Crystal structure of Bacillus stearothermophilus UvrA provides insight into ATP-modulated dimerization, UvrB interaction, and DNA binding

The nucleotide excision repair pathway corrects many structurally unrelated DNA lesions. Damage recognition in bacteria is performed by UvrA, a member of the ABC ATPase superfamily whose functional form is a dimer with four nucleotide-binding domains (NBDs), two per protomer. In the 3.2 A structure of UvrA from Bacillus stearothermophilus, we observe that the nucleotide-binding sites are formed in an intramolecular fashion and are not at the dimer interface as is typically found in other ABC ATPases. UvrA also harbors two unique domains; we show that one of these is required for interaction with UvrB, its partner in lesion recognition. In addition, UvrA contains three zinc modules, the number and ligand sphere of which differ from previously published models. Structural analysis, biochemical experiments, surface electrostatics, and sequence conservation form the basis for models of ATP-modulated dimerization, UvrA-UvrB interaction, and DNA binding during the search for lesions.     

Crystal structures of creatininase reveal the substrate binding site and provide an insight into the catalytic mechanism

Creatininase from Pseudomonas putida is a member of the urease-related amidohydrolase superfamily. The crystal structure of the Mn-activated enzyme has been solved by the single isomorphous replacement method at 1.8A resolution. The structures of the native creatininase and the Mn-activated creatininase-creatine complex have been determined by a difference Fourier method at 1.85 A and 1.6 A resolution, respectively. We found the disc-shaped hexamer to be roughly 100 A in diameter and 50 A in thickness and arranged as a trimer of dimers with 32 (D3) point group symmetry. The enzyme is a typical Zn2+ enzyme with a binuclear metal center (metal1 and metal2). Atomic absorption spectrometry and X-ray crystallography revealed that Zn2+ at metal1 (Zn1) was easily replaced with Mn2+ (Mn1). In the case of the Mn-activated enzyme, metal1 (Mn1) has a square-pyramidal geometry bound to three protein ligands of Glu34, Asp45, and His120 and two water molecules. Metal2 (Zn2) has a well-ordered tetrahedral geometry bound to the three protein ligands of His36, Asp45, and Glu183 and a water molecule. The crystal structure of the Mn-activated creatininase-creatine complex, which is the first structure as the enzyme-substrate/inhibitor complex of creatininase, reveals that significant conformation changes occur at the flap (between the alpha5 helix and the alpha6 helix) of the active site and the creatine is accommodated in a hydrophobic pocket consisting of Trp174, Trp154, Tyr121, Phe182, Tyr153, and Gly119. The high-resolution crystal structure of the creatininase-creatine complex enables us to identify two water molecules (Wat1 and Wat2) that are possibly essential for the catalytic mechanism of the enzyme. The structure and proposed catalytic mechanism of the creatininase are different from those of urease-related amidohydrolase superfamily enzymes. We propose a new two-step catalytic mechanism possibly common to creatininases in which the Wat1 acts as the attacking nucleophile in the water-adding step and the Wat2 acts as the catalytic acid in the ring-opening step.     

Solution structure of DinI provides insight into its mode of RecA inactivation

The Escherichia coli RecA protein triggers both DNA repair and mutagenesis in a process known as the SOS response. The 81-residue E. coli protein DinI inhibits activity of RecA in vivo. The solution structure of DinI has been determined by multidimensional triple resonance NMR spectroscopy, using restraints derived from two sets of residual dipolar couplings, obtained in bicelle and phage media, supplemented with J couplings and a moderate number of NOE restraints. DinI has an alpha/beta fold comprised of a three-stranded beta-sheet and two alpha-helices. The beta-sheet topology is unusual: the central strand is flanked by a parallel and an antiparallel strand and the sheet is remarkably flat. The structure of DinI shows that six negatively charged Glu and Asp residues on DinI's kinked C-terminal alpha-helix form an extended, negatively charged ridge. We propose that this ridge mimics the electrostatic character of the DNA phospodiester backbone, thereby enabling DinI to compete with single-stranded DNA for RecA binding. Biochemical data confirm that DinI is able to displace ssDNA from RecA.     

