15N backbone dynamics of the S-peptide from ribonuclease A in its free and S-protein bound forms: toward a site-specific analysis of entropy changes upon folding.

Backbone 15N relaxation parameters (R1, R2, 1H-15N NOE) have been measured for a 22-residue recombinant variant of the S-peptide in its free and S-protein bound forms. NMR relaxation data were analyzed using the "model-free" approach (Lipari & Szabo, 1982). Order parameters obtained from "model-free" simulations were used to calculate 1H-15N bond vector entropies using a recently described method (Yang & Kay, 1996), in which the form of the probability density function for bond vector fluctuations is derived from a diffusion-in-a-cone motional model. The average change in 1H-15N bond vector entropies for residues T3-S15, which become ordered upon binding of the S-peptide to the S-protein, is -12.6+/-1.4 J/mol.residue.K. 15N relaxation data suggest a gradient of decreasing entropy values moving from the termini toward the center of the free peptide. The difference between the entropies of the terminal and central residues is about -12 J/mol residue K, a value comparable to that of the average entropy change per residue upon complex formation. Similar entropy gradients are evident in NMR relaxation studies of other denatured proteins. Taken together, these observations suggest denatured proteins may contain entropic contributions from non-local interactions. Consequently, calculations that model the entropy of a residue in a denatured protein as that of a residue in a di- or tri-peptide, might over-estimate the magnitude of entropy changes upon folding.     

Understanding the structural and energetic basis of inhibitor and substrate bound to the full-length NS3/4A: insights from molecular dynamics simulation, binding free energy calculation and network analysis

Hepatitis C virus (HCV) bifunctional NS3/4A is an attractive anti-HCV drug target, as both the protease and helicase functions are required for viral infection and replication. Although the first generation of NS3/4A protease inhibitors (PIs) has focused almost exclusively on the interaction with the protease domain alone, recent studies have shown that PIs also inhibit the full-length NS3/4A protein. However, the detailed molecular mechanism of the interaction between protease inhibitors, as well as the peptide substance with the full-length NS3/4A protein, remains poorly understood. Herein, starting from the recently determined crystal structure of an inhibitor (inhibitor ) bound to the full-length NS3/4A protein, the structures of the full-length NS3/4A complexed with inhibitor ITMN-191 (by InterMune/Roche; Phase II) and substrate 4B5A (the viral cleavage product peptide) were built. Then, residue interaction network (RIN) analysis, molecular dynamics (MD) simulation, binding free energy calculation, decomposition of free energies on per-residue and dynamic substrate recognition pattern analysis were employed to uncover the structural and energetic basis of inhibitor and substrate binding mode in the binding cleft located at the interface of the protease and helicase domains of the full-length NS3/4A. The results from our study reveal that both the protease and helicase residues of the NS3/4A participate in the interactions with the inhibitor , ITMN-191 and 4B5A. Additional analysis of the NS3/4A substrate and inhibitor envelopes reveals the areas where the consensus inhibitor volume extended beyond the substrate envelope. These areas correspond to drug resistance mutations including Arg155, Ala156 and Asp168 at the protease active site as well as the two conserved helicase residues Gln526 and His528 that strongly interact with the inhibitors. Thus, the findings of this study will be very useful for understanding the interaction mechanism between the inhibitor (substrate) and NS3/4A and also for the rational design and development of new potent molecules targeting the full-length NS3/4A.     

An improved method for the structural profiling of keratan sulfates: analysis of keratan sulfates from brain and ovarian tumors

A previously developed method for the structural fingerprinting of keratan sulfates (Brown et al., Glycobiology, 5, 311-317, 1995) has been adapted for use with oligosaccharides fluorescently labeled with 2-aminobenzoic acid following keratanase II digestion. The oligosaccharides are separated by high-pH anion-exchange chromatography on a Dionex AS4A-SC column. This methodology permits quantitative analysis of labeled oligosaccharides which can be detected at the sub-nanogram ( approximately 100 fmol) level. Satisfactory calibration of this method can be achieved using commercial keratan sulfate standards. Keratan sulfates from porcine brain phosphocan and human ovarian tumors have been examined using this methodology, and their structural features are discussed.     

