Structure of rat aldose reductase-like protein AKR1B14 holoenzyme: Probing the role of His269 in coenzyme binding by site-directed mutagenesis

Rat aldose reductase-like protein (AKR1B14) is the ortholog of mouse vas deferens protein (AKR1B7) playing roles in detoxification of reactive aldehydes and synthesis of prostaglandin F_2α. The crystal structure of the binary complex (AKR1B14-NADPH) was determined at 1.86 Å resolution, and showed that the adenine ring and the 2′-phosphate group of the coenzyme formed π-stacking and electrostatic interactions with the imidazole ring and ND1 atom, respectively, of His269, which is not conserved in other aldose reductase-like proteins. The interactions were supported by site-directed mutagenesis of His269 to Arg, Phe and Met, which increased the K_m for NADPH by 4, 7 and 127-fold, respectively. This is the first report of the tertiary structure of a rodent AKR1B7 ortholog, which describes the role of a novel dual interaction for the non-conserved His269 in coenzyme binding.     

Structural insights into the autoinhibition and posttranslational activation of histone methyltransferase SmyD3

The SmyD family represents a new class of chromatin regulators that is important in heart and skeletal muscle development. However, the critical questions regarding how they are regulated posttranslationally remain largely unknown. We previously suggested that the histone methyltransferase activity of SmyD1, a vital myogenic regulator, appears to be regulated by autoinhibition and that the possible hinge motion of the conserved C-terminal domain (CTD) might be central to the maintenance and release of the autoinhibition. However, the lack of direct evidence of the hinge motion has limited our further understanding of this autoinhibitory mechanism. Here, we report the crystal structure of full-length SmyD3 in complex with the methyltransferase inhibitor sinefungin at 1.7 Å. SmyD3 has a two-lobed structure with the substrate binding cleft located at the bottom of a  15-Å-deep crevice formed between the N- and C-terminal lobes. Comparison of SmyD3 and SmyD1 clearly suggests that the CTD can undergo a large hinge-bending motion that defines two distinct conformations: SmyD3 adopts a closed conformation with the CTD partially blocking the substrate binding cleft; in contrast, SmyD1 appears to represent an open form, where the CTD swings out by ∼ 12 Å from the N-terminal lobe, forming an open cleft with the active site completely exposed. Overall, these findings provide novel structural insights into the mechanism that modulates the activity of the SmyD proteins and support the observation that a posttranslational activation, such as by molecular chaperon Hsp90, is required to potentiate the proteins.     

Dynamics may significantly influence the estimation of interatomic distances in biomolecular X-ray structures

Atomic positions obtained by X-ray crystallography are time and space averages over many molecules in the crystal. Importantly, interatomic distances, calculated between such average positions and frequently used in structural and mechanistic analyses, can be substantially different from the more appropriate time-average and ensemble-average interatomic distances. Using crystallographic B-factors, one can deduce corrections, which have so far been applied exclusively to small molecules, to obtain correct average distances as a function of the type of atomic motion. Here, using 4774 high-quality protein X-ray structures, we study the significance of such corrections for different types of atomic motion. Importantly, we show that for distances shorter than 5 Å, corrections greater than 0.5 Å may apply, especially for noncorrelated or anticorrelated motion. For example, 14% of the studied structures have at least one pair of atoms with a correction of ≥ 0.5 Å in the case of noncorrelated motion. Using molecular dynamics simulations of villin headpiece, ubiquitin, and SH3 domain unit cells, we demonstrate that the majority of average interatomic distances in these proteins agree with noncorrelated corrections, suggesting that such deviations may be truly relevant. Importantly, we demonstrate that the corrections do not significantly affect stereochemistry and the overall quality of final refined X-ray structures, but can provide marked improvements in starting unrefined models obtained from low-resolution X-ray data. Finally, we illustrate the potential mechanistic and biological significance of the calculated corrections for KcsA ion channel and show that they provide indirect evidence that motions in its selectivity filter are highly correlated.     

