Computational analyses of protein coded by rice (Oryza sativa japonica) cDNA (GI: 32984786) indicate lectin like Ca2+ binding properties for Eicosapenta Peptide Repeats (EPRs)

Eicosapenta peptide repeats (EPRs) occur exclusively in flowering plant genomes and exhibit very high amino acid residue conservation across occurrence. DNA and amino acid sequence searches yielded no indications about the function due to absence of similarity to known sequences. Tertiary structure of an EPR protein coded by rice (Oryza sativa japonica) cDNA (GI: 32984786) was determined based on ab initio methodology in order to draw clues on functional significance of EPRs. The resultant structure comprised of seven α-helices and thirteen anti-parallel β-sheets. Surface-mapping of conserved residues onto the structure deduced that (i) regions equivalent to β α4- the primary function of EPR protein could be Ca²⁺ binding, and (iii) the putative EPR Ca²⁺ binding domain is structurally similar to calcium-binding domains of plant lectins. Additionally, the phylogenetic analysis showed an evolving taxa-specific distribution of EPR proteins observed in some GNA-like lectins.     

Solution structure of the strawberry allergen Fra a 1

The PR10 family protein Fra a 1E from strawberry (Fragaria x ananassa) is down-regulated in white strawberry mutants, and transient RNAi (RNA interference)-mediated silencing experiments confirmed that Fra a 1 is involved in fruit pigment synthesis. In the present study, we determined the solution structure of Fra a 1E. The protein fold is identical with that of other members of the PR10 protein family and consists of a seven-stranded antiparallel β-sheet, two short V-shaped α-helices and a long C-terminal α-helix that encompass a hydrophobic pocket. Whereas Fra a 1E contains the glycine-rich loop that is highly conserved throughout the protein family, the volume of the hydrophobic pocket and the size of its entrance are much larger than expected. The three-dimensional structure may shed some light on its physiological function and may help to further understand the role of PR10 proteins in plants.     

Aromatic cluster mutations produce focal modulations of β-sheet structure

Site-directed mutagenesis is a powerful tool for altering the structure and function of proteins in a focused manner. Here, we examined how a model β-sheet protein could be tuned by mutation of numerous surface-exposed residues to aromatic amino acids. We designed these aromatic side chain “clusters” at highly solvent-exposed positions in the flat, single-layer β-sheet of Borrelia outer surface protein A (OspA). This unusual β-sheet scaffold allows us to interrogate the effects of these mutations in the context of well-defined structure but in the absence of the strong scaffolding effects of globular protein architecture. We anticipated that the introduction of a cluster of aromatic amino acid residues on the β-sheet surface would result in large conformational changes and/or stabilization and thereby provide new means of controlling the properties of β-sheets. Surprisingly, X-ray crystal structures revealed that the introduction of aromatic clusters produced only subtle conformational changes in the OspA β-sheet. Additionally, despite burying a large degree of hydrophobic surface area, the aromatic cluster mutants were slightly less stable than the wild-type scaffold. These results thereby demonstrate that the introduction of aromatic cluster mutations can serve as a means for subtly modulating β-sheet conformation in protein design.     

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III ε subunit (featuring 3’-5’ exonuclease activity) is highly improved when the same protein is co-expressed with the α and θ subunits (featuring DNA polymerase activity and stabilizing ε, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the α subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the ε subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type ε subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-β-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.     

Transmission of Stability Information through the N-domain of Tropomyosin Is Interrupted by a Stabilizing Mutation (A109L) in the Hydrophobic Core of the Stability Control Region (Residues 97–118)

