Gln Residues Research Articles

Physcomitrella PpORS, Basal to Plant Type III Polyketide Synthases in Phylogenetic Trees, Is a Very Long Chain 2′-Oxoalkylresorcinol Synthase

The plant type III polyketide synthases (PKSs), which produce diverse secondary metabolites with different biological activities, have successfully co-evolved with land plants. To gain insight into the roles that ancestral type III PKSs played during the early evolution of land plants, we cloned and characterized PpORS from the moss Physcomitrella. PpORS has been proposed to closely resemble the most recent common ancestor of the plant type III PKSs. PpORS condenses a very long chain fatty acyl-CoA with four molecules of malonyl-CoA and catalyzes decarboxylative aldol cyclization to yield the pentaketide 2'-oxoalkylresorcinol. Therefore, PpORS is a 2'-oxoalkylresorcinol synthase. Structure modeling and sequence alignments identified a unique set of amino acid residues (Gln(218), Val(277), and Ala(286)) at the putative PpORS active site. Substitution of the Ala(286) to Phe apparently constricted the active site cavity, and the A286F mutant instead produced triketide alkylpyrones from fatty acyl-CoA substrates with shorter chain lengths. Phylogenetic analysis and comparison of the active sites of PpORS and alkylresorcinol synthases from sorghum and rice suggested that the gramineous enzymes evolved independently from PpORS to have similar functions but with distinct active site architecture. Microarray analysis revealed that PpORS is exclusively expressed in nonprotonemal moss cells. The in planta function of PpORS, therefore, is probably related to a nonprotonemal structure, such as the cuticle.

Mitochondrial permeability transition pore induces mitochondria injury in Huntington disease

BackgroundMitochondrial impairment has been implicated in the pathogenesis of Huntington’s disease (HD). However, how mutant huntingtin impairs mitochondrial function and thus contributes to HD has not been fully elucidated. In this study, we used striatal cells expressing wild type (STHdhQ7/Q7) or mutant (STHdhQ111/Q111) huntingtin protein, and cortical neurons expressing the exon 1 of the huntingtin protein with physiological or pathological polyglutamine domains, to examine the interrelationship among specific mitochondrial functions.ResultsDepolarization induced by KCl resulted in similar changes in calcium levels without compromising mitochondrial function, both in wild type and mutant cells. However, treatment of mutant cells with thapsigargin (a SERCA antagonist that raises cytosolic calcium levels), resulted in a pronounced decrease in mitochondrial calcium uptake, increased production of reactive oxygen species (ROS), mitochondrial depolarization and fragmentation, and cell viability loss. The mitochondrial dysfunction in mutant cells was also observed in cortical neurons expressing exon 1 of the huntingtin protein with 104 Gln residues (Q104-GFP) when they were exposed to calcium stress. In addition, calcium overload induced opening of the mitochondrial permeability transition pore (mPTP) in mutant striatal cells. The mitochondrial impairment observed in mutant cells and cortical neurons expressing Q104-GFP was prevented by pre-treatment with cyclosporine A (CsA) but not by FK506 (an inhibitor of calcineurin), indicating a potential role for mPTP opening in the mitochondrial dysfunction induced by calcium stress in mutant huntingtin cells.ConclusionsExpression of mutant huntingtin alters mitochondrial and cell viability through mPTP opening in striatal cells and cortical neurons.