Square-planar copper(II) halide complexes of tridentate ligands with π-π stacking interactions and alternating short and long Cu ? Cu distances

The preparation of a new tridentate N₂O-donor ligand N-(2-pyridylmethyl)-3-methoxysalicylaldiminato (HL) is described, together with the corresponding copper(II) complexes [Cu(L)X] (X = Cl⁻, Br⁻). The compounds were characterized by elemental analysis, spectral, magnetic and crystallographic studies. In both compounds, the local molecular structure of the Cu(II) ion involves a square-planar CuN₂OX chromophore, consisting of a deprotonated phenolate oxygen, an imine nitrogen, the pyridine nitrogen and X. In the solid state, π-π stacking interactions are dominantly present, involving the pyridine and phenolate rings of neighboring molecules, which lead to a one-dimensional arrangement with alternating short and long Cu ? Cu distances of [3.720, 4.599 Å] for the bromo complex and of [3.698, 4.696 Å] for the chloro complex. The temperature-dependent magnetic measurements and EPR data of polycrystalline samples, as well as of frozen solutions in CHCl₃ show that there is no observable exchange interaction between the Cu ions. The EPR parameters (g_∥, A_∥) agree with a perfect planar geometry, just as found in the X-ray analysis.     

Conformational analysis of N,N-disubstituted-1,4-diazepane orexin receptor antagonists and implications for receptor binding

NMR spectroscopy, X-ray crystallography, and molecular modeling studies indicate that N,N-disubstituted-1,4-diazepane orexin receptor antagonists exist in an unexpected low-energy conformation that is characterized by an intramolecular π-stacking interaction and a twist-boat ring conformation. Synthesis and evaluation of a macrocycle that enforces a similar conformation suggest that this geometry mimics the bioactive conformation.     

Structure and Function of P19, a High-Affinity Iron Transporter of the Human Pathogen Campylobacter jejuni

Campylobacter jejuni, a major cause of acute bacterial diarrhea in humans, expresses numerous proteins to import diverse forms of essential iron. The expression of p19 and an adjacent iron transporter homologue (ftr1) is strongly induced upon iron limitation, suggesting a function in iron acquisition. Here, we show that the loss of P19 alone is detrimental to growth on iron-restricted media. Furthermore, metal binding analysis demonstrates that recombinant P19 has distinct copper and iron binding sites. Crystal structures of P19 have been solved to 1.41 Å resolution, revealing an immunoglobulin-like fold. A P19 homodimer in which both monomers contribute ligands to two equivalent copper sites located adjacent to methionine-rich patches is observed. Copper coordination occurs via three histidine residues (His42, His95, and His132) and Met88. A solvent channel lined with conserved acidic residues leads to the copper site. Soaking crystals with a solution of manganese as iron analog reveals a second metal binding site in this solvent channel (metal-metal distance, 7.7 Å). Glu44 lies between the metal sites and displays multiple conformations in the crystal structures, suggesting a role in regulating metal-metal interaction. Dimerization is shown to be metal dependent in vitro and is detected in vivo by cross-linking.     

Synthesis, crystallography, photoluminescence and electroluminescence of three polymorphs of dibenzoylmethane gallium complex

Three polymorphs of tris(dibenzoylmethane) gallium were characterized by optical microscopy, absorption, photoluminescence (PL) and used for electroluminescent devices (EL). PL spectrum of the thin film is composed of both γ-type yellow emission and β-type blue emission, but only γ-type yellow EL emission was observed, indicating the polymorphic counterpart β-type with weaker π-π interactions was defeated in competition for EL generation. A comparison of the three polymorphic crystal structures reveals that a zigzag channel from intermolecular π-π interaction at a distance of 3.6-3.7 Å to intramolecular bond-bond conjugation may dominate the charge transport and be responsible for the yellow EL emission.     