Tropomyosin (Tm) is an actin-binding, thin filament, two-stranded α-helical coiled-coil critical for muscle contraction and cytoskeletal function. We made the first identification of a stability control region (SCR), residues 97–118, in the Tm sequence that controls overall protein stability but is not required for folding. We also showed that the individual α-helical strands of the coiled-coil are stabilized by Leu-110, whereas the hydrophobic core is destabilized in the SCR by Ala residues at three consecutive d positions. Our hypothesis is that the stabilization of the individual α-helices provides an optimum stability and allows functionally beneficial dynamic motion between the α-helices that is critical for the transmission of stabilizing information along the coiled-coil from the SCR. We prepared three recombinant (rat) Tm(1–131) proteins, including the wild type sequence, a destabilizing mutation L110A, and a stabilizing mutation A109L. These proteins were evaluated by circular dichroism (CD) and differential scanning calorimetry. The single mutation L110A destabilizes the entire Tm(1–131) molecule, showing that the effect of this mutation is transmitted 165 Å along the coiled-coil in the N-terminal direction. The single mutation A109L prevents the SCR from transmitting stabilizing information and separates the coiled-coil into two domains, one that is ∼9 °C more stable than wild type and one that is ∼16 °C less stable. We know of no other example of the substitution of a stabilizing Leu residue in a coiled-coil hydrophobic core position d that causes this dramatic effect. We demonstrate the importance of the SCR in controlling and transmitting the stability signal along this rodlike molecule.     

High temperature unfolding of Bacillus anthracis amidase-03 by molecular dynamics simulations

The stability of amidase-03 structure (a cell wall hydrolase protein) from Bacillus anthracis was studied using classical molecular dynamics (MD) simulation. This protein (GenBank accession number: {"type":"entrez-protein","attrs":{"text":"NP_844822","term_id":"30262445","term_text":"NP_844822"}}NP_844822) contains an amidase-03 domain which is known to exhibit the catalytic activity of N-acetylmuramoyl-L-alanine amidase (digesting MurNAc-Lalanine linkage of bacterial cell wall). The amidase-03 enzyme has stability at high temperature due to the core formed by the combination of several secondary structure elements made of β-sheets. We used root-mean-square-displacement (RMSD) of the simulated structure from its initial state to demonstrate the unfolding of the enzyme using its secondary structural elements. Results show that amidase-03 unfolds in transition state ensemble (TSE). The data suggests that α-helices unfold before β-sheets from the core during simulation.     

Diversity of Channels Generated by Different Combinations of Epithelial Sodium Channel Subunits

The epithelial sodium channel is a multimeric protein formed by three homologous subunits: α, β, and γ; each subunit contains only two transmembrane domains. The level of expression of each of the subunits is markedly different in various Na⁺ absorbing epithelia raising the possibility that channels with different subunit composition can function in vivo. We have examined the functional properties of channels formed by the association of α with β and of α with γ in the Xenopus oocyte expression system using two-microelectrode voltage clamp and patch-clamp techniques. We found that αβ channels differ from αγ channels in the following functional properties: (a) αβ channels expressed larger Na⁺ than Li⁺ currents (I_Na+/I_Li+ 1.2) whereas αγ channels expressed smaller Na⁺ than Li⁺ currents (I_Na+/I_Li+ 0.55); (b) the Michaelis Menten constants (K _m) of activation of current by increasing concentrations of external Na⁺ and Li⁺ of αβ channels were larger (K _m > 180 mM) than those of αγ channels (K _m of 35 and 50 mM, respectively); (c) single channel conductances of αβ channels (5.1 pS for Na⁺ and 4.2 pS for Li⁺) were smaller than those of αγ channels (6.5 pS for Na⁺ and 10.8 pS for Li⁺); (d) the half-inhibition constant (K _i) of amiloride was 20-fold larger for αβ channels than for αγ channels whereas the K _i of guanidinium was equal for both αβ and αγ. To identify the domains in the channel subunits involved in amiloride binding, we constructed several chimeras that contained the amino terminus of the γ subunit and the carboxy terminus of the β subunit. A stretch of 15 amino acids, immediately before the second transmembrane domain of the β subunit, was identified as the domain conferring lower amiloride affinity to the αβ channels. We provide evidence for the existence of two distinct binding sites for the amiloride molecule: one for the guanidium moiety and another for the pyrazine ring. At least two subunits α with β or γ contribute to these binding sites. Finally, we show that the most likely stoichiometry of αβ and αγ channels is 1α:1β and 1α:1γ, respectively.     