Structure and mutational analysis of the PhoN protein of Salmonella typhimurium provide insight into mechanistic details

The Salmonella typhimurium PhoN protein is a nonspecific acid phosphatase and belongs to the phosphatidic acid phosphatase type 2 (PAP2) superfamily. We report here the crystal structures of phosphate-bound PhoN, the PhoN-tungstate complex, and the T159D mutant of PhoN along with functional characterization of three mutants: L39T, T159D, and D201N. Invariant active site residues, Lys-123, Arg-130, Ser-156, Gly-157, His-158, and Arg-191, interact with phosphate and tungstate oxyanions. Ser-156 also accepts a hydrogen bond from Thr-159. The T159D mutation, surprisingly, severely diminishes phosphatase activity, apparently by disturbing the active site scaffold: Arg-191 is swung out of the active site resulting in conformational changes in His-158 and His-197 residues. Our results reveal a hitherto unknown functional role of Arg-191, namely, restricting the active conformation of catalytic His-158 and His-197 residues. Consistent with the conserved nature of Asp-201 in the PAP2 superfamily, the D201N mutation completely abolished phosphatase activity. On the basis of this observation and in silico analysis we suggest that the crucial mechanistic role of Asp-201 is to stabilize the positive charge on the phosphohistidine intermediate generated by the transfer of phosphoryl to the nucleophile, His-197, located within hydrogen bond distance to the invariant Asp-201. This is in contrast to earlier suggestions that Asp-201 stabilizes His-197 and the His197-Asp201 dyad facilitates formation of the phosphoenzyme intermediate through a charge-relay system. Finally, the L39T mutation in the conserved polyproline motif (39LPPPP43) of dimeric PhoN leads to a marginal reduction in activity, in contrast to the nearly 50-fold reduction observed for monomeric Prevotella intermedia acid phosphatase, suggesting that the varying quaternary structure of PhoN orthologues may have functional significance.     

DNA barcode libraries provide insight into continental patterns of avian diversification

Background

The causes for the higher biodiversity in the Neotropics as compared to the Nearctic and the factors promoting species diversification in each region have been much debated. The refuge hypothesis posits that high tropical diversity reflects high speciation rates during the Pleistocene, but this conclusion has been challenged. The present study investigates this matter by examining continental patterns of avian diversification through the analysis of large-scale DNA barcode libraries.

Methodology and Principal Findings

Standardized COI datasets from the avifaunas of Argentina, the Nearctic, and the Palearctic were analyzed. Average genetic distances between closest congeners and sister species were higher in Argentina than in North America reflecting a much higher percentage of recently diverged species in the latter region. In the Palearctic genetic distances between closely related species appeared to be more similar to those of the southern Neotropics. Average intraspecific variation was similar in Argentina and North America, while the Palearctic fauna had a higher value due to a higher percentage of variable species. Geographic patterning of intraspecific structure was more complex in the southern Neotropics than in the Nearctic, while the Palearctic showed an intermediate level of complexity.

Conclusions and Significance

DNA barcodes can reveal continental patterns of diversification. Our analysis suggests that avian species are older in Argentina than in the Nearctic, supporting the idea that the greater diversity of the Neotropical avifauna is not caused by higher recent speciation rates. Species in the Palearctic also appear to be older than those in the Nearctic. These results, combined with the patterns of geographic structuring found in each region, suggest a major impact of Pleistocene glaciations in the Nearctic, a lesser effect in the Palearctic and a mild effect in the southern Neotropics.     