Surface charge engineering of a Bacillus gibsonii subtilisin protease

In proteins, a posttranslational deamidation process converts asparagine (Asn) and glutamine (Gln) residues into negatively charged aspartic (Asp) and glutamic acid (Glu), respectively. This process changes the protein net charge affecting enzyme activity, pH optimum, and stability. Understanding the principles which affect these enzyme properties would be valuable for protein engineering in general. In this work, three criteria for selecting amino acid substitutions of the deamidation type in the Bacillus gibsonii alkaline protease (BgAP) are proposed and systematically studied in their influence on pH-dependent activity and thermal resistance. Out of 113 possible surface amino acids, 18 (11 Asn and 7 Gln) residues of BgAP were selected and evaluated based on three proposed criteria: (1) The Asn or Gln residues should not be conserved, (2) should be surface exposed, and (3) neighbored by glycine. "Deamidation" in five (N97, N253, Q37, Q200, and Q256) out of eight (N97, N154, N250, N253, Q37, Q107, Q200, and Q256) amino acids meeting all criteria resulted in increased proteolytic activity. In addition, pH activity profiles of the variants N253D and Q256E and the combined variant N253DQ256E were dramatically shifted towards higher activity at lower pH (range of 8.5-10). Variant N253DQ256E showed twice the specific activity of wild-type BgAP and its thermal resistance increased by 2.4 °C at pH 8.5. These property changes suggest that mimicking surface deamidation by substituting Gln by Glu and/or Asn by Asp might be a simple and fast protein reengineering approach for modulating enzyme properties such as activity, pH optimum, and thermal resistance.

Local Unfolding Is Required for the Site-Specific Protein Modification by Transglutaminase

The transglutaminase (TGase) from Streptomyces mobaraensis catalyzes transamidation reactions in a protein substrate leading to the modification of the side chains of Gln and Lys residues according to the A-CONH(2) + H(2)N-B → A-CONH-B + NH(3) reaction, where both A and B can be a protein or a ligand. A noteworthy property of TGase is its susbstrate specificity, so that often only a few specific Gln or Lys residues can be modified in a globular protein. The molecular features of a globular protein dictating the site-specific reactions mediated by TGase are yet poorly understood. Here, we have analyzed the reactivity toward TGase of apomyoglobin (apoMb), α-lactalbumin (α-LA), and fragment 205-316 of thermolysin. These proteins are models of protein structure and folding that have been studied previously using the limited proteolysis technique to unravel regions of local unfolding in their amino acid sequences. The three proteins were modified by TGase at the level of Gln or Lys residues with dansylcadaverine or carbobenzoxy-l-glutaminylglycine, respectively. Despite these model proteins containing several Gln and Lys residues, the sites of TGase derivatization occur over restricted chain regions of the protein substrates. In particular, the TGase-mediated modifications occur in the "helix F" region in apoMb, in the β-domain in apo-α-LA in its molten globule state, and in the N-terminal region in fragment 205-316 of thermolysin. Interestingly, the sites of limited proteolysis are located in the same chain regions of these proteins, thus providing a clear-cut demonstration that chain flexibility or local unfolding overwhelmingly dictates the site-specific modification by both TGase and a protease.

Structural differences between thermophilic and mesophilic membrane proteins

The evolutionary adaptations of thermophilic water-soluble proteins required for maintaining stability at high temperature have been extensively investigated. Little is known about the adaptations in membrane proteins, however. Here, we compare many properties of mesophilic and thermophilic membrane protein structures, including side-chain burial, packing, hydrogen bonding, transmembrane kinks, loop lengths, hydrophobicity, and other sequence features. Most of these properties are quite similar between mesophiles and thermophiles although we observe a slight increase in side-chain burial and possibly a slight decrease in the frequency of transmembrane kinks in thermophilic membrane protein structures. The most striking difference is the increased hydrophobicity of thermophilic transmembrane helices, possibly reflecting more stringent hydrophobicity requirements for membrane partitioning at high temperature. In agreement with prior work examining transmembrane sequences, we find that thermophiles have an increase in small residues (Gly, Ala, Ser, and Val) and a strong suppression of Cys. We also find a relative dearth of most strongly polar residues (Asp, Asn, Glu, Gln, and Arg). These results suggest that in thermophiles, there is significant evolutionary pressure to offload destabilizing polar amino acids, to decrease the entropy cost of side chain burial, and to eliminate thermally sensitive amino acids.