X-ray structure analyses of photosynthetic reaction center variants from Rhodobacter sphaeroides: structural changes induced by point mutations at position L209 modulate electron and proton transfer

The structures of the reaction center variants Pro L209 --> Tyr, Pro L209 --> Phe, and Pro L209 --> Glu from the photosynthetic purple bacterium Rhodobacter sphaeroides have been determined by X-ray crystallography to 2.6-2.8 A resolution. These variants were constructed to interrupt a chain of tightly bound water molecules that was assumed to facilitate proton transfer from the cytoplasm to the secondary quinone Q(B) [Baciou, L., and Michel, H. (1995) Biochemistry 34, 7967-7972]. However, the amino acid exchanges Pro L209 --> Tyr and Pro L209 --> Phe do not interrupt the water chain. Both aromatic side chains are oriented away from this water chain and interact with three surrounding polar side chains (Asp L213, Thr L226, and Glu H173) which are displaced by up to 2.6 A. The conformational changes induced by the bulky aromatic rings of Tyr L209 and Phe L209 lead to unexpected displacements of Q(B) compared to the wild-type protein. In the structure of the Pro L209 --> Tyr variant, Q(B) is shifted by approximately 4 A and is now located at a position similar to that reported for the wild-type reaction center after illumination [Stowell, M. H. B., et al. (1997) Science 276, 812-816]. In the Pro L209 --> Phe variant, the electron density map reveals an intermediate Q(B) position between the binding sites of the wild-type protein in the dark and the Pro L209 --> Tyr protein. In the Pro L209 --> Glu reaction center, the carboxylic side chain of Glu L209 is located within the water chain, and the binding site of Q(B) remains unchanged compared to the wild-type structure.     

Influence of the pathogenic mutations T188K/R/A on the structural stability and misfolding of human prion protein: Insight from molecular dynamics simulations

Background

Prion diseases are associated with a conformational switch for PrP from PrP^C to PrP^Sc. Many genetic mutations are linked with prion diseases, such as mutations T188K/R/A with fCJD.

Scope of review

MD simulations for the WT PrP and its mutants were performed to explore the underlying dynamic effects of T188 mutations on human PrP. Although the globular domains are fairly conserved, the three mutations have diverse effects on the dynamics properties of PrP, including the shift of H1, the elongation of native β-sheet and the conversion of S2-H2 loop to a 3₁₀ helix.

Major conclusions

Our present study indicates that the three mutants for PrP may undergo different pathogenic mechanisms and the realistic atomistic simulations can provide insights into the effects of disease-associated mutations on PrP dynamics and stability, which can enhance our understanding of how mutations induce the conversion from PrP^C to PrP^Sc.General significanceOur present study helps to understand the effects of T188K/R/A mutations on human PrP: despite the three pathogenic mutations almost do not alter the native structure of PrP, but perturb its stability. This instability may further modulate the oligomerization pathways and determine the features of the PrP^Sc assemblies.     

Concerted bifunctionality of the dCTP deaminase-dUTPase from Methanocaldococcus jannaschii: A structural and pre-steady state kinetic analysis

Two mutant dCTP deaminase-dUTPases from Methanocaldococcus jannaschii were crystallised and the crystal structures were solved: E145A in complex with the substrate analogue α,β-imido-dUTP and E145Q in complex with diphosphate. Both mutant enzymes were defect in the deaminase reaction and had reduced dUTPase activity. In the structure of E145Q in complex with diphosphate, the diphosphate occupied the same position as the β- and γ-phosphoryls of the nucleotide analogue in the E145A complex. The C-terminal region that is unresolved in the apo-form of the enzyme was ordered in both complexes and closed over the active site by interacting with the phosphate backbone of the nucleotide or with the diphosphate. A magnesium ion was readily observed to complex with all three phosphoryls in the nucleotide complex or with the diphosphate. A water molecule that is likely to be involved in the nucleotidyl diphosphorylase reaction was observed in the E145A:α,β-imido-dUTP complex and positioned similarly as in the monofunctional trimeric dUTPase. A comparison of the active sites of the bifunctional enzyme and the monofunctional family members, dCTP deaminase and dUTPase, suggests similar reaction mechanisms. The similar side chain conformations in the deaminase site between the nucleotide and diphosphate complexes indicated a concerted re-arrangement, or induced fit, of the whole active site promoted by enzyme and nucleotide phosphoryl interactions. A pre-steady state kinetic analysis of the bifunctional reaction and the dUTPase half-reaction supported a conformational change upon substrate binding in both reactions and a concerted catalytic step for the bifunctional reaction.     