Electron microscopy of dental enamel: Analysis of crystal lattice images

Summary Thin sections of rat incisor enamel were studied with the electron microscope. Fringe patterns having repeat periods in the range 3.1–8.2 Å were seen in individual enamel crystals. These images were interpreted as representing the resolution of corresponding planes in the hydroxyapatite crystal lattice. The lattice spacings and interplanar angles were identified by comparing the observations with available data derived from X-ray diffraction analysis.     

Analysis of Flavivirus NS5 Methyltransferase Cap Binding

The flavivirus 2′-O-nucleoside N-terminal RNA methyltransferase (MTase) enzyme is responsible for methylating the viral RNA cap structure. To increase our understanding of the mechanism of viral RNA cap binding we performed a detailed structural and biochemical characterization of the guanosine cap-binding pocket of the dengue (DEN) and yellow fever (YF) virus MTase enzymes. We solved an improved 2.1 Å resolution crystal structure of DEN2 Mtase, new 1.5 Å resolution crystal structures of the YF virus MTase domain in apo form, and a new 1.45 Å structure in complex with guanosine triphosphate and RNA cap analog. Our structures clarify the previously reported DEN MTase structure, suggest novel protein-cap interactions, and provide a detailed view of guanine specificity. Furthermore, the structures of the DEN and YF proteins are essentially identical, indicating a large degree of structural conservation amongst the flavivirus MTases. Guanosine triphosphate analog competition assays and mutagenesis analysis, performed to analyze the biochemical characteristics of cap binding, determined that the major interaction points are (i) guanine ring via π−π stacking with Phe24, N1 hydrogen interaction with the Leu19 backbone carbonyl via a water bridge, and C2 amine interaction with Leu16 and Leu19 backbone carbonyls; (ii) ribose 2′ hydroxyl interaction with Lys13 and Asn17; and (iii) α-phosphate interactions with Lys28 and Ser215. Based on our mutational and analog studies, the guanine ring and α-phosphate interactions provide most of the energy for cap binding, while the combination of the water bridge between the guanine N1 and Leu19 carbonyl and the hydrogen bonds between the C2 amine and Leu16/Leu19 carbonyl groups provide for specific guanine recognition. A detailed model of how the flavivirus MTase protein binds RNA cap structures is presented.     

The crystal structure of saporin SO6 from Saponaria officinalis and its interaction with the ribosome

The 2.0 Å resolution crystal structure of the ribosome inactivating protein saporin (isoform 6) from seeds of Saponaria officinalis is presented. The fold typical of other plant toxins is conserved, despite some differences in the loop regions. The loop between strands β7 and β8 in the C-terminal region which spans over the active site cleft appears shorter in saporin, suggesting an easier access to the substrate. Furthermore we investigated the molecular interaction between saporin and the yeast ribosome by differential chemical modifications. A contact surface inside the C-terminal region of saporin has been identified. Structural comparison between saporin and other ribosome inactivating proteins reveals that this region is conserved and represents a peculiar motif involved in ribosome recognition.     

Cyclodextrin inclusion compounds of vanadium complexes: Structural characterization and catalytic sulfoxidation

Reaction of potassium vanadate with the hydrazone ligand derived from Schiff-base condensation of salicylaldehyde and biphenyl-4-carboxylic acid hydrazide (H₂salhybiph) in the presence of two equivalents α-cyclodextrin (α-CD) in water yields the 1:2 inclusion compound K[VO₂(salhybiph)@(α-CD)₂]. Characterization in solution confirmed the integrity of the inclusion compound in the polar solvent water. The inclusion compound crystallizes together with additional water molecules as K[VO₂(salhybiph)@(α-CD)₂] · 18H₂O in the monoclinic space group P2(1). Two α-CD rings forming a hydrogen bonded head to head dimer are hosting the hydrophobic biphenyl side chain of the complex K[VO₂(salhybiph)]. The supramolecular aggregation of the inclusion compound in the solid state is established through hydrogen bonding interactions among adjacent α-CD hosts and with vanadate moieties of the guest complexes as well as ionic interactions with the potassium counterions. In contrast the supramolecular structure of the guest complex K[VO₂(salhybiph)] without the presence of CD host molecules is governed by π-π-stacking interactions and additional CH/π interactions. The new inclusion complex K[VO₂(salhybiph)@(α-CD)₂] and the analogous 1:1 inclusion compound with β-CD were tested as catalyst in the oxidation of methyl phenyl sulfide (thioanisol) using hydrogen peroxide as oxidant in a water/ethanol mixture, under neutral as well as acidic conditions.     