Age‐dependent deamidation of glutamine residues in human γS crystallin: Deamidation and unstructured regions

Human aging is associated with the deterioration of long-lived proteins. Gradual cumulative modifications to the life-long proteins of the lens may ultimately be responsible for the pronounced alterations to the optical and physical properties that characterize lenses from older people. γS crystallin, a major human lens protein, is known to undergo several age-dependent changes. Using proteomic techniques, a site of deamidation involving glutamine 92 has been characterized and its time course established. The proportion of deamidation increased from birth to teen-age years and then plateaud. Deamidation at this site increased again in the eighth decade of life. There was no significant difference in the extent of deamidation between cataract and age-matched normal lenses. Gln92 is located in the linker region between the two domains, and the introduction of a negative charge at this site may alter the interaction between the two regions of the protein. Gln170, which is located in another unstructured part of γS crystallin, showed a similar deamidation profile to that of Gln92. As the other Gln residues in β-sheet regions of γS crystallin appear to remain as amides, modification of Gln92 and Gln170 thus conforms to a pattern whereby deamidation is localized to the unstructured regions of long-lived proteins.

Cascade Dissociations of Peptide Cation-Radicals. Part 1. Scope and Effects of Amino Acid Residues in Penta-, Nona-, and Decapeptides

Amino acid residue-specific backbone and side-chain dissociations of peptide z ions in MS(3) spectra were elucidated for over 40 pentapeptides with arginine C-terminated sequences of the AAXAR and AAHXR type, nonapeptides of the AAHAAXX"AR and AAHAXAX"AR type, and AAHAAXX"AAR decapeptides. Peptide z(n) ions containing amino acid residues with readily transferrable benzylic or tertiary β-hydrogen atoms (Phe, Tyr, His, Trp, Val) underwent facile backbone cleavages to form dominant z(n-2) or z(n-3) ions. These backbone cleavages are thought to be triggered by a side-chain β-hydrogen atom transfer to the z ion C(α) radical site followed by homolytic dissociation of the adjacent C(α)-CO bond, forming x(n-2) cation-radicals that spontaneously dissociate by loss of HNCO. Amino acid residues that do not have readily transferrable β-hydrogen atoms (Gly, Ala) do not undergo the z(n) → z(n-2) dissociations. The backbone cleavages compete with side-chain dissociations in z ions containing Asp and Asn residues. Side-chain dissociations are thought to be triggered by α-hydrogen atom transfers that activate the C(β)-C(γ) or C(β)-heteroatom bonds for dissociations that dominate the MS(3) spectra of z ions from peptides containing Leu, Cys, Lys, Met, Ser, Arg, Glu, and Gln residues. The Lys, Arg, Gln, and Glu residues also participate in γ-hydrogen atom transfers that trigger other side-chain dissociations.

Characterization and expression analysis of phospholipid hydroperoxide glutathione peroxidase cDNA from Chironomus riparius on exposure to cadmium

Phospholipid-hydroperoxide glutathione peroxidase (PHGPx) is an antioxidant enzyme in the glutathione peroxidases (GPx) family that reduces hydroperoxides of phospholipids and maintains the integrity of biomembranes. Here, we report the identification and characterization of a full length cDNA of PHGPx from the ecotoxicologically important aquatic midge Chironomus riparius (CrPHGPx1) from the Expressed Sequence Tags (ESTs) database generated through pyrosequencing. The 837 base pair (bp) cDNA contained an open reading frame of 597 bp, and a 75 bp 5' and a 159 bp 3'untranslated region. The theoretical molecular mass of the deduced amino acid (aa) sequence (197 aa) was 22.40 kDa with an estimated pI of 8.77. The Cys-codon was present at residue 74 and also the active site residues Gln(91) and Trp(164). The active-site motifs and GPx family signature motifs LAFPCNQF(101-108) and WNFTK(163-168) were also found. Phylogenetic analysis showed that CrPHGPx1 is grouped with PHGPx1 from other species and is more closely related to insects belonging to the dipteran order. The mRNA of CrPHGPx1 was detected in larvae, pupae and adults. The expression of CrPHGPx1 is induced by cadmium exposure indicating that the mRNA expression of CrPHGPx1 is differently regulated in response to oxidative stress caused by environmental stressors.

