NMR and computational studies of interactions between remote residues in gangliosides

Conformational preferences of the gangliosides GM1, GM1b, and GD1a have been investigated by using a systematic combination of NMR distance constraints and molecular mechanics calculations. These gangliosides share a common four-sugar core but differ in the number or placement of sialic acid residues attached to the core. Placement of the sialic acid residues is shown to influence the preferred core conformation. The origin of these effects is postulated to be intramolecular interactions of the sialic acid residues with other remote residues. In the case of GM1, hydrogen bonds between the internal sialic acid and an N-acetyl group on GalNAc are suggested. In the case of GD1a, a hydrogen-bonding network between the terminal and internal sialic acids is suggested to play a role.     

Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

Comparison of structural effects in 1,4 DNA-DNA interstrand cross-links formed by dinuclear and trinuclear platinum complexes

The novel anticancer drug ([[trans-PtCl(NH(3))(2)](2)-mu-[trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)NH(2))(2)]](NO(3))(4)) (BBR3464, 1,0,1/t,t,t, TPC) forms a 1,4-interstrand cross-linked adduct with the self-complementary DNA octamer 5'-d(ATG*TACAT)(2)-3', with the two platinum atoms coordinated in the major groove at N7 positions of guanines four base pairs apart on opposite DNA strands [Y. Qu, N.J. Scarsdale, M.-C. Tran, N. Farrell, J. Biol. Inorg. Chem. 8 (2003) 19-28]. The structure of the identical cross-link formed by the dinuclear [[trans-PtCl(NH(3))(2)](2)-mu-NH(2)(CH(2))(6)NH(2)]](NO(3))(2) (BBR3005, 1,1/t,t, DPC) was examined for comparison. The adduct was characterized and analyzed by MS, UV and NMR spectroscopy. NMR analysis of the adduct shows platination of the unique guanine residues. The strong H8/H1' intraresidue cross-peaks observed for all purine residues (A1, G3, A5 and A7) are consistent with a syn-conformation of the nucleoside unit in all cases. Thus, the structure resembles closely that formed by the trinuclear compound. Further confirmation of this similarity comes from the increase in melting temperature (66 degrees for DPC, 60 degrees for TPC, 22 degrees for free oligonucleotide). Since DNA is the principal target in vivo for these Pt cross-linking agents, the unique structural perturbations induced by these cross-links may be related to the increased cytotoxicity and antitumor activity of polynuclear platinum compounds as compared to cisplatin (cis-DDP). The similarity in the structures suggests opportunities to "deliver" the cross-link in a more efficient manner than the current clinically tested drug.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

3d interaction homology: The structurally known rotamers of tyrosine derive from a surprisingly limited set of information‐rich hydropathic interaction environments described by maps

Sidechain rotamer libraries are obtained through exhaustive statistical analysis of existing crystallographic structures of proteins and have been applied in multiple aspects of structural biology, for example, crystallography of relatively low‐resolution structures, in homology model building and in biomolecular NMR. Little is known, however, about the driving forces that lead to the preference or suitability of one rotamer over another. Construction of 3D hydropathic interaction maps for nearly 30,000 tyrosines reveals the environment around each, in terms of hydrophobic (π–π stacking, etc.) and polar (hydrogen bonding, etc.) interactions. After partitioning the tyrosines into backbone‐dependent (?, ψ) bins, a map similarity metric based on the correlation coefficient was applied to each map‐map pair to build matrices suitable for clustering with k‐means. The first bin (?200° ≤ ? < –155°; ?205° ≤ ψ < –160°), representing 631 tyrosines, reduced to 14 unique hydropathic environments, with most diversity arising from favorable hydrophobic interactions with many different residue partner types. Polar interactions for tyrosine include surprisingly ubiquitous hydrogen bonding with the phenolic OH and a handful of unique environments surrounding the tyrosine backbone. The memberships of all but one of the 14 environments are dominated (>50%) by a single χ₁/χ₂ rotamer. The last environment has weak or no interactions with the tyrosine ring and its χ₁/χ₂ rotamer is indeterminate, which is consistent with it being composed of mostly surface residues. Each tyrosine residue attempts to fulfill its hydropathic valence and thus, structural water molecules are seen in a variety of roles throughout protein structure. Proteins 2015; 83:1118–1136. © 2015 Wiley Periodicals, Inc.     

Applying an empirical hydropathic forcefield in refinement may improve low-resolution protein X-ray crystal structures

Background

The quality of X-ray crystallographic models for biomacromolecules refined from data obtained at high-resolution is assured by the data itself. However, at low-resolution, >3.0 Å, additional information is supplied by a forcefield coupled with an associated refinement protocol. These resulting structures are often of lower quality and thus unsuitable for downstream activities like structure-based drug discovery.

