The TIM Barrel Architecture Facilitated the Early Evolution of Protein-Mediated Metabolism.

The triosephosphate isomerase (TIM) barrel protein fold is a structurally repetitive architecture that is present in approximately 10% of all enzymes. It is generally assumed that this ubiquity in modern proteomes reflects an essential historical role in early protein-mediated metabolism. Here, we provide quantitative and comparative analyses to support several hypotheses about the early importance of the TIM barrel architecture. An information theoretical analysis of protein structures supports the hypothesis that the TIM barrel architecture could arise more easily by duplication and recombination compared to other mixed α/ß structures. We show that TIM barrel enzymes corresponding to the most taxonomically broad superfamilies also have the broadest range of functions, often aided by metal and nucleotide-derived cofactors that are thought to reflect an earlier stage of metabolic evolution. By comparison to other putatively ancient protein architectures, we find that the functional diversity of TIM barrel proteins cannot be explained simply by their antiquity. Instead, the breadth of TIM barrel functions can be explained, in part, by the incorporation of a broad range of cofactors, a trend that does not appear to be shared by proteins in general. These results support the hypothesis that the simple and functionally general TIM barrel architecture may have arisen early in the evolution of protein biosynthesis and provided an ideal scaffold to facilitate the metabolic transition from ribozymes, peptides, and geochemical catalysts to modern protein enzymes.

Properties of ecdysteroid receptors from diverse insect species in a heterologous cell culture system--a basis for screening novel insecticidal candidates.

Insect development is driven by the action of ecdysteroids on morphogenetic processes. The classic ecdysteroid receptor is a protein heterodimer composed of two nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle (USP), the insect ortholog of retinoid X receptor. The functional properties of EcR and USP vary among insect species, and provide a basis for identifying novel and species-specific insecticidal candidates that disrupt this receptor's normal activity. A heterologous mammalian cell culture assay was used to assess the transcriptional activity of the heterodimeric ecdysteroid receptor from species representing two major insect orders: the fruit fly, Drosophila melanogaster (Diptera), and the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera). Several nonsteroidal agonists evoked a strong response with the L. decemlineata heterodimer that was consistent with biochemical and in vivo evidence, whereas the D. melanogaster receptor's response was comparatively modest. Conversely, the phytoecdysteroid muristerone A was more potent with the D. melanogaster heterodimer. The additional presence of juvenile hormone III potentiated the inductive activity of muristerone A in the receptors from both species, but juvenile hormone III was unable to potentiate the inductive activity of the diacylhydrazine methoxyfenozide (RH2485) in the receptor of either species. The effects of USP on ecdysteroid-regulated transcriptional activity also varied between the two species. When it was tested with D. melanogaster EcR isoforms, basal activity was lower and ligand-dependent activity was higher with L. decemlineata USP than with D. melanogaster USP. Generally, the species-based differences validate the use of the cell culture assay screen for novel agonists and potentiators as species-targeted insecticidal candidates.

Functional studies on the ligand-binding domain of Ultraspiracle from Drosophila melanogaster.

The functional insect ecdysteroid receptor is comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP). The ligand-binding domain (LBD) of USP was fused to the GAL4 DNA-binding domain (GAL4-DBD) and characterized by analyzing the effect of site-directed mutations in the LBD. Normal and mutant proteins were tested for ligand and DNA binding, dimerization, and their ability to induce gene expression. The presence of helix 12 proved to be essential for DNA binding and was necessary to confer efficient ecdysteroid binding to the heterodimer with the EcR (LBD), but did not influence dimerization. The antagonistic position of helix 12 is indispensible for interaction between the fusion protein and DNA, whereas hormone binding to the EcR (LBD) was only partially reduced if fixation of helix 12 was disturbed. The mutation of amino acids, which presumably bind to a fatty acid evoked a profound negative influence on transactivation ability, although enhanced transactivation potency and ligand binding to the ecdysteroid receptor was impaired to varying degrees by mutation of these residues. Mutations of one fatty acid-binding residue within the ligand-binding pocket, 1323, however, evoked enhanced transactivation. The results confirmed that the LBD of Ultraspiracle modifies ecdysteroid receptor function through intermolecular interactions and demonstrated that the ligand-binding pocket of USP modifies the DNA-binding and transactivation abilities of the fusion protein.