DataSheet1_Co-crystalization reveals the interaction between AtYchF1 and ppGpp.docx

AtYchF1 is an unconventional G-protein in Arabidopsis thaliana that exhibits relaxed nucleotide-binding specificity. The bindings between AtYchF1 and biomolecules including GTP, ATP, and 26S rRNA have been reported. In this study, we demonstrated the binding of AtYchF1 to ppGpp in addition to the above molecules. AtYchF1 is a cytosolic protein previously reported as a negative regulator of both biotic and abiotic stresses while the accumulation of ppGpp in the cytoplasm induces retarded plant growth and development. By co-crystallization, in vitro pull-down experiments, and hydrolytic biochemical assays, we demonstrated the binding and hydrolysis of ppGpp by AtYchF1. ppGpp inhibits the binding of AtYchF1 to ATP, GTP, and 26S rRNA. The ppGpp hydrolyzing activity of AtYchF1 failed to be activated by AtGAP1. The AtYchF1-ppGpp co-crystal structure suggests that ppGpp might prevent His136 from executing nucleotide hydrolysis. In addition, upon the binding of ppGpp, the conformation between the TGS and helical domains of AtYchF1 changes. Such structural changes probably influence the binding between AtYchF1 and other molecules such as 26S rRNA. Since YchF proteins are conserved among different kingdoms of life, the findings advance the knowledge on the role of AtYchF1 in regulating nucleotide signaling as well as hint at the possible involvement of YchF proteins in regulating ppGpp level in other species.</p

Zwitterion-Immobilized Imprinted Polymers for Promoting the Crystallization of Proteins

Zwitterion additives have been used in protein crystallization to prevent the appearance of crystal clusters. Herein, we have developed a novel approach for the immobilization of zwitterion onto molecularly imprinted polymers (MIPs) to yield high-quality single protein crystals. For lysozyme, trypsin, catalase, proteinase K, concanavalin A-type IV, and thaumatin, simply adding the selected zwitterion (3-(methacryloylamino)­propyl)-dimethyl­(3-sulfopropyl) ammonium hydroxide) into the free solution, the crystallization was improved. When further using the zwitterion-immobilized molecularly imprinted polymers (ziMIPs) developed in the current study, the formation of higher quality crystals was facilitated in a shorter time compared with regular MIPs and traditional crystallization trials. Most notably, concanavalin A-type IV, which has nonunique ordered assembly, gave only the form IV structure with higher resolution in the presence of ziMIPs, justifying the superior function of ziMIPs for the ordered assembly of protein molecules. Thus, the ziMIPs could be widely used in protein crystallization

Structural characterizations and bioactivity of PYL9-(+)-ABA.

<p>(<b>A</b>) Two protomers of PYL9-(+)-ABA in each asymmetric unit. 2<i>F_o</i>−<i>F_c</i> electron density map of (+)-ABA at 1.0σ. (<b>B</b>) One protomer of PYL9-(+)-ABA with five cysteine residues (green) and a disulphide formed between C34 and C159. (<b>C</b>) The monomeric state of PYL9 in solution was confirmed by the sedimentation velocity. The sample purity for sedimentation velocity experiments was detected by SDS-PAGE and then Coomassie Brilliant Blue staining in the subplot. (<b>D</b>) The C159 was the key residue in disulphide bond, while the C29 competed with the C34 to form the disulphide bond. PYL9 and its mutants were under different oxidation-reduction conditions for SDS-PAGE. 1% βme or 1% βme +100 mM DTT was used as reducing agents to disrupt the disulphide bond. Only C159S mutant was not affected by the reducing agents (black arrow).</p

Characterizations of PYL5 and PYL3-(−)-ABA.

<p>(A). Chain A together with L2 of chain B and L4 of chain C displayed a whole protomer of apo-PYL5 (also seen <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067477#pone.0067477.s004" target="_blank">Fig. S4C</a>). (<b>B</b>) Four PYL3-<b>(−)</b>-ABA molecules in an asymmetric unit, organized as two trans-dimers. (<b>C</b>) Close-up view of <b>(−)</b>-ABA (yellow) in the binding pocket. 2<i>F_o</i> − <i>F_c</i> electron density map of <b>(−)</b>-ABA at 1.0σ. (<b>D</b>) Superposition of PYL3-<b>(−)</b>-ABA and PYL3-(+)-ABA (PDB: 4DSC). There were partially rotation and shift between the rings in both ABA. <b>(−)</b>-ABA was constrained in a hydrophobic cavity with little flexibility in PYL3. (<b>E</b>) The binding affinity of <b>(−)</b>-ABA to PYL3, assessed by ITC assays, was less than that of (+)-ABA or (±)-ABA, (also seen <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067477#pone.0067477.s005" target="_blank">Fig. S5D,E</a>). (<b>F</b>) PP2C activity (upper panel) and GST-mediated pulldown of PYL3 mutants protein in the presence of <b>(−)</b>-ABA (lower panel). GST-HAB1 and PYL3, highlighted by red arrows, were visualized by Coomassie Brilliant Blue staining.</p