Side‐chain cyclization of Gln or Asn residues coupled to peptide bond cleavage in an intein and a model peptide

Protein splicing is a post‐translational event by which an intervening polypeptide, or intein, facilitates its own excision from the flanking polypeptides, or exteins, and the ligation of the exteins. An epimerase found in Trichodesmium erythraeum contains two inteins, Ndse1 and Ndse2. The first intein, Ndse1, can facilitate protein splicing efficiently in E. coli, even with a native C‐terminal Gln replacing the highly conserved Asn. In addition, activity of the intein may be influenced two downstream Cys residues and the presence of disulfide bonds, as we observed differential reactivity in inteins expressed under reducing and non‐reducing conditions. Substitution of Gln for Asn results in an increase in the rate of C‐terminal bond cleavage for the PolII intein from Pyrococcus abyssi. To study side chain cyclization in a model system, we obtained three pentapeptides of Abz‐His‐Xxx‐ Src‐Lys, where Xxx is Gln, Asn or Ala. Dnp, dinitrophenyl, is attached to the C‐terminal Lys and ortho‐aminobenzoic acid (Abz) serves as the N‐terminal residue. Peptide bond cleavage separates the Abz fluorophore from the Dnp quencher.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245, the Camille and Henry Dreyfus Foundation (KM), and a Holy Cross Alumni/Parents Summer Research Scholarship (LU).

Functional Evaluation Of Angiotensin‐Converting Enzyme (ACE) Amino Acid Residues important for the interaction with its inhibitors

Hypertension is a worldwide health problem considered to be one of the greatest public health challenges. Since hypertension is a major risk factor for cardiovascular and renal disease, many studies have focused on the Renin‐Angiotensin System (RAS), an endocrine system where renin cleaves angiotensinogen and releases Angiotensin I (AI) which is converted into Angiotensin II (AII) by the action of Angiotensin Converting Enzyme I (ACE). The ACE has a large physiological importance being a target of several therapeutic studies. This work aims to investigate the role of amino acids residues Asp140, Gln259, Ala332, Ser 333, Gln 355, Thr 358, Phe435 and Arg 500, in the interaction with the ACE inhibitor lisinopril as well as other ones.. An analysis of sequence homology to the ACEs of Xenopus laevis, mouse, rat and human, human isoform N‐domain (ND) and isoforms of 65 and 90 kDa of rat showed a high conservation of amino acid residues proposed to be mutated. Primers containing the mutation were synthesized. Following, we used a vector containing the cDNA with the human ACE gene (pACE) to perform PCR reactions; then the products obtained from PCR reactions were digested with the enzyme DpnI and transformed in E. coli DH5α. Positive colonies had the plasmid extracted, sequenced and analyzed concerning the desired mutation. The ACE mutants will be analyzed related to enzyme‐substrate interaction as well as enzyme activity.

Structure of a dominant-negative helix-loop-helix transcriptional regulator suggests mechanisms of autoinhibition

Helix-loop-helix (HLH) family transcription factors regulate numerous developmental and homeostatic processes. Dominant-negative HLH (dnHLH) proteins lack DNA-binding ability and capture basic HLH (bHLH) transcription factors to inhibit cellular differentiation and enhance cell proliferation and motility, thus participating in patho-physiological processes. We report the first structure of a free-standing human dnHLH protein, HHM (Human homologue of murine maternal Id-like molecule). HHM adopts a V-shaped conformation, with N-terminal and C-terminal five-helix bundles connected by the HLH region. In striking contrast to the common HLH, the HLH region in HHM is extended, with its hydrophobic dimerization interfaces embedded in the N- and C-terminal helix bundles. Biochemical and physicochemical analyses revealed that HHM exists in slow equilibrium between this V-shaped form and the partially unfolded, relaxed form. The latter form is readily available for interactions with its target bHLH transcription factors. Mutations disrupting the interactions in the V-shaped form compromised the target transcription factor specificity and accelerated myogenic cell differentiation. Therefore, the V-shaped form of HHM may represent an autoinhibited state, and the dynamic conformational equilibrium may control the target specificity.