Functional insights by comparison of modeled structures of 18kDa small heat shock protein and its mutant in Mycobacterium leprae

In this work we are proposing Homology modeled structures of Mycobacterium leprae 18kDa heat shock protein and its mutant. The more closely related structure of the small heat shock protein (sHSP) belonging to the eukaryotic species from wheat sHSP16.9 and 16.3kDa ACR1 protein from Mycobacterium tuberculosis were used as template structures. Each model contains an N-terminal domain, alpha-crystalline domain and a C-terminal tail. The models showed that a single point mutation from serine to proline at 52^nd position causes structural changes. The structural changes are observed in N-terminal region and alpha-crystalline domains. Serine in 52^nd position is observed in β4 strand and Proline in 52^nd position is observed in loop. The number of residues contributing α helix at N-terminal region varies in both models. In 18S more number of residues is present in α helix when compared to 18P. The loop regions between β3 and β4 strands of both models vary in number of residues present in it. Number of residues contributing β4 strand in both models vary. β6 strand is absent in both models. Major functional peptide region of alpha crystalline domains of both models varies. These differences observed in secondary structures support their distinct functional roles. It also emphasizes that a point mutation can cause structural variation.     

β-Filagenin, a Newly Identified Protein Coassembling with Myosin and Paramyosin in Caenorhabditis elegans

Muscle thick filaments are stable assemblies of myosin and associated proteins whose dimensions are precisely regulated. The mechanisms underlying the stability and regulation of the assembly are not understood. As an approach to these problems, we have studied the core proteins that, together with paramyosin, form the core structure of the thick filament backbone in the nematode Caenorhabditis elegans. We obtained partial peptide sequences from one of the core proteins, β-filagenin, and then identified a gene that encodes a novel protein of 201–amino acid residues from databases using these sequences. β-Filagenin has a calculated isoelectric point at 10.61 and a high percentage of aromatic amino acids. Secondary structure algorithms predict that it consists of four β-strands but no α-helices. Western blotting using an affinity-purified antibody showed that β-filagenin was associated with the cores. β-Filagenin was localized by immunofluorescence microscopy to the A bands of body–wall muscles, but not the pharynx. β-filagenin assembled with the myosin homologue paramyosin into the tubular cores of wild-type nematodes at a periodicity matching the 72-nm repeats of paramyosin, as revealed by immunoelectron microscopy. In CB1214 mutants where paramyosin is absent, β-filagenin assembled with myosin to form abnormal tubular filaments with a periodicity identical to wild type. These results verify that β-filagenin is a core protein that coassembles with either myosin or paramyosin in C. elegans to form tubular filaments.     

Molecular structure of an N-formyltransferase from Providencia alcalifaciens O30

The existence of N-formylated sugars in the O-antigens of Gram-negative bacteria has been known since the middle 1980s, but only recently have the biosynthetic pathways for their production been reported. In these pathways, glucose-1-phosphate is first activated by attachment to a dTMP moiety. This step is followed by a dehydration reaction and an amination. The last step in these pathways is catalyzed by N-formyltransferases that utilize N¹⁰-formyltetrahydrofolate as the carbon source. Here we describe the three-dimensional structure of one of these N-formyltransferases, namely VioF from Providencia alcalifaciens O30. Specifically, this enzyme catalyzes the conversion of dTDP-4-amino-4,6-dideoxyglucose (dTDP-Qui4N) to dTDP-4,6-dideoxy-4-formamido-d-glucose (dTDP-Qui4NFo). For this analysis, the structure of VioF was solved to 1.9 Å resolution in both its apoform and in complex with tetrahydrofolate and dTDP-Qui4N. The crystals used in the investigation belonged to the space group R32 and demonstrated reticular merohedral twinning. The overall catalytic core of the VioF subunit is characterized by a six stranded mixed β-sheet flanked on one side by three α-helices and on the other side by mostly random coil. This N-terminal domain is followed by an α-helix and a β-hairpin that form the subunit:subunit interface. The active site of the enzyme is shallow and solvent-exposed. Notably, the pyranosyl moiety of dTDP-Qui4N is positioned into the active site by only one hydrogen bond provided by Lys 77. Comparison of the VioF model to that of a previously determined N-formyltransferase suggests that substrate specificity is determined by interactions between the protein and the pyrophosphoryl group of the dTDP-sugar substrate.     

