Protein adaptation to high hydrostatic pressure: Computational analysis of the structural proteome

Hydrostatic pressure has a vital role in the biological adaptation of the piezophiles, organisms that live under high hydrostatic pressure. However, the mechanisms by which piezophiles are able to adapt their proteins to high hydrostatic pressure is not well understood. One proposed hypothesis is that the volume changes of unfolding (ΔV_Tot) for proteins from piezophiles is distinct from those of nonpiezophilic organisms. Since ΔV_Tot defines pressure dependence of stability, we performed a comprehensive computational analysis of this property for proteins from piezophilic and nonpiezophilic organisms. In addition, we experimentally measured the ΔV_Tot of acylphosphatases and thioredoxins belonging to piezophilic and nonpiezophilic organisms. Based on this analysis we concluded that there is no difference in ΔV_Tot for proteins from piezophilic and nonpiezophilic organisms. Finally, we put forward the hypothesis that increased concentrations of osmolytes can provide a systemic increase in pressure stability of proteins from piezophilic organisms and provide experimental thermodynamic evidence in support of this hypothesis.     

Interactions of Meso-tetra-(4-N-oxyethylpyridyl) Porphyrin,Its 3-N Analog and Their Metallocomplexes with Duplex DNA

Abstract

Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA have been investigated by uv/visible absorbance and circular dichrosim spectroscopies. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by intercalation) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni. Results indicate that (4N-oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins.     

Melting of Cross-Linked DNA IV. Methods for Computer Modeling of Total Influence on DNA Melting of Monofunctional Adducts,Intrastrand and Interstrand Cross-Links Formed by Molecules of an Antitumor Drug

Abstract

A theoretical method is developed for calculation of melting curves of covalent complexes of DNA with antitumor drugs. The method takes into account all the types of chemical modifications of the double helix caused by platinum compounds and DNA alkylating agents: 1) monofunctional adducts bound to one nucleotide; 2) intrastrand cross-links which appear due to bidentate binding of a drug molecule to two nucleotides that are included into the same DNA strand; 3) interstrand cross-links caused by bidentate binding of a molecule to two nucleotides of different strands. The developed calculation method takes into account the following double helix alterations at sites of chemical modifications: 1) a change in stability of chemically modified base pairs and neighboring ones, that is caused by all the types of chemical modifications; 2) a change in the energy of boundaries between helical and melted regions at sites of chemical modification (local alteration of the factor of cooperativity of DNA melting), that is caused by all the types of chemical modifications, too; 3) a change in the loop entropy factor of melted regions that include interstrand cross-links; 4) the prohibition of divergence of DNA strands in completely melted DNA molecules, which is caused by interstrand cross-links only. General equations are derived, and three calculation methods are proposed to calculate DNA melting curves and the parameters that characterize the helix-coil transition.     

Melting of Cross-Linked DNA V. Cross-Linking Effect Caused by Local Stabilization of the Double Helix

Abstract

DNA interstrand cross-links are usually formed due to bidentate covalent or coordination binding of a cross-linking agent to nucleotides of different strands. However interstrand linkages can be also caused by any type of chemical modification that gives rise to a strong local stabilization of the double helix. These stabilized sites conserve their helical structure and prevent local and total strand separation at temperatures above the melting of ordinary AT and GC base pairs. This local stabilization makes DNA melting fully reversible and independent of strand concentration like ordinary covalent interstrand cross-links. The stabilization can be caused by all the types of chemical modifications (interstrand cross-links, intrastrand cross-links or monofunctional adducts) if they give rise to a strong enough local stabilization of the double helix. Our calculation demonstrates that an increase in stability by 25 to 30 kcal in the free energy of a single base pair of the double helix is sufficient for this “cross-linking effect” (i.e. conserving the helicity of this base pair and preventing strand separation after melting of ordinary base pairs). For the situation where there is more then one stabilized site in a DNA duplex (e.g., 1 stabilized site per 1000 bp), a lower stabilization per site is sufficient for the “cross-linking effect” (18–20 kcal). A substantial increase in DNA stability was found in various experimental studies for some metal-based anti-tumor compounds. These compounds may give rise to the effect described above. If ligand induced stabilization is distributed among several neighboring base pairs, a much lower minimum increase per stabilized base pair is sufficient to produce the cross-linking effect (1 bp- 24.4 kcal; 5 bp- 5.3 kcal; 10 bp- 2.9 kcal, 25 bp- 1.4 kcal; 50 bp- 1.0 kcal). The relatively weak non-covalent binding of histones or protamines that cover long regions of DNA (20–40 bp) can also cause this effect if the salt concentration of the solution is sufficiently low to cause strong local stabilization of the double helix. Stretches of GC pairs more than 25 bp in length inserted into poly(AT) DNA also exhibit properties of stabilizing interstrand cross-links.     