Synthesis and structural characterization of two new polynuclear metal thiosaccharinates: Hexakis(thiosaccharinato)hexasilver(I) and tetrakis(thiosaccharinato)tetracopper(I)

The title compounds, for short Ag₆(tsac)₆ (1) and [Cu₄(tsac)₄(MeCN)₂] · 2MeCN (2), were prepared by the reaction of thiosaccharin with Ag(I) or Cu(II) salts in different solvents. The new complexes were characterized by FT-IR, Raman, UV-Vis and NMR spectroscopy. Their crystal and molecular structures were determined by X-ray diffraction methods. The structures were solved from 1621 (1) and 7080 (2) reflections with I > 2σ(I) and refined to agreement R1-factors of 0.0261 (1) and 0.0456 (2). Ag₆(tsac)₆ molecule derives from the clustering of six Ag(tsac) moieties related to each other through the crystallographic 3-bar (S₆) symmetry operations of the space group. This results in a highly regular molecular structure where the silver atoms are at the corners of an octahedral core slightly compressed along one of its three-fold axis [inter-metallic Ag?Ag contacts of 3.1723(4) and 3.1556(4) Å]. The six thiosaccharinate ligands bridge neighboring Ag atoms along the C₃-axis through Ag-N bonds [d(Ag-N) = 2.285(2) Å] at one end and bifurcated Ag-S(thione)-Ag bonds [Ag-S distances of 2.4861(7) and 2.5014(8) Å] at the other end. In contrast, the 2 compound is arranged in the lattice as an irregular tetrameric copper complex [Cu₄(tsac)₄(MeCN)₂] where the metals show different environments. Two copper ions are four-fold coordinated to three tsac ions through the N-atom of one tsac [Cu-N distances of 2.112(3) and 2.064(3) Å] and the thione sulfur atom of the other two [Cu-S distances in the range from 2.284(1) to 2.358(1) Å] and to a MeCN solvent molecule [Cu-N distances of 1.983(4) and 2.052(3) Å]. The other two copper ions are in three-fold environment, one trans-coordinated to two tsac ions [Cu-N distances of 1.912(3) and 1.920(3) Å] and to the thione S-atom of a third ligand [d(Cu-S) = 2.531(1) Å], the other one to the thione sulfur atom of three tsac ligands [Cu-S distances in the range from 2.229(1) to 2.334(1) Å]. The clustering renders the metals to short distances from each other, the shorter Cu?Cu distance being 2.6033(7) Å, as to presume the existence of weak inter-metallic interaction within the cluster.     

Identification of nonplanar small molecule for G-quadruplex grooves: molecular docking and molecular dynamic study

DNA G-quadruplex is an attractive drug target for anticancer therapy. Most G-quadruplex ligands have little selectivity, due to π-stacking interaction with common G-tetrads surface. Thanks to the varieties of G-quadruplex grooves, the groove-binding ligand is expected to create high selectivity. Therefore, developing novel molecular geometries that target G-quadruplex groove has been paid growing attention. In this work, steroid FG, a special nonplanar and nonaromatic small molecule, interacting with different conformations of G-quadruplexes has been studied by molecular docking and molecular dynamics simulations. The results showed the selectivity of the hydrophobic group of steroid FG for the wide groove of antiparallel G-quadruplex. The methyl groups on the tetracyclic ring of steroid represent the specific binding ability for the small hydrophobic cavity formed by reversed stacking of G-tetrads in antiparallel G-quadruplex groove. This work provides new insight for developing new classes of G-quadruplex groove-binding ligands.     

