A comparative structural analysis of the ADF/cofilin family

Actin-depolymerizing factor (ADF) and cofilin define a family of actin-binding proteins essential for the rapid turnover of filamentous actin in vivo. Here we present the 2.0 A crystal structure of Arabidopsis thaliana ADF1 (AtADF1), the first plant crystal structure from the ADF/cofilin (AC) family. Superposition of the four AC isoform structures permits an accurate sequence alignment that differs from previously reported data for the location of vertebrate-specific inserts and reveals a contiguous, vertebrate-specific surface opposite the putative actin-binding surface. Extending the structure-based sequence alignment to include 30 additional isoforms indicates three major groups: vertebrates, plants, and "other eukaryotes." Within these groups, several structurally conserved residues that are not conserved throughout the entire AC family have been identified. Residues that are highly conserved among all isoforms tend to cluster around the tryptophan at position 90 and a structurally conserved kink in alpha-helix 3. Analysis of surface character shows the presence of a hydrophobic patch and a highly conserved acidic cluster, both of which include several residues previously implicated in actin binding.     

Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase

Chalcone synthase (CHS) catalyzes formation of the phenylpropanoid chalcone from one p-coumaroyl-CoA and three malonyl-coenzyme A (CoA) thioesters. The three-dimensional structure of CHS [Ferrer, J.-L., Jez, J. M., Bowman, M. E., Dixon, R. A., and Noel, J. P. (1999) Nat. Struct. Biol. 6, 775-784] suggests that four residues (Cys164, Phe215, His303, and Asn336) participate in the multiple decarboxylation and condensation reactions catalyzed by this enzyme. Here, we functionally characterize 16 point mutants of these residues for chalcone production, malonyl-CoA decarboxylation, and the ability to bind CoA and acetyl-CoA. Our results confirm Cys164's role as the active-site nucleophile in polyketide formation and elucidate the importance of His303 and Asn336 in the malonyl-CoA decarboxylation reaction. We suggest that Phe215 may help orient substrates at the active site during elongation of the polyketide intermediate. To better understand the structure-function relationships in some of these mutants, we also determined the crystal structures of the CHS C164A, H303Q, and N336A mutants refined to 1.69, 2.0, and 2.15 A resolution, respectively. The structure of the C164A mutant reveals that the proposed oxyanion hole formed by His303 and Asn336 remains undisturbed, allowing this mutant to catalyze malonyl-CoA decarboxylation without chalcone formation. The structures of the H303Q and N336A mutants support the importance of His303 and Asn336 in polarizing the thioester carbonyl of malonyl-CoA during the decarboxylation reaction. In addition, both of these residues may also participate in stabilizing the tetrahedral transition state during polyketide elongation. Conservation of the catalytic functions of the active-site residues may occur across a wide variety of condensing enzymes, including other polyketide and fatty acid synthases.     

Crystal structure of the oligomerization domain of NSP4 from rotavirus reveals a core metal-binding site

During the maturation of rotaviral particles, non-structural protein 4 (NSP4) plays a critical role in the translocation of the immature capsid into the lumen of the endoplasmic reticulum. Full-length NSP4 and a 22 amino acid peptide (NSP4(114-135)) derived from this protein have been shown to induce diarrhea in young mice in an age-dependent manner, and may therefore be the agent responsible for rotavirally-induced symptoms. We have determined the crystal structure of the oligomerization domain of NSP4 which spans residues 95 to 137 (NSP4(95-137)). NSP4(95-137) self-associates into a parallel, tetrameric coiled-coil, with the hydrophobic core interrupted by three polar layers occupying a and d-heptad positions. Side-chains from two consecutive polar layers, consisting of four Gln123 and two of the four Glu120 residues, coordinate a divalent cation. Two independent structures built from MAD-phased data indicated the presence of a strontium and calcium ion bound at this site, respectively. This metal-binding site appears to play an important role in stabilizing the homo-tetramer, which has implications for the engagement of NSP4 as an enterotoxin.     