Dual anti-inflammatory and selective inhibition mechanism of leukotriene A4 hydrolase/aminopeptidase: insights from comparative molecular dynamics and binding free energy analyses

Human leukotriene A₄ hydrolase/aminopeptidase (LTA₄H) is a zinc metalloenzyme with a dual catalytic activity; conversion of LTA₄ into LTB₄ and degradation of chemotactic tripeptide Pro-Gly-Pro (PGP). Existing inhibitors, such as SC-57461A, block both catalytic activities of the enzyme, leading to drug failures. Recently, a novel compound, ARM1, was reported to selectively inhibit the hydrolase activity of LTA₄H while sparing its aminopeptidase activity. However, the molecular understanding of such preferential inhibitory mechanism remains obscure. The discovery of ARM1 prompted us to further explore its binding theme and provide more insight into the structural and dual mechanistic features of LTA₄H protein. To accomplish this, we embarked on wide range of computational tools, including comparative molecular dynamics (MDs) simulations and postdynamic analyses for LTA₄H and in complex with ARM1, PGP, ARM1-PGP, and SC-57461A. MD analysis reveals that the binding of ARM1 exhibits a more stable active site and overall stable protein conformation when compared to the nonselective inhibitor SC-57461A. In addition, MM/GBSA-binding free energy calculation also reveals that ARM1 exhibit a lower binding affinity, when compared to the nonselective inhibitor SC-57461A – which is in a great agreement with experimental data. Per residue energy decomposition analysis showed that Phe314, Val367, Tyr378, Trp311, Pro382, and Leu369 are key residues critical for the selective inhibition of the epoxide hydrolase activity of LTA₄H by ARM1. Findings from this report will not only provide more understanding into the structural, dynamic, and mechanistic features of LTA₄H but would also assist toward the rational design of novel and selective hydrolase inhibitors of LTA₄H as anti-inflammatory drugs.     

MuSICA, a CO2, water and energy multilayer, multileaf pine forest model: evaluation from hourly to yearly time scales and sensitivity analysis

The current emphasis on global climate studies has led the scientific community to set up a number of sites for measuring long‐term biospheric fluxes, and to develop a wide range of biosphere–atmosphere exchange models. This paper presents a new model of this type, which has been developed for a pine forest canopy. In most coniferous species the canopy layer is well separated from the understorey and several cohorts of needles coexist. It was therefore found necessary to distinguish several vegetation layers and, in each layer, several leaf classes defined not only by their light regime and wetness status but also by their age. This model, named MuSICA, is a multilayer, multileaf process‐based model. Each submodel is first independently parameterized using data collected at a EUROFLUX site near Bordeaux (Southwestern France). Particular care is brought to identify the seasonal variations in the various physiological parameters. The full model is then evaluated using a two‐year long data set, split up into 12 day‐type classes defined by the season, the weather type and the soil water status. Beyond the good overall agreement obtained between measured and modelled values at various time scales, several points of further improvement are identified. They concern the seasonal variations in the stomatal response of needles and the soil/litter respiration, as well as their interaction with soil or litter moisture. A sensitivity analysis to some of the model features (in‐canopy turbulent transfer scheme, leaf age classes, water retention, distinction between shaded and sunlit leaves, number of layers) is finally performed in order to evaluate whether significant simplifications can be brought to such a model with little loss in its predictive quality. The distinction between several leaf classes is crucial if one is to compute biospheric fluxes accurately. It is also evidenced that accounting for in‐canopy turbulent transfer leads to better estimates of the sensible heat flux.     