Strandings provide insight into social group structure of Atlantic white-sided dolphins

Atlantic white-sided dolphins (Lagenorhynchus acutus) are highly social odontocetes with a poorly understood tendency to mass strand. With limited capacity to study social ecology in the open ocean, mass strandings provide an opportunity to improve our understanding of group structure. Our study of 32 mass stranding events that occurred on Cape Cod, Massachusetts, between 1999 and 2009 identifies aspects of social ecology that vary across the year. A greater number of mass stranding events occurred outside of the breeding season and there was evidence of age-structuring during the breeding season. We find generally low average intragroup pairwise relatedness assessed across eight microsatellite loci in a subset of 16 mass stranding events. Mass stranded groups do not show higher than expected relatedness when compared to a baseline estimate derived from single-stranded individuals. Overall, our integration of genetic estimates of relatedness with data on sex and maturity-class from stranded specimens suggests that Atlantic white-sided dolphins fall near the more fluid end of the continuum from short-term, highly fluid social associations to long-term, stable groups represented among the odontocetes. Despite their tendency to mass strand, stable, kin-based associations are not a defining feature of social group structure in this species.     

The first structure of dipeptidyl-peptidase III provides insight into the catalytic mechanism and mode of substrate binding

Dipeptidyl-peptidases III (DPP III) are zinc-dependent enzymes that specifically cleave the first two amino acids from the N terminus of different length peptides. In mammals, DPP III is associated with important physiological functions and is a potential biomarker for certain types of cancer. Here, we present the 1.95-A crystal structure of yeast DPP III representing the prototype for the M49 family of metallopeptidases. It shows a novel fold with two domains forming a wide cleft containing the catalytic metal ion. DPP III exhibits no overall similarity to other metallopeptidases, such as thermolysin and neprilysin, but zinc coordination and catalytically important residues are structurally conserved. Substrate recognition is accomplished by a binding site for the N terminus of the peptide at an appropriate distance from the metal center and by a series of conserved arginine residues anchoring the C termini of different length substrates.     

Substitutions of Thr30 provide mechanistic insight into tryptophan-mediated activation of TRAP binding to RNA

TRAP is an 11 subunit RNA binding protein that regulates expression of genes involved in tryptophan biosynthesis and transport in Bacillus subtilis. TRAP is activated to bind RNA by binding up to 11 molecules of l-tryptophan in pockets formed by adjacent subunits. The precise mechanism by which tryptophan binding activates TRAP is not known. Thr30 is in the tryptophan binding pocket. A TRAP mutant in which Thr30 is substituted with Val (T30V) does not bind tryptophan but binds RNA constitutively, suggesting that Thr30 plays a key role in the activation mechanism. We have examined the effects of other substitutions of Thr30. TRAP proteins with small beta-branched aliphatic side chains at residue 30 bind RNA constitutively, whereas those with a small polar side chain show tryptophan-dependent RNA binding. Several mutant proteins exhibited constitutive RNA binding that was enhanced by tryptophan. Although the tryptophan and RNA binding sites on TRAP are distinct and are separated by approximately 7.5 A, several substitutions of residues that interact with the bound RNA restored tryptophan binding to T30V TRAP. These observations support the hypothesis that conformational changes in TRAP relay information between the tryptophan and RNA binding sites of the protein.     

An insight into the structure of DNA through melting studies

Helix-coil transition of calf thymus (CT) DNA in 0.1 M NaCl was observed under different environmental conditions for studying structural and conformational changes, if any. Pre-exposure of the DNA to different types of radiations - UV and gamma-brought different degrees of change (lowering) in the melting temperature (Tm). Interaction of drugs (daunomycin and actinomycin-D) increased the Tm inducing structural stability. There was 50% more destabilisation in case of polynucleotide-drug complex as compared to DNA-drug complex exposed to UV radiation. Thermodynamic studies on DNA in D2O (0.1 M NaCl prepared in D2O) were carried out and compared with the corresponding results in H2O. Presence of D2O increased the stability of the DNA structure. Structural and conformational aspects have been discussed in the light of the results obtained.     

