.

High-resolution crystal structure of cytochrome P450cam

The crystal structure of Pseudomonas putida cytochrome P450cam with its substrate, camphor, bound has been refined to R = 0.19 at a normal resolution of 1.63 Å. While the 1.63 Å model confirms our initial analysis based on the 2.6 Å model, the higher resolution structure has revealed important new details. These include a more precise assignment of sequence to secondary structure, the identification of three cis-proline residues, and a more detailed picture of substrate-protein interactions. In addition, 204 ordered solvent molecules have been found, one of which appears to be a cation. The cation stabilizes an unfavorable polypeptide conformation involved in forming part of the active site pocket, suggesting that the cation may be the metal ion binding site associated with the well-known ability of metal ions to enhance formation of the enzyme-substrate complex. Another unusual polypeptide conformation forms the proposed oxygen-binding pocket. A localized distortion and widening of the distal helix provides a pocket for molecular oxygen. An intricate system of side-chain to backbone hydrogen bonds aids in stabilizing the required local disruption in helical geometry. Sequence homologies strongly suggest a common oxygen-binding pocket in all P450 species. Further sequence comparisons between P450 species indicate common three-dimensional structures with changes focused in a region of the molecule postulated to be associated with the control of substrate specificity.

The crystal structure of d(G-G-G-G-C-C-C-C) a model for poly(dG) · poly(dC)

The structure of the DNA oligomer d(G-G-G-G-C-C-C-C) has been determined at a resolution of 2.5 Å by single-crystal X-ray methods. There are two strands in the asymmetric unit, and these coil about each other to form a right-handed double-helix of the A-type with Watson-Crick hydrogen bonds between base-pairs. The helix has a shallow minor groove and a deep, water-filled major groove; almost all exposed functional groups on the DNA are hydrated, and 106 ordered solvent molecules have been found. The two d(G-G-G-G) · d(C-C-C-C) segments in the octamer exhibit similar and uniform structures, but there is a slight discontinuity at the GpC step between them. A recurring feature of the structure is the overlap of adjacent guanine bases in each GpG step, with the five-membered ring of one guanine stacking on the six-membered ring of its neighbour. There is little or no overlap between adjacent cytosine rings. Conformational parameters for these GpG steps are compared with those from other single-crystal X-ray analyses. In general, GpG steps exhibit high slide, low roll and variable twist. Models for poly(dG) · poly(dC) were generated by applying a simple rotation and translation to each of the unmodified d(G-G-G-G) · d(C-C-C-C) units. Detailed features of these models are shown to be compatible with various assays of poly(dG) · poly(dC) in solution, and are useful in understanding the polymorphic behaviour of this sequence under a variety of experimental conditions.

Solute-solvent interactions bioinorganic chemistry

The role of solvation in the electron transfer reactions of metalloproteins has been examined. Measurements of the rates of electro transfer reactions between metalloproteins and various inorganic complexes at different temperatures show that the mechanisms employed by hydrophobic and hydrophilic substrates vary significantly. Reactions of blue copper proteins and cytochromes with oxidants such as Co(dipic) − 2 (dipic = dipicolinate) and Co(phen) 3+ 3 (phen = 1,10-phenanthroline) exhibit large positive activation enthalpies and small (i some cases positive) activation entropies. One interpretation of these results is that substantial solvation changes accompany the interaction of the protein with the substrate in the activated complex. Specifically, penetration of hydrophobic substrates into hydrophobic protein interiors is thought to lead to displacement of ordered solvent molecules, thereby increasing entropy. This effect is apparently even larger in electron transfer reactions involving proteins that are believed to be physiological partners. Solvation of metalloproteins is believed to play a critical role in preventing close approach to reagents with hydrophilic electron transfer surfaces to the protein redox centers. In several cases electron tunneling between redox centers occurs over distances that are determined by the primary solvation shell. These distances have been calculated from electron transfer rate constants determined for several proteins. Measurements of the redox potentials and the standard enthalpies and entropies of several electron transfer reactions of cytochromes, iron-sulfur proteins, and blue proteins have been made. The determination of thermodynamic parameters for protein redox couples was accomplished by measuring the temperature dependence of the electrode potential of suitably designed electrochemical cells. If the electrochemical cell is in a nonisothermal configuration, the experimental temperature dependence, dE°/dt, of the electrode potential can be separated into the following components. dE°/dT = dφ tlj/dT + dφ tc/dT + dφ m f/dT φ tlj is the Galvani potential differences across the thermal liquid junction within the KCl salt bridge, φ tc is the ‘thermocouple’ potential difference between the hot and cold regions of the working electrode, and φ m f is the Galvani metal-solution potential difference at the working electrode. Since S° red - S° ox = F·(dφ m f/dT) where F si the Faraday. of dφ tc/dT and dφ tlj/dT are constant or can be neglected, partial molal entropy differences for the redox couples of interest can be obtained directly from measurements of dE°/dT. It has been proposed that in the nonisothermal electrochemical cell configurations under consideration dφ tc/dT < 14 μV° and dφ tlj/dT < 20μV°. Since these values are well below the experimental precision of the dE°/dT measurements, we neglect dφ tc/dT and dφ tlj/dT in nonisothemal experiments. Furthermore, since dφ tc/dT and dφ tlj/dT are constant for a given experimental arrangements, the relative values of dE°/dT for various systems will be unaffected even if these two sources of the observed temperature dependences are not negligible. We have determined the enthalpy and entropy differences for oxidation states changes in metalloproteins using an optically transparent thin-layer electrode (OTTLE) in a nonisothermal electrochemical cell configuration. The most striking result is that large negative values of Δ S° are obtained for proteins with buried copper or iron sites. Possible interpretations of these Δ S° values include conformational effects as well as solvation changes in the oxidized and reduced states.

The structure of triclinic lysozyme at 2·5 Å resolution

The gross similarity of the conformation of the hen egg-white lysozyme molecule in the triclinic and tetragonal crystal forms is known from an earlier study. In this work we have established the detailed conformation of the molecule in the triclinic form and compared the two structures using appropriately weighted difference maps. An independent model of the triclinic structure has been obtained by use of the real-space refinement technique. There are appreciable conformational differences of main chain, as well as side-chains, but these all occur in surface regions involved in intermolecular contacts. The locations of ordered solvent molecules have been determined.