The interactions of square platinum(II) complexes with guanine and adenine: a quantum-chemical ab initio study of metalated tautomeric forms

The influence of binding of square planar platinum complexes on tautomeric equilibria of the DNA bases guanine and adenine was investigated using the density functional B3LYP method. Neutral trans-dichloro(amine)-, +1 charged chloro(diamine)-, and +2 charged triamine-platinum(II) species were chosen for coordination to bases. Only the N₇ interaction site of the bases was considered. The calculations demonstrate that the neutral platinum adduct does not change the tautomeric equilibria of the bases. Furthermore, N₇ binding of the neutral Pt adduct moderately reduces the probability of protonation of the N₁ position of adenine. Larger effects can be observed for +1 and mainly +2 adducts, but these can be rationalized by electrostatic effects. Since the electrostatic effects are expected to be efficiently compensated for by a charged backbone of DNA and counterions in a polar solvent, no dramatic increase in mispair formation is predicted for Pt(II) adducts, which is in agreement with experiment. The interaction energies between Pt adducts and the nucleobases were also evaluated. These interaction energies range from ca. 210 kJ/mol for neutral adducts, interacting with both bases and their tautomers, up to 500 kJ/mol for the +2 charged adducts, interacting with the keto-guanine tautomer and the anti-imino-adenine tautomer. The surprisingly large interaction energy for the latter structure is due to the strong H-bond between the NH₃ ligand group of the metal adduct and the N₆ nitrogen atom of the base. Received: 6 July 1999 / Accepted: 7 December 1999     

NMR diffusion as a novel tool for measuring the association constant between cyclodextrin and guest molecules

In this paper we introduce the use of diffusion measurements by nuclear magnetic resonance (NMR) spectroscopy for determining association constants of weak and very weak interactions between cyclodextrin and guest molecules, as long as both the free and complexed guest molecules are soluble to an extent that allows good sensitivity in the NMR experiment. The experimental setup and data analysis is discussed for three different guest molecules: L-phenylalanine, L-leucine and L-valine, representing different strengths of interaction. The underlying assumptions are discussed and the scope of the method (range of K(a) values, requirements to the guest molecule) are discussed. The method's main advantage is its general applicability independent of chromogenic or electrochemical properties of the guest molecule. Whereas calorimetric methods that exhibit a similar generality, are applicable mainly to strong interactions, NMR diffusion measurements are applicable to weaker interactions down to the theoretical limit of 1 M(-1), the upper limit for K(a) values to be determined by it is approximately 200. A further advantage of the method is the low amount of sample needed. The method is in principle applicable to any case of molecular recognition between a host and guest molecule leading to weak interactions.     

Electrocatalytic reduction of carbon dioxide induced by bis(N-R-2-hydroxy-1-naphthaldiminato)-copper(II) (R = n-octyl, n-dodecyl): Magnetic and theoretical studies and the X-ray structure of bis(N-n-octyl-2-hydroxy-1-naphthaldiminato)-copper(II)