Structural characterization of the late competence protein ComFB from Bacillus subtilis

Many bacteria take up DNA from their environment as part of the process of natural transformation. DNA uptake allows microorganisms to gain genetic diversity and can lead to the spread of antibiotic resistance or virulence genes within a microbial population. Development of genetic competence (Com) in Bacillus subtilis is a highly regulated process that culminates in expression of several late competence genes and formation of the DNA uptake apparatus. The late competence operon comF encodes a small protein of unknown function, ComFB. To gain insight into the function of ComFB, we determined its 3D structure via X-ray crystallography. ComFB is a dimer and each subunit consists of four α-helices connected by short loops and one extended β-strand-like stretch. Each subunit contains one zinc-binding site formed by four cysteines, which are unusually spaced in the primary sequence. Using structure- and bioinformatics-guided substitutions we analyzed the inter-subunit interface of the ComFB dimer. Based on these analyses, we conclude that ComFB is an obligate dimer. We also characterized ComFB in vivo and found that this protein is produced in competent cells and is localized to the cytosol. Consistent with previous reports, we showed that deletion of ComFB does not affect DNA uptake function. Combining our results, we conclude that ComFB is unlikely to be a part of the DNA uptake machinery under tested conditions and instead may have a regulatory function.     

Insights from the molecular docking of curcumin to the virulent factors of Helicobacter pylori

The domains of virulent (Ureα/β, VacA-p55, and CagA) factors of Helicobacter pylori play a pivotal role in developmental processes of numerous diseases including gastric cancer. The pharmacological role of curcumin indicates that it could regulate the signaling of virulent factors by interacting with active domains. However, the controlling mechanism of the curcumin interactions and the binding diversity on structural basis of virulent (Ureα/β, VacA-p55, and CagA) factors are unknown. Curcumin as therapeutic agent was filtered by using Lipinski rule׳s five and the druglikeness property for assessment of pharmacological properties. Here outcome of molecular docking presented the 3-D structure of curcumin complex, that interacted with especially conserved residues of target domains. The structure revealed that the curcumin complexation with domains of these proteins provided structural insight into the diverse nature of proteins (Ureα/β, VacA-p55, and CagA) recognition. In silico study elucidated that the broad specificity of curcumin was achieved by multiple binding mode mechanisms such as distinct hydrogen and hydrophobic interactions with involvement of binding energy. The higher score of curcumin in complexation with both subunits Ureα/β showed the stable binding, and less stability with VacA-p55 complexation with lower score. Curcumin exhibited good interaction with these targeted virulent factors, although extensive interactions of curcumin with Ureα/β subunits could have an important implication to prevent survival and colonisation of H. pylori in stomach.     

Molecular modeling and docking characterization of CzR1, a CC-NBS-LRR R-gene from Curcuma zedoaria Loeb. that confers resistance to Pythium aphanidermatum

Plant NBS-LRR R-genes recognizes several pathogen associated molecular patterns (PAMPs) and limit pathogen infection through a multifaceted defense response. CzR1, a coiled-coil-nucleotide-binding-site-leucine-rich repeat R-gene isolated from Curcuma zedoaria L exhibit constitutive resistance to different strains of P. aphanidermatum. Majority of the necrotrophic oomycetes are characterized by the presence of carbohydrate PAMPs β-glucans in their cell walls which intercat with R-genes. In the present study, we predicted the 3D (three dimensional) structure of CzR1 based on homology modeling using the homology module of Prime through the Maestro interface of Schrodinger package ver 2.5. The docking investigation of CzR1 with β-glucan using the Glide software suggests that six amino acid residues, Ser186, Glu187, Ser263, Asp264, Asp355 and Tyr425 act as catalytic residues and are involved in hydrogen bonding with ligand β-(1,3)-D-Glucan. The calculated distance between the carboxylic oxygen atoms of Glu187–Asp355 pair is well within the distance of 5Å suggesting a positive glucanase activity of CzR1. Elucidation of these molecular characteristics will help in in silico screening and understanding the structural basis of ligand binding to CzR1 protein and pave new ways towards a broad spectrum rhizome rot resistance development in the cultivated turmeric.     