The Upper Palaeolithic site of Kalavan 1 (Armenia): An Epigravettian settlement in the Lesser Caucasus

The open-air site of Kalavan 1 is located in the Aregunyats mountain chain (at 1640 m above sea level) on the northern bank of Lake Sevan. It is the first Upper Palaeolithic site excavated in Armenia. Led by an Armenian-French team, several excavations (2005–2009) have revealed a well preserved palaeosoil, dated to around 14,000 BP (years before present), containing fauna, lithic artefacts, as well as several hearths and activity areas that structure the settlement. The initial studies enable placement of the site in its environment and justify palaeoethnological analysis of the Epigravettian human groups of the Lesser Caucasus.     

Adsorption of ligands on macromolecules in the fluctuating medium

In the present work fluctuations of number of ligands adsorbed on macromolecule are investigated. We have taken into account the adsorption and desorption of ligands under the circumstance of some adsorption centers fluctuations affected by medium fluctuation. The correlation function and spectral density of number of ligands adsorbed on macromolecule are calculated. The properties of these fluctuations which allow identifying a noisemaker are determined. It has been shown, thatfas andsluggis adsorption can be distinguished by properties of dispersion and spectral density. It has been also shown, that comparison of experimental and theoretical correlation functions (or spectral densities) allows to calculate constants of ligand - adsorption center binding and unbinding.     

A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats

Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure of SSO1404 was solved at 1.6A resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.     

Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli

Gluconeogenesis is an important metabolic pathway, which produces glucose from noncarbohydrate precursors such as organic acids, fatty acids, amino acids, or glycerol. Fructose-1,6-bisphosphatase, a key enzyme of gluconeogenesis, is found in all organisms, and five different classes of these enzymes have been identified. Here we demonstrate that Escherichia coli has two class II fructose-1,6-bisphosphatases, GlpX and YggF, which show different catalytic properties. We present the first crystal structure of a class II fructose-1,6-bisphosphatase (GlpX) determined in a free state and in the complex with a substrate (fructose 1,6-bisphosphate) or inhibitor (phosphate). The crystal structure of the ligand-free GlpX revealed a compact, globular shape with two α/β-sandwich domains. The core fold of GlpX is structurally similar to that of Li⁺-sensitive phosphatases implying that they have a common evolutionary origin and catalytic mechanism. The structure of the GlpX complex with fructose 1,6-bisphosphate revealed that the active site is located between two domains and accommodates several conserved residues coordinating two metal ions and the substrate. The third metal ion is bound to phosphate 6 of the substrate. Inorganic phosphate strongly inhibited activity of both GlpX and YggF, and the crystal structure of the GlpX complex with phosphate demonstrated that the inhibitor molecule binds to the active site. Alanine replacement mutagenesis of GlpX identified 12 conserved residues important for activity and suggested that Thr⁹⁰ is the primary catalytic residue. Our data provide insight into the molecular mechanisms of the substrate specificity and catalysis of GlpX and other class II fructose-1,6-bisphosphatases.Fructose-1,6-bisphosphatase (FBPase,² EC 3.1.3.11), a key enzyme of gluconeogenesis, catalyzes the hydrolysis of fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. It is the reverse of the reaction catalyzed by phosphofructokinase in glycolysis, and the product, fructose 6-phosphate, is an important precursor in various biosynthetic pathways (1). In all organisms, gluconeogenesis is an important metabolic pathway that allows the cells to synthesize glucose from noncarbohydrate precursors, such as organic acids, amino acids, and glycerol. FBPases are members of the large superfamily of lithium-sensitive phosphatases, which includes three families of inositol phosphatases and FBPases (the phosphoesterase clan CL0171, 3167 sequences, Pfam data base). These enzymes show metal-dependent and lithium-sensitive phosphomonoesterase activity and include inositol polyphosphate 1-phosphatases, inositol monophosphatases (IMPases), 3′-phosphoadenosine 5′-phosphatases (PAPases), and enzymes acting on both inositol 1,4-bisphosphate and PAP (PIPases) (2). They possess a common structural core with the active site lying between α+β and α/β domains (3). Li⁺-sensitive phosphatases are putative targets for lithium therapy in the treatment of manic depressive patients (4), whereas FBPases are targets for the development of drugs for the treatment of noninsulin-dependent diabetes (5, 6). In addition, FBPase is required for virulence in Mycobacterium tuberculosis and Leishmania major and plays an important role in the production of lysine and glutamate by Corynebacterium glutamicum (7, 8).Presently, five different classes of FBPases have been proposed based on their amino acid sequences (FBPases I to V) (9–11). Eukaryotes contain only the FBPase I-type enzyme, but all five types exist in various prokaryotes. Types I, II, and III are primarily in bacteria, type IV in archaea (a bifunctional FBPase/inositol monophosphatase), and type V in thermophilic prokaryotes from both domains (11). Many organisms have more than one FBPase, mostly the combination of types I + II or II + III, but no bacterial genome has a combination of types I and III FBPases (9). The type I FBPase is the most widely distributed among living organisms and is the primary FBPase in Escherichia coli, most bacteria, a few archaea, and all eukaryotes (9, 11–15). The type II FBPases are represented by the E. coli GlpX and FBPase F-I from Synechocystis PCC6803 (9, 16); type III is represented by the Bacillus subtilis FBPase (17); type IV is represented by the dual activity FBPases/inosine monophosphatases FbpA from Pyrococcus furiosus (18), MJ0109 from Methanococcus jannaschii (19), and AF2372 from Archaeoglobus fulgidus (20); and type V is represented by the FBPases TK2164 from Pyrococcus (Thermococcus) kodakaraensis and ST0318 from Sulfolobus tokodai (10, 21).Three-dimensional structures of the type I (from pig kidney, spinach chloroplasts, and E. coli), type IV (MJ0109 and AF2372), and type V (ST0318) FBPases have been solved (10, 11, 19, 20, 22, 23). FBPases I and IV and inositol monophosphatases share a common sugar phosphatase fold organized in five layered interleaved α-helices and β-sheets (α-β-α-β-α) (2, 19, 24). ST0318 (an FBPase V enzyme) is composed of one domain with a completely different four-layer α-β-β-α fold (10). The FBPases from these three classes (I, IV, and V) require divalent cations for activity (Mg²⁺, Mn²⁺, or Zn²⁺), and their structures have revealed the presence of three or four metal ions in the active site.E. coli has five Li⁺-sensitive phosphatases as follows: CysQ (a PAPase), SuhB (an IMPase), Fbp (a FBPase I enzyme), GlpX (a FBPase II), and YggF (an uncharacterized protein) (see the Pfam data base). CysQ is a 3′-phosphoadenosine 5′-phosphatase involved in the cysteine biosynthesis pathway (25, 26), whereas SuhB is an inositol monophosphatase (IMPase) that is also known as a suppressor of temperature-sensitive growth phenotypes in E. coli (27, 28). Fbp is required for growth on gluconeogenic substrates and probably represents the main gluconeogenic FBPase (12). This enzyme has been characterized both biochemically and structurally and shown to be inhibited by low concentrations of AMP (IC₅₀ 15 μm) (11, 29, 30). The E. coli GlpX, a class II enzyme FBPase, has been shown to possess a Mn²⁺-dependent FBPase activity (9). The increased expression of glpX from a multicopy plasmid complemented the Fbp^- phenotype; however, the glpX knock-out strain grew normally on gluconeogenic substrates (succinate or glycerol) (9).In this study, we present the first structure of a class II FBPase, the E. coli GlpX, in a free state and in the complex with FBP + metals or phosphate. We have demonstrated that the fold of GlpX is similar to that of the lithium-sensitive phosphatases. We have identified the GlpX residues important for activity and proposed a catalytic mechanism. We have also showed that YggF is a third FBPase in E. coli, which has distinct catalytic properties and is more sensitive than GlpX to the inhibition by lithium or phosphate.     

Heightened endotoxin susceptibility of monocytes and neutrophils during familial Mediterranean fever

Familial Mediterranean fever (FMF) is a relapsing autoinflammatory disorder, caused by various mutations in the MEFV gene, which encodes a protein called pyrin, expressed in neutrophils and activated monocytes. Induction of monocyte endotoxin tolerance is observed in FMF patients during attack, whereas monocytes from patients in the attack-free period failed to induce lipopolysaccharide tolerance and exhibited heightened sensitivity to bacterial endotoxin. In this study, we demonstrated that impaired lipopolysaccharide tolerance induction in attack-free FMF patients correlates with both increased lipopolysaccharide-induced proinflammatory cytokine synthesis polarization and a different time-course pattern of lipopolysaccharide-induced changes on monocytic surface expression of CD14 and CD11b coreceptors. We found that this pattern is characterized either by delayed turnover of CD14 or increased surface retention of CD11b receptors on monocytes during stimulation with lipopolysaccharide. In addition, enhancement of lipopolysaccharide-induced apoptosis of neutrophils was observed in FMF patients, and was confirmed based on the fact that neutrophils from FMF patients previously unexposed to Salmonella enteritidis exhibited heightened susceptibility to the lipopolysaccharide of this pathogen similar to that of patients infected with this species.