Copper(II) complexes of n-alkyl-2-hydroxy-1-naphthaldimine Schiff bases (with n-alkyl: n-octyl, and n-dodecyl) have been synthesized, to study steric and electronic effects of long alkyl chain substituents on their structure and properties. These complexes have been characterized with FT-IR, UV-Vis, magnetic susceptibility and cyclic voltammetry both in nitrogen and carbon dioxide atmosphere. Metal-ligand coordination is inferred from the shifting of the ν_CN stretching vibration mode in the 1610-1620 cm⁻¹ region when compared to that of the free ligand. The UV-Vis spectra show one band around 640 nm typical for square planar Cu(II) complexes. Results obtained from cyclic voltammetry indicate electrocatalytic reduction of carbon dioxide around −0.90 V (versus Ag/AgCl). Bis(N-n-octyl-2-hydroxy-1-naphthaldiminato)-copper(II) has been studied with X-ray diffraction. The molecular structure shows the copper atom in a planar environment and the n-octyl chains having thermal disorder. The crystal packing shows stacked units intermolecularly separated by 3.33 Å, probably due to π-π interactions between naphthyl groups, and Cu-O and O-O separations of 3.95 and 3.42 Å, respectively. The magnetic susceptibility data between 10 and 300 K are indicative of diluted paramagnetic behavior. Density functional theory calculations of spin density for the n-octyl complex shows the unpaired electron localized along the planar CuO₂N₂ moiety. The calculated electrostatic potential show electron rich regions on the oxygen atoms.     

Solid-state structures and thermal properties of inclusion complexes of the organophosphate insecticide fenitrothion with permethylated cyclodextrins

The X-ray crystal structures and thermal stabilities of the inclusion complexes formed between the organophosphate insecticide fenitrothion [O,O-dimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate] and the host compounds TRIMEA and TRIMEB (permethylated α- and β-cyclodextrins, respectively) are reported. In the complex (TRIMEA)₂·fenitrothion 1, the guest phosphate ester group is disordered and the molecule is fully encapsulated within a novel TRIMEA dimer in which the secondary rims of the two host molecules are in close contact. In contrast, the complex TRIMEB·fenitrothion 2 is monomeric and the guest molecule is statistically disordered over two positions, with the phosphate group inserted in the host cavity in both cases. Thermal analysis indicated gradual and partial loss of the guest in 1 during heating between 130 °C and the melting point of the complex (∼200 °C), whereas complex 2 displayed significant mass loss only after fusion of the complex at 161 °C.     

Structure of the complex of heptakis(2,6-di-O-methyl)-beta-cyclodextrin with (2,4-dichlorophenoxy)acetic acid

The structure of the complex formed by heptakis(2,6-di-O-methyl)-beta-cyclodextrin and (2,4-dichlorophenoxy)acetic acid was studied by X-ray diffraction. The dichlorophenyl moiety of the guest molecule was found outside the host hydrophobic cavity in the primary methoxy groups region whereas the oxyacetic acid chain penetrates the cavity from the primary face. The host molecules stacks along the a crystal axis forming a column. In the space between three successive hosts of the column, a guest molecule is accommodated.     

Asymmetric chlorine-bridged zig-zag Cu(II) chains linked together in 2D sheets by strong Watson-Crick type intermolecular double hydrogen bonds of bis(pyrimidin-2-yl)amine ligands: synthesis, structure and antiferromagnetism

A novel polymeric, asymmetric chloro-bridged copper chain with general formula [μ-Cl-CuCl(dipm]_n (in which dipm = bis(pyrimidin-2-yl)amine) has been synthesized and characterised by X-ray crystallography and infrared spectroscopy. The chains are organized in 2D sheets by intermolecular double H bonds between pairs of dipm molecules. In addition, EPR and magnetic measurements have been performed, and these have been related to the dinuclear structural details.The geometry around the copper(II) ion is distorted square pyramidal with the basal plane formed by the two nitrogen atoms of the dipm ligand and two chloride atoms, one of which is bridging. The Cu-N distances are 2.0342(15) and 2.0125(15) Å and Cu-Cl distances are 2.2899(6) and 2.2658(6) Å. The apical position of Cu is occupied by a chloride atom of a neighbouring unit atom at a distance of 2.6520(6) Å, resulting in the polynuclear array in which one chloride anion and the copper ion forms a zig-zag chain. The magnetic interaction between the Cu-Cu atoms is weak antiferromagnetic with a singlet-triplet separation (J) of −3.2(1) cm⁻¹.