From red to blue to far-red in Lhca4: How does the protein modulate the spectral properties of the pigments?

The first event of photosynthesis is the harvesting of solar energy by a large array of pigments. These pigments are coordinated to proteins that organize them to assure efficient excitation energy transfer. The protein plays an essential role in tuning the spectroscopic properties of the pigments, by determining their site energy and/or by favoring pigment–pigments interactions. Here we investigate how the protein modulates the pigment properties by using a single-point-mutation approach. We monitor changes in the low-energy absorption/emission band of Lhca4, which is well separated from the bulk absorption and thus represents an attractive model system. Moreover, it was recently shown that Lhca4 exists in at least two conformations, a dominating one emitting at 720nm and a second one emitting at 685nm (Kruger et al. PNAS 2011). Here we show that a single amino-acid substitution (from Asn to Gln, which are both chlorophyll-binding residues and only differ for one C―C bond), moves the equilibrium almost completely towards the 685-nm conformation. This indicates that small changes in the protein can have a large effect on the properties of the pigments. We show that His99, which was suggested to coordinate a red-absorbing chlorophyll (Melkozernov and Blankenship, JBC 2003), is not a chlorophyll ligand. We also show that single amino-acid substitutions nearby the chlorophylls allow to tune the emission spectrum of the pigments over a wide range of wavelengths and to modulate the excited-state lifetimes of the complex. These findings are discussed in the light of previously proposed non-photochemical quenching models.

67 Influence of Transglutaminase and the Reducing Agent Glutathione in the Gastric Digestion and Immunogenicity of Beta-Lactoglobulin

BackgroundAmong the milk whey proteins, β-lactoglobulin (β-Lg) is the main allergen. In native state, β-Lg is resistant to pepsin, which is considered an indicator of its allergenic potential. Protein antigenicity can be reduced by treatment with transglutaminase (TG), an enzyme which catalyses inter or intra crosslink between Lys and Gln residues. Reducing agents have been used to increase the access of TG to the Lys and Gln residues. This study aimed at investigating the antigenicity and digestibility by pepsin of β-Lg modified by TG in the presence of the reducing agent glutathione (GSH).Methods and ResultsThe polymerization of β-Lg (Davisco, MN, USA) by microbial TG (WM, Ajinomoto Ltda, Brazil) was studied using a central-composite experimental design with 2 independent variables, GSH concentration [GSH] (0–0.2 mmol L−1) and enzyme:substrate ratio (E/S) (0–44.1 U g−1). The dependent variable was the response of specific IgE obtained from the serum of BALB/c mice sensitized with native β-Lg (IgE anti-β-Lg) against the modified protein. Polymerization was carried out at 50°C/180 minutes and stopped by heating at 80°C/5 min. Digestion was simulated using pepsin (E/S 1:20 w/w, pH 2, 37°C, 1 hour), and SDS-PAGE and the ELISA assay used to characterize and evaluate the antigenicity of the samples, respectively. The linear and quadratic factors of [GSH] and E/S, and their interaction, showed no significant effect on the response of lgE. No treatment resulted in a decrease in the response of IgE anti-β-Lg, but a significant (P < 0.05) increase was observed in the response of IgE against treatments 1, 7 and center point. Intact β-Lg was observed in the electrophoretic profile of the digested samples, indicating resistance to pepsin. After digestion, the samples not presented difference with respect to the digested native β-Lg sample (213.06 ± 9.59 ng mL−1).ConclusionPolymerization of the β-Lg with TG in the presence of the GSH did not alter its digestibility by pepsin or decrease the antigenicity. Under some of the conditions studied the antigenicity of the β-Lg increased, indicating that the treatment could have created or exposed epitopes.