Methodology

An X-ray crystallography refinement protocol that enhances standard methodology by incorporating energy terms from the HINT (Hydropathic INTeractions) empirical forcefield is described. This protocol was tested by refining synthetic low-resolution structural data derived from 25 diverse high-resolution structures, and referencing the resulting models to these structures. The models were also evaluated with global structural quality metrics, e.g., Ramachandran score and MolProbity clashscore. Three additional structures, for which only low-resolution data are available, were also re-refined with this methodology.

Results

The enhanced refinement protocol is most beneficial for reflection data at resolutions of 3.0 Å or worse. At the low-resolution limit, ≥4.0 Å, the new protocol generated models with Cα positions that have RMSDs that are 0.18 Å more similar to the reference high-resolution structure, Ramachandran scores improved by 13%, and clashscores improved by 51%, all in comparison to models generated with the standard refinement protocol. The hydropathic forcefield terms are at least as effective as Coulombic electrostatic terms in maintaining polar interaction networks, and significantly more effective in maintaining hydrophobic networks, as synthetic resolution is decremented. Even at resolutions ≥4.0 Å, these latter networks are generally native-like, as measured with a hydropathic interactions scoring tool.     

Crystal structure of a substrate complex of Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III (FabH) with lauroyl-coenzyme A

Beta-ketoacyl-acyl carrier protein synthase III (FabH) catalyzes a two step reaction that initiates the pathway of fatty acid biosynthesis in plants and bacteria. In Mycobacterium tuberculosis, FabH catalyzes extension of lauroyl, myristoyl and palmitoyl groups from which cell wall mycolic acids of the bacterium are formed. The first step of the reaction is an acyl group transfer from acyl-coenzyme A to the active-site cysteine of the enzyme; the second step is acyl chain extension by two carbon atoms through Claisen condensation with malonyl-acyl carrier protein. We have previously determined the crystal structure of a type II, dissociated M.tuberculosis FabH, which catalyzes extension of lauroyl, myristoyl and palmitoyl groups. Here we describe the first long-chain Michaelis substrate complex of a FabH, that of lauroyl-coenzyme A with a catalytically disabled Cys-->Ala mutant of M.tuberculosis FabH. An elongated channel extending from the mutated active-site cysteine defines the acyl group binding locus that confers unique acyl substrate specificity on M.tuberculosis FabH. CoA lies in a second channel, bound primarily through interactions of its nucleotide group at the enzyme surface. The apparent weak association of CoA in this complex may play a role in the binding and dissociation of long chain acyl-CoA substrates and products and poses questions pertinent to the mechanism of this enzyme.     

O-acetylated gangliosides in bovine buttermilk. Characterization of 7-O-acetyl, 9-O-acetyl, and 7,9-di-O-acetyl GD3.

Three O-acetylated gangliosides, G1, G2, and G3, were purified from bovine buttermilk by using chloroform/methanol extraction, Folch partitioning, chromatography on DEAE-Sephadex A-25, and Iatrobeads columns. The final yields of gangliosides G1, G2, and G3 were 2 mg, 37 mg, and 40 mg per 1.7 kg of the buttermilk powder, respectively. On the basis of immunostaining on high performance thin layer chromatography with specific monoclonal antibodies, mild alkaline treatment, gas-liquid chromatographic analysis, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance studies, G1 and G2 are characterized as O-acetylated GD3 and G3 as O-acetylated GT3, and the structures of these gangliosides are as follows: [formula: see text] The major fatty acids of these gangliosides were C18:0, C22:0, C23:0, and C24:0, and the long chain base was C18-sphingosine.     

Identification and hydropathic characterization of structural features affecting sequence specificity for doxorubicin intercalation into DNA double-stranded polynucleotides.

The computer molecular modeling program HINT (Hydropathic INTeractions), an empirical hydropathic force field function that includes hydrogen bonding, coulombic and hydrophobic terms, was used to study sequence-selective doxorubicin binding/intercalation in the 64 unique CAxy, CGxy, TAxy, TGxy base pair quartet combinations. The CAAT quartet sequence is shown to have the highest binding score of the 64 combinations. Of the two regularly alternating polynucleotides, d(CGCGCG)2and d(TATATA)2, the HINT calculated binding scores reveal doxorubicin binds preferentially to d(TATATA)2. Although interactions of the chromophore with the DNA base pairs defining the intercalation site [I-1] [I+1] and the neighboring [I+2] base pair are predominant, the results obtained with HINT indicate that the base pair [I+3] contributes significantly to the sequence selectivity of doxorubicin by providing an additional hydrogen bonding opportunity for the N3' ammonium of the daunosamine sugar moiety in approximately 25% of the sequences. This observation, that interactions involving a base pair [I+3] distal to the intercalation site play a significant role in stabilizing/destabilizing the intercalation of doxorubicin into the various DNA sequences, has not been previously reported. In general terms, this work shows that molecular modeling and careful analysis of molecular interactions can have a significant role in designing and evaluating nucleotides and antineoplastic agents.