Density of an intraguild predator mediates feeding group size, intraguild egg predation, and intra- and interspecific competition

Intraguild predation (IGP) is common in most communities, but many aspects of density-dependent interactions of IG predators with IG prey are poorly resolved. Here, we examine how the density of an IG predator can affect feeding group size, IG egg predation, and the growth responses of IG prey. We used laboratory feeding trials and outdoor mesocosm experiments to study interactions between a social intraguild predator (larvae of the wood frog; Rana sylvatica) and its prey (spotted salamander; Ambystoma maculatum). Larvae of R. sylvatica could potentially affect A. maculatum by consuming shared larval food resources or by consuming eggs and hatchlings. However, successful egg predation requires group feeding by schooling tadpoles. We established from five to 1,190 hatchlings of R. sylvatica in mesocosms, then added either 20 A. maculatum hatchlings to study interspecific competition, or a single egg mass to examine IGP. Crowding strongly suppressed the growth of R. sylvatica, and IGP was restricted to the egg stage. In the larval competition experiment, growth of A. maculatum was inversely proportional to R. sylvatica density. In the predation experiment, embryonic mortality of A. maculatum was directly proportional to the initial density of R. sylvatica and the mean number of tadpoles foraging on egg masses. IGP on eggs reduced A. maculatum hatchling density, which accelerated larval growth. Surprisingly, the density of R. sylvatica had no overall effect on A. maculatum growth because release from intraspecific competition via egg predation was balanced by increased interspecific competition. Our results demonstrate that the density of a social IG predator can strongly influence the nature and intensity of interactions with a second guild member by simultaneously altering the intensity of IGP and intra- and interspecific competition.L . A. Burley and A. T. Moyer contributed equally to this work.     

Engaging the ribosome: universal IFs of translation

Eukaryotic initiation factor 1A (eIF1A) and the GTPase IF2/eIF5B are the only universally conserved translation initiation factors. Recent structural, biochemical and genetic data indicate that these two factors form an evolutionarily conserved structural and functional unit in translation initiation. Based on insights gathered from studies of the translation elongation factor GTPases, we propose that these factors occupy the aminoacyl-tRNA site (A site) on the ribosome, and promote initiator tRNA binding and ribosomal subunit joining. These processes yield a translationally competent ribosome with Met-tRNA in the ribosomal peptidyl-tRNA site (P site), base-paired to the AUG start codon of a mRNA.     

Recognition of the rotavirus mRNA 3' consensus by an asymmetric NSP3 homodimer.

Rotaviruses, the cause of life-threatening diarrhea in humans and cattle, utilize a functional homolog of poly(A) binding protein (PABP) known as nonstructural protein 3 (NSP3) for translation of viral mRNAs. NSP3 binds to viral mRNA 3' consensus sequences and circularizes the mRNA via interactions with eIF4G. The X-ray structure of the NSP3 RNA binding domain bound to a rotaviral mRNA 3' end has been determined. NSP3 is a novel, heart-shaped homodimer with a medial RNA binding cleft. The homodimer is asymmetric, and contains two similar N-terminal segments plus two structurally different C-terminal segments that intertwine to create a tunnel enveloping the mRNA 3' end. Biophysical studies demonstrate high affinity binding leading to increased thermal stability and slow dissociation kinetics, consistent with NSP3 function.     

X-Ray structures of the universal translation initiation factor IF2/eIF5B: conformational changes on GDP and GTP binding

X-ray structures of the universal translation initiation factor IF2/eIF5B have been determined in three states: free enzyme, inactive IF2/eIF5B.GDP, and active IF2/eIF5B.GTP. The "chalice-shaped" enzyme is a GTPase that facilitates ribosomal subunit joining and Met-tRNA(i) binding to ribosomes in all three kingdoms of life. The conserved core of IF2/eIF5B consists of an N-terminal G domain (I) plus an EF-Tu-type beta barrel (II), followed by a novel alpha/beta/alpha-sandwich (III) connected via an alpha helix to a second EF-Tu-type beta barrel (IV). Structural comparisons reveal a molecular lever, which amplifies a modest conformational change in the Switch 2 region of the G domain induced by Mg(2+)/GTP binding over a distance of 90 A from the G domain active center to domain IV. Mechanisms of GTPase function and ribosome binding are discussed.     

