Identification of two Amino Acids in the C-terminal Domain of Mouse CRY2 Essential for PER2 Interaction

<p>Abstract</p> <p>Background</p> <p>Cryptochromes (CRYs) are a class of flavoprotein blue-light signaling receptors found in plants and animals, and they control plant development and the entrainment of circadian rhythms. They also act as integral parts of the central circadian oscillator in humans and other animals. In mammals, the CLOCK-BMAL1 heterodimer activates transcription of the <it>Per </it>and <it>Cry </it>genes as well as clock-regulated genes. The PER2 proteins interact with CRY and CKIε, and the resulting ternary complexes translocate into the nucleus, where they negatively regulate the transcription of <it>Per </it>and <it>Cry </it>core clock genes and other clock-regulated output genes. Recent studies have indicated that the extended C-termini of the mammalian CRYs, as compared to photolyase proteins, interact with PER proteins.</p> <p>Results</p> <p>We identified a region on mCRY2 (between residues 493 and 512) responsible for direct physical interaction with mPER2 by mammalian two-hybrid and co-immunoprecipitation assays. Moreover, using oligonucleotide-based degenerate PCR, we discovered that mutation of Arg-501 and Lys-503 of mCRY2 within this C-terminal region totally abolishes interaction with PER2.</p> <p>Conclusions</p> <p>Our results identify mCRY2 amino acid residues that interact with the mPER2 binding region and suggest the potential for rational drug design to inhibit CRYs for specific therapeutic approaches.</p

Investigation of the Interaction between the Large and Small Subunits of Potato ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase), a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Current evidence indicates that the two subunit types play distinct roles in enzyme function. Recently the heterotetrameric structure of potato AGPase has been modeled. In the current study, we have applied the molecular mechanics generalized born surface area (MM-GBSA) method and identified critical amino acids of the potato AGPase LS and SS subunits that interact with each other during the native heterotetrameric structure formation. We have further shown the role of the LS amino acids in subunit-subunit interaction by yeast two-hybrid, bacterial complementation assay and native gel. Comparison of the computational results with the experiments has indicated that the backbone energy contribution (rather than the side chain energies) of the interface residues is more important in identifying critical residues. We have found that lateral interaction of the LS-SS is much stronger than the longitudinal one, and it is mainly mediated by hydrophobic interactions. This study will not only enhance our understanding of the interaction between the SS and the LS of AGPase, but will also enable us to engineer proteins to obtain better assembled variants of AGPase which can be used for the improvement of plant yield

Back to the plant: overcoming roadblocks to the microbial production of pharmaceutically important plant natural products

Microbial fermentation platforms offer a cost-effective and sustainable alternative to plant cultivation and chemical synthesis for the production of many plant-derived pharmaceuticals. Plant alkaloids, particularly benzylisoquinoline alkaloids and monoterpene indole alkaloids, and recently cannabinoids have become attractive targets for microbial biosynthesis owing to their medicinal importance. Recent advances in the discovery of pathway components, together with the application of synthetic biology tools, have facilitated the assembly of plant alkaloid and cannabinoid pathways in the microbial hosts Escherichia coli and Saccharomyces cerevisiae. This review highlights key aspects of these pathways in the framework of overcoming bottlenecks in microbial production to further improve end-product titers. We discuss the opportunities that emerge from a better understanding of the pathway components by further study of the plant, and strategies for generation of new and advanced medicinal compounds.</p

ΔG_binding values of important residues in SS.

§<p>Standard error of mean. These residues are reported by Jin et al (19) in our AGPase model. Values are in kcal/mol. Note that interface residues in A and C chains are not listed since these chains are occupied with LSs in our AGPase model. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000546#s2" target="_blank">Results</a> were obtained from the free energy decomposition of LS-SS interaction (D1 and D2 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000546#pcbi-1000546-g001" target="_blank">Fig. 1b</a>).</p

Functional analysis of selected hot-spot residues with comparison to backbone and total ΔG_binding energy values.

<p>Functional analysis of selected hot-spot residues with comparison to backbone and total ΔG_binding energy values.</p

Snapshots of MD simulations from the final structures heterotetrameric.

<p>AGPase. MD analyses indicate the hot-spot residues (A) in D2 and (B) in D5. LS is cyan and SS is yellow in color. Hot-spots are shown in spheres.</p

Free energy decomposition of hot spot residues in Dimer 1 (Values are in kcal/mol).

<p>Free energy decomposition of hot spot residues in Dimer 1 (Values are in kcal/mol).</p

Binding free energy components (kcal/mol) for each of the dimers averaged over the 200 snapshots.

<p>Values in parentheses are standard errors of the means. Explanation for the abbreviations can be found in materials and methods. ΔG_elec corresponds to sum of gas-phase electrostatic energy and polar solvation energy.</p

Oligonucleotide primers used for amplification of the LS cDNA and generation of site-directed mutations.

<p>Underlined and bold nucleotides indicate the nucleotides used to replace the wild-type amino acid. F, forward and R, antisense.</p