Asparagine and glutamine differ in their propensities to form specific side chain‐backbone hydrogen bonded motifs in proteins

Short range side chain-backbone hydrogen bonded motifs involving Asn and Gln residues have been identified from a data set of 1370 protein crystal structures (resolution ≤ 1.5 Å). Hydrogen bonds involving residues i - 5 to i + 5 have been considered. Out of 12,901 Asn residues, 3403 residues (26.4%) participate in such interactions, while out of 10,934 Gln residues, 1780 Gln residues (16.3%) are involved in these motifs. Hydrogen bonded ring sizes (C(n), where n is the number of atoms involved), directionality and internal torsion angles are used to classify motifs. The occurrence of the various motifs in the contexts of protein structure is illustrated. Distinct differences are established between the nature of motifs formed by Asn and Gln residues. For Asn, the most highly populated motifs are the C(10)(CO(δ)(i) …NH(i + 2)), C(13)(CO(δ)(i) …NH(i + 3)) and C(17)(N(δ)H(i) …CO(i - 4)) structures. In contrast, Gln predominantly forms C(16)(CO(ε)(i) …NH(i - 3)), C(12)(N(ε)H(i) …CO(i - 2)), C(15)(N(ε)H(i) …CO(i - 3)) and C(18)(N(ε)H(i) …CO(i - 4)) motifs, with only the C(18) motif being analogous to the Asn C(17) structure. Specific conformational types are established for the Asn containing motifs, which mimic backbone β-turns and α-turns. Histidine residues are shown to serve as a mimic for Asn residues in side chain-backbone hydrogen bonded ring motifs. Illustrative examples from protein structures are considered.

Structure and stability of cyclic peptide based nanotubes: a molecular dynamics study of the influence of amino acid composition

The stability of self-assembling cyclic peptides (CPs) is attained by the intermolecular backbone-backbone hydrogen bonding (H-bonding) interactions. In addition to these H-bonding interactions, the self-assembled CPs are further stabilized by various intermolecular side chain-side chain interactions. This study investigates the role of amino acids on the structure and stability of self-assembled CPs using classical molecular dynamics (MD) simulations and molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method. The amino acids considered for the construction of model structures of cyclic peptide nanotubes (CPNTs) are Ala, Leu, Phe, Gln, Glu, and Trp. The calculated structural parameters from classical MD simulations reveal that the backbone flexibility of CPNTs composed of non-Ala residues results from an intrinsic property of the amino acids. The presence of an Ala residue at the alternate position increases the solvation of side chains of Gln residue. The occurrence of Glu residue does not favour the formation of intermolecular side chain-side chain H-bonding interactions in aqueous medium. It is evident from the calculated free energy of binding that CPNTs composed of non-polar residues are highly stable in aqueous medium. At the same time, CPNTs with polar side chains are less stable in aqueous medium. Results obtained from this study demonstrate the role played by amino acid side chains on the structure and stability of CPNTs and provide valuable suggestions for the design of CPNTs with moderate stability in various solvent environments.

Rho/ROCK-dependent inhibition of 3T3-L1 adipogenesis by G-protein-deamidating dermonecrotic toxins: differential regulation of Notch1, Pref1/Dlk1, and β-catenin signaling