Ultrastructural analysis of progressive endometrial hypercytolipidemia induced by obese (ob/ob) and diabetes (db/db) genotype mutations: structural basis of female reproductive tract involution

The diabetes (db/db) and obese (ob/ob) genotype mutations induce a progressive, hypercytolipidemic condition within the endometrium of the female reproductive tract that promotes sterility and premature organ involution in C57BL/KsJ mice. The current studies focus on the ultrastructural changes that occur within the epithelial and stromal layers of the uterine endometrium during the progressive expression of these mutations, which induce a hyperglycemic-hyperinsulinemic metabolic state and promote tissue cytolipidemia and organoinvolution. Control (normal: +/-), diabetes (db/db) and obese (ob/ob) genotype groups were prepared for high resolution light (LM) and transmission (TEM) microscopic analysis of endometrial tissue samples collected from 4 (young)- to 20 (aged)-week-old mice, allowing for the progressive influences of the mutational aberrations on uterine structure to be evaluated. Compared to controls, both (ob/ob) and (db/db) mutations induced a dramatic increase in endometrial epithelial cytolipid vacuole accumulation, which increased in density between 4 and 20 weeks of age. Lipid vacuoles aggregated at the baso-polar regions of epithelial cells in response to the hyperglycemic-hyperlipidemic conditions typical of both (ob/ob) and (db/db) groups. Progressive cytoplasmic movement of the lipid pools induced a perinuclear isolation from surrounding cytoplasmic organelles. Apical lipid accumulations forced cytoplasmic organelles into peripheral cell compartments and altered the periepithelial stromal cell profile relative to controls. These studies define the progressive, intracellular accumulation of hypercytolipidemic pools which induce a transformation of normal endometrial cell types into adipocyte-like entities. The lipidemia-induced alterations in cell structure disrupt normal tissue continuity and function, culminating in organoinvolution and overt female reproductive sterility.     

Extension of the in-gel release method for structural analysis of neutral and sialylated N-linked glycans to the analysis of sulfated glycans: application to the glycans from bovine thyroid-stimulating hormone

This paper reports an extension of the in-gel technique for releasing N-linked glycans from glycoproteins for analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry reported by B. Küster, S. F. Wheeler, A. P. Hunter, R. A. Dwek, and D. J. Harvey (1997, Anal. Biochem. 250, 82-101) to allow it to be used for sulfated glycans. The method was used to identify N-linked glycans from bovine thyroid-stimulating hormone. Following glycan release, either in gel or in solution, the glycans were fractionated directly with a porous graphatized carbon column. The sulfated glycans were examined by MALDI mass spectrometry in negative ion mode with d-arabinosazone as the matrix and both neutral and acidic glycans were examined in positive ion mode from 2,5-dihydroxybenzoic acid. Negative ion post-source decay spectra were also obtained. Twenty-two neutral and fifteen sulfated N-linked glycans were identified and it was shown that negligible loss of sulfate groups occurred even though these groups are often readily lost during MALDI analysis. The glycans were mainly sulfated hybrid and biantennary complex structures. Negative ion post-source decay and positive ion collision-induced fragmentation spectra were obtained.     

The system of sulfated galactans from the red seaweed Gymnogongrus torulosus (Phyllophoraceae, Rhodophyta): Location and structural analysis

Sulfated polysaccharides were localized in the cuticle, cortex and medulla of the gametophyte thallus, being more concentrated in the intercellular matrix than in the cell walls. During the water extraction sequence, a small percentage of galactan sulfates (5.1% of dry seaweed) with average low M_r (6–11.4 kDa) were extracted at room temperature without disturbing the cellular arrangement, while sulfated galactans of average medium M_r (18–45 kDa) were obtained by further hot-water extractions (52.4% of dry seaweed), with diorganization of the tissue. The residue (40.0% of dry seaweed) still contained carrageenan-type (major) and agaran-type (minor) galactans. Part of these galactans was extracted with 8.4% LiCl solution in DMSO, from which “pure” κ/ι-carrageenans were isolated.Carrageenans and agarans were extracted in a ratio 1:0.5, showing the highest amount of agaran-structures for a carrageenophyte. The galactans comprise alternating 4-sulfated (major) and non-sulfated (minor) 3-linked β-d-galactopyranose units, and 4-linked α-galactopyranose units with the following substitutions: (i) non-sulfated and 2-sulfated 3,6-anhydro-α-d-galactopyranose residues in the carrageenan-structures, which belong to the κ-family (κ/ι-carrageenans); (ii) 3-sulfated α-l-galactopyranose units and 2-sulfated 3,6-anhydro-α-l-galactopyranose residues in the agaran-structures.Alkaline treatment and alkaline dialysis of the main extracts gave “pure” κ/ι-carrageenans, showing that carrageenan molecules are extracted together with low M_r agarans or agaran-dl-hybrids.     