HDEA, a periplasmic protein that supports acid resistance in pathogenic enteric bacteria

The X-ray crystal structure of the Escherichia coli stress response protein HDEA has been determined at 2.0 A resolution. The single domain alpha-helical protein is found in the periplasmic space, where it supports an acid resistance phenotype essential for infectivity of enteric bacterial pathogens, such as Shigella and E. coli. Functional studies demonstrate that HDEA is activated by a dimer-to-monomer transition at acidic pH, leading to suppression of aggregation by acid-denatured proteins. We suggest that HDEA may support chaperone-like functions during the extremely acidic conditions.     

Crystal structure of shikimate 5-dehydrogenase (SDH) bound to NADP: insights into function and evolution

The crystal structure of Methanococcus jannaschii shikimate 5-dehydrogenase (MjSDH) bound to the cofactor nicotinamide adenine dinucleotide phosphate (NADP) has been determined at 2.35 A resolution. Shikimate 5-dehydrogenase (SDH) is responsible for NADP-dependent catalysis of the fourth step in shikimate biosynthesis, which is essential for aromatic amino acid metabolism in bacteria, microbial eukaryotes, and plants. The structure of MjSDH is a compact alpha/beta sandwich with two distinct domains, responsible for binding substrate and the NADP cofactor, respectively. A phylogenetically conserved deep cleft on the protein surface corresponds to the enzyme active site. The structure reveals a topologically new domain fold within the N-terminal segment of the polypeptide chain, which binds substrate and supports dimerization. Insights gained from homology modeling and sequence/structure comparisons suggest that the SDHs represent a unique class of dehydrogenases. The structure provides a framework for further investigation to discover and develop novel inhibitors targeting this essential enzyme.     

Inhibition of mandelate racemase by the substrate-intermediate-product analogue 1,1-diphenyl-1-hydroxymethylphosphonate

Mandelate racemase has been studied as a paradigm for enzyme-catalyzed abstraction of a proton from carbon acids with relatively high pKa values. 1,1-Diphenyl-1-hydroxymethylphosphonate is a substrate-intermediate-product analogue and is a modest competitive inhibitor of the enzyme (Ki=1.41+/-0.09 mM), suggesting that simultaneous binding of the two phenyl groups obviates mimicry of the aci-carboxylate function of the intermediate by the phosphonate group.     

High-throughput Limited Proteolysis/Mass Spectrometry for Protein Domain Elucidation

High-resolution structural information is important for improving our understanding of protein function in vitro and in vivo and providing information to enable drug discovery. The process leading to X-ray structure determination is often time consuming and labor intensive. It requires informed decisions in expression construct design, expression host selection, and strategies for protein purification, crystallization and structure determination. Previously published studies have demonstrated that compact globular domains defined by limited proteolysis represent good candidates for production of diffraction quality crystals [1–7]. Integration of mass spectrometry and proteolysis experiments can provide accurate definition of domain boundaries at unprecedented rates. We have conducted a critical evaluation of this approach with 400 target proteins produced by SGX (Structural GenomiX, Inc.) for the New York Structural GenomiX Research Consortium (NYSGXRC; ) under the auspices of the National Institute of General Medical Sciences Protein Structure Initiative (). The objectives of this study were to develop parallel/automated protocols for proteolytic digestion and data acquisition for multiple proteins, and to carry out a systematic study to correlate domain definition via proteolysis with outcomes of crystallization and structure determination attempts. Initial results from this work demonstrate that proteins yielding diffraction quality crystals are typically resistant to proteolysis. Large-scale sub cloning and subsequent testing of expression, solubility, and crystallizability of proteolytically defined truncations is currently underway.     

Structural investigation of the hedamycin:d(ACCGGT)2 complex by NMR and restrained molecular dynamics

Hedamycin, a member of the pluramycin family of drugs, displays a range of biological responses including antitumor and antimicrobial activity. The mechanism of action is via direct interaction with DNA through intercalation between the bases of the oligonucleotide and alkylation of a guanine residue at 5'-PyG-3' sites. There appears to be some minor structural differences between two earlier studies on the interaction of hedamycin with 5'-PyG-3' sites. In this study, a high-resolution NMR analysis of the hedamycin:d(ACCGGT)2 complex was undertaken in order to investigate the effect of replacing the thymine with a guanine at the preferred 5'-CGT-3' site. The resultant structure was compared with earlier work, with particular emphasis placed on the drug conformation. The structure of the hedamycin:d(ACCGGT)2 complex has many features in common with the two previous NMR structures of hedamycin:DNA complexes but differed in the conformation and orientation of the N,N-dimethylvancosamine saccharide of hedamycin in one of these structures. The preferential binding of hedamycin to 5'-CG-3' over 5'-TG-3' binding sites is explained in terms of the orientation and location of the N,N-dimethylvancosamine saccharide in the minor groove.