The dermonecrotic toxins from Pasteurella multocida (PMT), Bordetella (DNT), Escherichia coli (CNF1-3), and Yersinia (CNFY) modulate their G-protein targets through deamidation and/or transglutamination of an active site Gln residue, which results in activation of the G protein and its cognate downstream signaling pathways. Whereas DNT and the CNFs act on small Rho GTPases, PMT acts on the α subunit of heterotrimeric Gq, Gi, and G12/13 proteins. We previously demonstrated that PMT potently blocks adipogenesis and adipocyte differentiation in a calcineurin-independent manner through downregulation of Notch1 and stabilization of β-catenin and Pref1/Dlk1, key proteins in signaling pathways strongly linked to cell fate decisions, including fat and bone development. Here, we report that similar to PMT, DNT, and CNF1 completely block adipogenesis and adipocyte differentiation by preventing upregulation of adipocyte markers, PPARγ and C/EBPα, while stabilizing the expression of Pref1/Dlk1 and β-catenin. We show that the Rho/ROCK inhibitor Y-27632 prevented or reversed these toxin-mediated effects, strongly supporting a role for Rho/ROCK signaling in dermonecrotic toxin-mediated inhibition of adipogenesis and adipocyte differentiation. Toxin treatment was also accompanied by downregulation of Notch1 expression, although this inhibition was independent of Rho/ROCK signaling. We further show that PMT-mediated downregulation of Notch1 expression occurs primarily through G12/13 signaling. Our results reveal new details of the pathways involved in dermonecrotic toxin action on adipocyte differentiation, and the role of Rho/ROCK signaling in mediating toxin effects on Wnt/β-catenin and Notch1 signaling, and in particular the role of Gq and G12/13 in mediating PMT effects on Rho/ROCK and Notch1 signaling.

The Antibacterial Activity of LL-37 against Treponema denticola Is Dentilisin Protease Independent and Facilitated by the Major Outer Sheath Protein Virulence Factor

Host defense peptides are innate immune effectors that possess both bactericidal activities and immunomodulatory functions. Deficiency in the human host defense peptide LL-37 has previously been correlated with severe periodontal disease. Treponema denticola is an oral anaerobic spirochete closely associated with the pathogenesis of periodontal disease. The T. denticola major surface protein (MSP), involved in adhesion and cytotoxicity, and the dentilisin serine protease are key virulence factors of this organism. In this study, we examined the interactions between LL-37 and T. denticola. The three T. denticola strains tested were susceptible to LL-37. Dentilisin was found to inactivate LL-37 by cleaving it at the Lys, Phe, Gln, and Val residues. However, dentilisin deletion did not increase the susceptibility of T. denticola to LL-37. Furthermore, dentilisin activity was found to be inhibited by human saliva. In contrast, a deficiency of the T. denticola MSP increased resistance to LL-37. The MSP-deficient mutant bound less fluorescently labeled LL-37 than the wild-type strain. MSP demonstrated specific, dose-dependent LL-37 binding. In conclusion, though capable of LL-37 inactivation, dentilisin does not protect T. denticola from LL-37. Rather, the rapid, MSP-mediated binding of LL-37 to the treponemal outer sheath precedes cleavage by dentilisin. Moreover, in vivo, saliva inhibits dentilisin, thus preventing LL-37 restriction and ensuring its bactericidal and immunoregulatory activities.

Structure of Hydrogenase Maturation Protein HypF with Reaction Intermediates Shows Two Active Sites

[NiFe]-hydrogenases are multimeric proteins. Thelarge subunit contains the NiFe(CN)(2)CO bimetallic active center and the small subunit contains Fe-S clusters. Biosynthesis and assembly of the NiFe(CN)(2)CO active center requires six Hyp accessory proteins. The synthesis of the CN(-) ligands iscatalyzed by the combined actions of HypF andHypE using carbamoylphosphate as a substrate.We report the structure of Escherichia coli HypF(92-750) lacking the N-terminal acylphosphatase domain. HypF(92-750) comprises the novel Zn-finger domain, the nucleotide-binding YrdC-like domain, and the Kae1-like universal domain, also binding a nucleotide and a Zn(2+) ion. The two nucleotide-binding sites are sequestered in an internal cavity, facing each other and separated by ∼14Å. The YrdC-like domain converts carbamoyl moiety to a carbamoyl adenylate intermediate, which is channeled to the Kae1-like domain. Mutations within either nucleotide-binding site compromise hydrogenase maturation but do not affect the carbamoylphosphate phosphatase activity.