Novel Structural Components Contribute to the High Thermal Stability of Acyl Carrier Protein from Enterococcus faecalis

Enterococcus faecalis is a Gram-positive, commensal bacterium that lives in the gastrointestinal tracts of humans and other mammals. It causes severe infections because of high antibiotic resistance. E. faecalis can endure extremes of temperature and pH. Acyl carrier protein (ACP) is a key element in the biosynthesis of fatty acids responsible for acyl group shuttling and delivery. In this study, to understand the origin of high thermal stabilities of E. faecalis ACP (Ef-ACP), its solution structure was investigated for the first time. CD experiments showed that the melting temperature of Ef-ACP is 78.8 °C, which is much higher than that of Escherichia coli ACP (67.2 °C). The overall structure of Ef-ACP shows the common ACP folding pattern consisting of four α-helices (helix I (residues 3–17), helix II (residues 39–53), helix III (residues 60–64), and helix IV (residues 68–78)) connected by three loops. Unique Ef-ACP structural features include a hydrophobic interaction between Phe⁴⁵ in helix II and Phe¹⁸ in the α₁α₂ loop and a hydrogen bonding between Ser¹⁵ in helix I and Ile²⁰ in the α₁α₂ loop, resulting in its high thermal stability. Phe⁴⁵-mediated hydrophobic packing may block acyl chain binding subpocket II entry. Furthermore, Ser⁵⁸ in the α₂α₃ loop in Ef-ACP, which usually constitutes a proline in other ACPs, exhibited slow conformational exchanges, resulting in the movement of the helix III outside the structure to accommodate a longer acyl chain in the acyl binding cavity. These results might provide insights into the development of antibiotics against pathogenic drug-resistant E. faecalis strains.     

A Proposed Mechanism for the Promotion of Prion Conversion Involving a Strictly Conserved Tyrosine Residue in the β2-α2 Loop of PrPC

The transmission of infectious prions into different host species requires compatible prion protein (PrP) primary structures, and even one heterologous residue at a pivotal position can block prion infection. Mapping the key amino acid positions that govern cross-species prion conversion has not yet been possible, although certain residue positions have been identified as restrictive, including residues in the β₂-α₂ loop region of PrP. To further define how β₂-α₂ residues impact conversion, we investigated residue substitutions in PrP^C using an in vitro prion conversion assay. Within the β₂-α₂ loop, a tyrosine residue at position 169 is strictly conserved among mammals, and transgenic mice expressing mouse PrP having the Y169G, S170N, and N174T substitutions resist prion infection. To better understand the structural requirements of specific residues for conversion initiated by mouse prions, we substituted a diverse array of amino acids at position 169 of PrP. We found that the substitution of glycine, leucine, or glutamine at position 169 reduced conversion by ∼75%. In contrast, replacing tyrosine 169 with either of the bulky, aromatic residues, phenylalanine or tryptophan, supported efficient prion conversion. We propose a model based on a requirement for tightly interdigitating complementary amino acid side chains within specific domains of adjacent PrP molecules, known as “steric zippers,” to explain these results. Collectively, these studies suggest that an aromatic residue at position 169 supports efficient prion conversion.     