Symbiosis between hydra and chlorella: Molecular phylogenetic analysis and experimental study provide insight into its origin and evolution

Although many physiological studies have been reported on the symbiosis between hydra and green algae, very little information from a molecular phylogenetic aspect of symbiosis is available. In order to understand the origin and evolution of symbiosis between the two organisms, we compared the phylogenetic relationships among symbiotic green algae with the phylogenetic relationships among host hydra strains. To do so, we reconstructed molecular phylogenetic trees of several strains of symbiotic chlorella harbored in the endodermal epithelial cells of viridissima group hydra strains and investigated their congruence with the molecular phylogenetic trees of the host hydra strains. To examine the species specificity between the host and the symbiont with respect to the genetic distance, we also tried to introduce chlorella strains into two aposymbiotic strains of viridissima group hydra in which symbiotic chlorella had been eliminated in advance. We discussed the origin and history of symbiosis between hydra and green algae based on the analysis.     

Global and local molecular dynamics of a bacterial carboxylesterase provide insight into its catalytic mechanism

Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of xenobiotics. In humans, substrates for these enzymes are far-ranging, and include the street drug heroin and the anticancer agent irinotecan. Hence, their ability to bind and metabolize substrates is of broad interest to biomedical science. In this study, we focused our attention on dynamic motions of a CE from B. subtilis (pnbCE), with emphasis on the question of what individual domains of the enzyme might contribute to its catalytic activity. We used a 10 ns all-atom molecular dynamics simulation, normal mode calculations, and enzyme kinetics to understand catalytic consequences of structural changes within this enzyme. Our results shed light on how molecular motions are coupled with catalysis. During molecular dynamics, we observed a distinct C-C bond rotation between two conformations of Glu310. Such a bond rotation would alternately facilitate and impede protonation of the active site His399 and act as a mechanism by which the enzyme alternates between its active and inactive conformation. Our normal mode results demonstrate that the distinct low-frequency motions of two loops in pnbCE, coil_5 and coil_21, are important in substrate conversion and seal the active site. Mutant CEs lacking these external loops show significantly reduced rates of substrate conversion, suggesting this sealing motion prevents escape of substrate. Overall, the results of our studies give new insight into the structure-function relationship of CEs and have implications for the entire family of α/β fold family of hydrolases, of which this CE is a member.     

The solution structure and dynamics of human pancreatic ribonuclease determined by NMR spectroscopy provide insight into its remarkable biological activities and inhibition

Human pancreatic ribonuclease (RNase 1) is expressed in many tissues; has several important enzymatic and biological activities, including efficient cleavage of single-stranded RNA, double-stranded RNA and double-stranded RNA-DNA hybrids, digestion of dietary RNA, regulation of vascular homeostasis, inactivation of the HIV, activation of immature dendritic cells and induction of cytokine production; and furthermore shows potential as an anti-tumor agent. The solution structure and dynamics of uncomplexed, wild-type RNase 1 have been determined by NMR spectroscopy methods to better understand these activities. The family of 20 structures determined on the basis of 6115 unambiguous nuclear Overhauser enhancements is well resolved (pairwise backbone RMSD = 1.07 Å) and has the classic RNase A type of tertiary structure. Important structural differences compared with previously determined crystal structures of RNase 1 variants or inhibitor-bound complexes are observed in the conformation of loop regions and side chains implicated in the enzymatic as well as biological activities and binding to the cytoplasmic RNase inhibitor. Multiple side chain conformations observed for key surface residues are proposed to be crucial for membrane binding as well as translocation and efficient RNA hydrolysis. ¹⁵N-¹H relaxation measurements interpreted with the standard and our extended Lipari-Szabo formalism reveal rigid regions and identify more dynamic loop regions. Some of the most dynamic areas are key for binding to the cytoplasmic RNase inhibitor. This finding and the important differences observed between the structure in solution and that bound to the inhibitor are indications that RNase 1 to inhibitor binding can be better described by the “induced fit” model rather than the rigid “lock-into-key” mechanism. Translational diffusion measurements reveal that RNase 1 is predominantly dimeric above 1 mM concentration; the possible implications of this dimeric state for the remarkable biological properties of RNase 1 are discussed.     

Transcriptome and DNA methylome analyses provide insight into the heterosis in flag leaf of inter-subspecific hybrid rice

Plant Molecular Biology - Flag leaf heterosis of inter-subspecific hybrid rice is suggested to be related to leaf area, gene expression pattern and allele-specific expression, putatively related to...