Functionally important sites in the elongation factor EF-Tu from Thermus aquaticus: analysis of fine structural changes upon binding of guanosine-3'-triphosphate and guanosine-3'-diphosphate]

High-resolution data were used to analyze conformational changes of the main chain in two functional states of the ribosome elongation factor EF-Tu from Thermus aquaticus: the inactive state with guanosine-3'-diphosphate and the active state with guanosine-3'-triphosphate. Earlier only major changes in the effector loop of the domain I were determined. In this paper, all rearrangements in the main chain were observed upon shifting of C alpha-atoms from 1 to 8 A for each of the three protein domains. It was shown that these changes occur in numerous regions. New regions of changes were found, and they were located mostly in the loops of protein domains. Some of them are in the regions of interdomain interactions, others correlate with the known functionally important regions of EF-Tu binding with EF-Ts, aminoacyl-tRNA and the ribosome. Most changes induced by the conformational signal transfer from the guanosine-3'-triphosphate binding site occur just in the regions that are important for further stages of the factor functioning. The signal is transferred from domain I to domains II and III via interdomain contacts, predetermining fine fitting of functionally important regions to be involved in the following stages of the elongation cycle. The greatest part of the detected changes occurs in conservative residues of the whole family of bacterial factors, and only some of them are specific. This approach may prove useful for predetermining potential functionally important sites in other proteins.     

An electron spin resonance study on the free radicals produced from aclacinomycin a and its derivatives: analysis of hyperfine structure of the spectra by means of molecular orbital method

Quinone-containing carcinostatics, aclacinomycin A and its derivatives were investigated on the convertibility to free radical under a mild reducing condition. The hyperfine structures of electron spin resonance (ESR) spectra were satisfactorily reproduced by computer simulations, using the hyperfine coupling constants calculated by the Intermediate Neglect of Differential Overlap Molecular Orbital (INDO MO) method. This verifies the reliability of molecular orbital calculations and opens a way to analyze theoretically the correlation between chemical structures and carcinostatic activities. By analyzing hyperfine structures of ESR spectra, the free radical produced from aclacinomycin was identified as a neutral form of semiquinone radical of intact aclacinomycin. Taking into account the previous finding that 7-deoxyaklavinone (C1) is formed reductively by cytochrome P-450 reductase (EC 1.6.2.4; Komiyama et al., 1979), it is postulated that two types of semi-quinone radicals exist in vivo.     

Relationships of Cysteine and Lysine residues with the substrate binding site of the mitochondrial ornithine/citrulline carrier: an inhibition kinetic approach combined with the analysis of the homology structural model

To gain insights in the relationships of specific amino acid residues with the active site of the mitochondrial ornithine/citrulline carrier, we studied the effect of specific protein modifying reagents on the transport catalysed by the carrier reconstituted into liposomes. It was found that, besides the sulfhydryl reagents NEM, MTSEA, p-hydroxymercuribenzoate, diamide also the lysine reagents PLP, DIDS, SITS, the carboxyl reagents WRK, EDC and the arginine reagent methylglyoxal inhibited the carrier. NEM, MTSEA and PLP inhibited the ornithine/citrulline carrier with a completely competitive type of mechanism. A 1:1 interaction of NEM with the carrier molecule has been demonstrated. The results are in agreement with the localization of one sulfhydryl and at least one amino group in the substrate binding site. On the basis of the interferences between SH reagents and PLP in the transport inhibition, it has been deduced that the distance between the SH and the NH(2) residues of the active site should be comparable to the distance between the gamma-NH(2) and COOH residues of the ornithine molecule. The structural model of the ornithine/citrulline carrier has been obtained by homology modelling using as template the ADP/ATP carrier structure. The combined analysis of the experimental data and the structural model allows to deduce that Cys-132 is located in the substrate binding site, flanked by at least one Lys residue.