Identification of ribonuclease P activity from chick embryos

RNAase P (EC 3.1.26.5) activity has been identified in chick embryo thigh tissue on the basis of specific cleavage of Escherichia coli 129 nucleotide tRNATyr precursor and has been partially purified by the procedure used for human tissue culture KB cell RNAase P. RNAase P from chick resembles the KB cell RNAase P in substrate specificity, requirement for a divalent cation (Mg2+) and a monovalent cation (K+, Na+ or NH4+) for activity, inhibition by bulk tRNA, ready inactivation by proteases, and increasing instability; with purification. RNAase P activity is also present in whole chick embryos, as well as in liver and heart tissues. Furthermore, crude preparations of RNAase P from chick embryo heart tissue are relatively free of contaminating nucleases.     

Fine‐scale habitat selection by sympatric Canada lynx and bobcat

The Canada lynx (Lynx canadensis) and the bobcat (Lynx rufus) are closely related species with overlap at their range peripheries, but the factors that limit each species and the interactions between them are not well understood. Habitat selection is a hierarchical process, in which selection at higher orders (geographic range, home range) may constrain selection at lower orders (within the home range). Habitat selection at a very fine scale within the home range has been less studied for both lynx and bobcat compared to selection at broader spatiotemporal scales. To compare this fourth‐order habitat selection by the two species in an area of sympatry, we tracked lynx and bobcat during the winters of 2017 and 2018 on the north shore of Lake Huron, Ontario. We found that both lynx and bobcat selected shallower snow, higher snowshoe hare abundance, and higher amounts of coniferous forest at the fourth order. However, the two species were spatially segregated at the second order, and lynx were found in areas with deeper snow, more snowshoe hare, and more coniferous forest. Taken together, our findings demonstrate that the lynx and bobcat select different resources at the second order, assorting along an environmental gradient in the study area, and that competition is unlikely to be occurring between the two species at finer scales.     

Purification and characterization of the Tetrahymena pyriformis P-C bond forming enzyme phosphoenolpyruvate phosphomutase

In this paper the purification and characterization of the Tetrahymena pyriformis enzyme phosphoenolpyruvate phosphomutase are described. PEP phosphomutase was first fractionated from T. pyriformis cellular extract by using 70% ammonium sulfate. Chromatography of the crude protein fraction on a DEAE-cellulose column followed by phenyl-Sepharose column chromatography and then Bio-Gel P-200 column chromatography afforded pure PEP phosphomutase in an approximate overall yield of 70 units/150 g of cells. The maximum turnover number observed for PEP phosphomutase catalysis of the phosphonopyruvate----PEP reaction is 38 s-1 (25 degrees C). The enzyme was shown to be a homodimer of 38,000-dalton subunits and to require a divalent metal ion for activity. Mg2+ (relative Vm = 1), Co2+ (rel Vm = 0.5), Zn2+ (rel Vm = 0.4), and Mn2+ (rel Vm = 0.3) each satisfied the cofactor requirement. Binding of the physiological cofactor, Mg2+ (Ki = 0.3 mM at pH 7.5), and phosphonopyruvate (Km = 2 microM at pH 7.5) was found to be ordered, with cofactor binding preceding substrate binding. Within the pH range of 5-9 catalysis (Vm) was found to be pH independent, while phosphonopyruvate binding dropped at acidic and basic pH.     

A slide down a slippery slope – alpine ecosystem responses to nitrogen deposition

Background: Nitrogen (N) deposition in the Front Range of the southern Rocky Mountains has been increasing for several decades, and has exceeded the critical load for several ecological metrics.

Aims: Our objective was to predict potential future ecological changes in alpine zones in response to anthropogenic N deposition based on a review of research from Niwot Ridge, Colorado.

Results: Empirical observations and experimental studies indicate that plant, algal and soil microbe species compositions are changing in response to N deposition, with nitrophilic species increasing in abundance. Biotic sequestration of N deposition is insufficient to compensate for greater nitrate production, leading to the potential for acidification and base cation loss.

Conclusions: Changes in biotic composition in both terrestrial and aquatic ecosystems have important impacts on ecosystem functioning, including a lower capacity to take up and neutralise the acidifying effect of anthropogenic N, increasing phosphorus limitation of production in terrestrial and aquatic systems, and shifts in rates of N and carbon cycling. Continued elevated N deposition rates coupled with ongoing climate change, including warmer summer temperatures and lower snow cover of shorter duration, will influence the ecological thresholds for biotic and functional changes. We suggest that these thresholds will occur at lower inputs of N deposition under future climate change, meriting reconsideration of current N critical loads to protect sensitive alpine ecosystems.