Molecular characterization and functional analysis by heterologous expression in E. coli under diverse abiotic stresses for OsLEA5, the atypical hydrophobic LEA protein from Oryza sativa L.

In this study, we report the molecular characterization and functional analysis of OsLEA5 gene, which belongs to the atypical late embryogenesis abundant (LEA) group 5C from Oryza sativa L. The cDNA of OsLEA5 contains a 456bp ORF encoding a polypeptide of 151 amino acids with a calculated molecular mass of 16.5kDa and a theoretical pI of 5.07. The OsLEA5 polypeptide is rich in Leu (10%), Ser (8.6%), and Asp (8.6%), while Cys, Trp, and Gln residue contents are very low, which are 2, 1.3, and 1.3%, respectively. Bioinformatic analysis revealed that group 5C LEA protein subfamily contains a Pfam:LEA_2 domain architecture and is highly hydrophobic, intrinsically ordered with largely β-sheet and specific amino acid composition and distribution. Real-time PCR analysis showed that OsLEA5 was expressed in different tissue organs during different development stages of rice. The expression levels of OsLEA5 in the roots and panicles of full ripe stage were dramatically increased. The results of stress tolerance and cell viability assay demonstrated that recombinant E. coli cells producing OsLEA5 fusion protein exhibited improved resistance against diverse abiotic stresses: high salinity, osmotic, freezing, heat, and UV radiation. The OsLEA5 protein confers stabilization of the LDH under different abiotic stresses, such as heating, freeze-thawing, and drying in vitro. The combined results indicated that OsLEA5 protein was a hydrophobic atypical LEA and closely associated with resistance to multiple abiotic stresses. This research offered the valuable information for the development of crops with enhanced resistance to diverse stresses.

Non-nucleosidic inhibition of Herpes simplex virus DNA polymerase: mechanistic insights into the anti-herpetic mode of action of herbal drug withaferin A

BackgroundHerpes Simplex Virus 1 and 2 causes several infections in humans including cold sores and encephalitis. Previous antiviral studies on herpes viruses have focussed on developing nucleoside analogues that can inhibit viral polymerase and terminate the replicating viral DNA. However, these drugs bear an intrinsic non-specificity as they can also inhibit cellular polymerase apart from the viral one. The present study is an attempt to elucidate the action mechanism of naturally occurring withaferin A in inhibiting viral DNA polymerase, thus providing an evidence for its development as a novel anti-herpetic drug.ResultsWithaferin A was found to bind very similarly to that of the previously reported 4-oxo-DHQ inhibitor. Withaferin A was observed binding to the residues Gln 617, Gln 618, Asn 815 and Tyr 818, all of which are crucial to the proper functioning of the polymerase. A comparison of the conformation obtained from docking and the molecular dynamics simulations shows that substantial changes in the binding conformations have occurred. These results indicate that the initial receptor-ligand interaction observed after docking can be limited due to the receptor rigid docking algorithm and that the conformations and interactions observed after simulation runs are more energetically favoured.ConclusionsWe have performed docking and molecular dynamics simulation studies to elucidate the binding mechanism of prospective herbal drug withaferin A onto the structure of DNA polymerase of Herpes simplex virus. Our docking simulations results give high binding affinity of the ligand to the receptor. Long de novo MD simulations for 10 ns performed allowed us to evaluate the dynamic behaviour of the system studied and corroborate the docking results, as well as identify key residues in the enzyme-inhibitor interactions. The present MD simulations support the hypothesis that withaferin A is a potential ligand to target/inhibit DNA polymerase of the Herpes simplex virus. Results of these studies will also guide the design of selective inhibitors of DNA POL with high specificity and potent activity in order to strengthen the therapeutic arsenal available today against the dangerous biological warfare agent represented by Herpes Simplex Virus.