Doxorubicin bypasses the cytoprotective effects of eIF2α phosphorylation and promotes PKR-mediated cell death

The eukaryotic cell responds to various forms of environmental stress by adjusting the rates of mRNA translation thus facilitating adaptation to the assaulting stress. One of the major pathways that control protein synthesis involves the phosphorylation of the α-subunit of eukaryotic initiation factor eIF2 at serine 51. Different forms of DNA damage were shown to induce eIF2α phosphorylation by using PERK, GCN2 or PKR. However, the specificity of the eIF2α kinases and the biological role of eIF2α phosphorylation pathway in the DNA damage response (DDR) induced by chemotherapeutics are not known. Herein, we show that PKR is the eIF2α kinase that responds to DDR induced by doxorubicin. We show that activation of PKR integrates two signaling pathways with opposing biological outcomes. More specifically, induction of eIF2α phosphorylation has a cytoprotective role, whereas activation of c-jun N-terminal kinase (JNK) by PKR promotes cell death in response to doxorubicin. We further show that the proapoptotic effects of JNK activation prevail over the cytoprotection mediated by eIF2α phosphorylation. These findings reveal that PKR can be an important inducer of cell death in response to chemotherapies through its ability to act independently of eIF2α phosphorylation.     

Specific Conserved C-terminal Amino Acids of Caenorhabditis elegans HMP-1/α-Catenin Modulate F-actin Binding Independently of Vinculin

Stable intercellular adhesions formed through the cadherin-catenin complex are important determinants of proper tissue architecture and help maintain tissue integrity during morphogenetic movements in developing embryos. A key regulator of this stability is α-catenin, which connects the cadherin-catenin complex to the actin cytoskeleton. Although the C-terminal F-actin-binding domain of α-catenin has been shown to be crucial for its function, a more detailed in vivo analysis of discrete regions and residues required for actin binding has not been performed. Using Caenorhabditis elegans as a model system, we have characterized mutations in hmp-1/α-catenin that identify HMP-1 residues 687–742 and 826–927, as well as amino acid 802, as critical to the localization of junctional proximal actin during epidermal morphogenesis. We also find that the S823F transition in a hypomorphic allele, hmp-1(fe4), decreases actin binding in vitro. Using hmp-1(fe4) animals in a mutagenesis screen, we were then able to identify 11 intragenic suppressors of hmp-1(fe4) that revert actin binding to wild-type levels. Using homology modeling, we show that these amino acids are positioned at key conserved sites within predicted α-helices in the C terminus. Through the use of transgenic animals, we also demonstrate that HMP-1 residues 315–494, which correspond to a putative mechanotransduction domain that binds vinculin in vertebrate αE-catenin, are not required during epidermal morphogenesis but may aid efficient recruitment of HMP-1 to the junction. Our studies are the first to identify key conserved amino acids in the C terminus of α-catenin that modulate F-actin binding in living embryos of a simple metazoan.     

Structure-Based Analysis of the Ligand-Binding Mechanism for DhelOBP21, a C-minus Odorant Binding Protein,from Dastarcus helophoroides (Fairmaire; Coleoptera: Bothrideridae)

Odorant binding proteins (OBPs) transport hydrophobic odor molecules across the sensillar lymph to trigger a neuronal response. Herein, the Minus-C OBP (DhelOBP21) was characterized from Dastarcus helophoroides, the most important natural parasitic enemy insect that targets Monochamus alternatus. Homology modeling and molecular docking were conducted on the interaction between DhelOBP21 and 17 volatile molecules (including volatiles from pine bark, the larva of M. alternatus, and the faeces of the larva). The predicted three-dimensional structure showed only two disulfide bridges and a hydrophobic binding cavity with a short C-terminus. Ligand-binding experiments using N-phenylnaphthylamine (1-NPN) as a fluorescent probe showed that DhelOBP21 exhibited better binding affinities against those ligands with a molecular volume between 100 and 125 ų compared with ligands with a molecular volume between 160 and 185 ų. Molecules that are too big or too small are not conducive for binding. We mutated the amino acid residues of the binding cavity to increase either hydrophobicity or hydrophilia. Ligand-binding experiments and cyber molecular docking assays indicated that hydrophobic interactions are more significant than hydrogen-bonding interactions. Although hydrogen-bond interactions could be predicted for some binding complexes, the hydrophobic interactions had more influence on binding following hydrophobic changes that affected the cavity. The orientation of ligands affects binding by influencing hydrophobic interactions. The binding process is controlled by multiple factors. This study provides a basis to explore the ligand-binding mechanisms of Minus-C OBP.