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Low- and room-temperature X-ray structures of protein kinase A ternary complexes shed new light on its activity.

ABSTRACT: Post-translational protein phosphorylation by protein kinase A (PKA) is a ubiquitous signalling mechanism which regulates many cellular processes. A low-temperature X-ray structure of the ternary complex of the PKA catalytic subunit (PKAc) with ATP and a 20-residue peptidic inhibitor (IP20) at the physiological Mg(2+) concentration of ?0.5?mM (LT PKA-MgATP-IP20) revealed a single metal ion in the active site. The lack of a second metal in LT PKA-MgATP-IP20 renders the ?- and ?-phosphoryl groups of ATP very flexible, with high thermal B factors. Thus, the second metal is crucial for tight positioning of the terminal phosphoryl group for transfer to a substrate, as demonstrated by comparison of the former structure with that of the LT PKA-Mg(2)ATP-IP20 complex obtained at high Mg(2+) concentration. In addition to its kinase activity, PKAc is also able to slowly catalyze the hydrolysis of ATP using a water molecule as a substrate. It was found that ATP can be readily and completely hydrolyzed to ADP and a free phosphate ion in the crystals of the ternary complex PKA-Mg(2)ATP-IP20 by X-ray irradiation at room temperature. The cleavage of ATP may be aided by X-ray-generated free hydroxyl radicals, a very reactive chemical species, which move rapidly through the crystal at room temperature. The phosphate anion is clearly visible in the electron-density maps; it remains in the active site but slides about 2?Å from its position in ATP towards Ala21 of IP20, which mimics the phosphorylation site. The phosphate thus pushes the peptidic inhibitor away from the product ADP, while resulting in dramatic conformational changes of the terminal residues 24 and 25 of IP20. X-ray structures of PKAc in complex with the nonhydrolysable ATP analogue AMP-PNP at both room and low temperature demonstrated no temperature effects on the conformation and position of IP20.

SUBMITTER: Kovalevsky AY

PROVIDER: S-EPMC3388813 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Low- and room-temperature X-ray structures of protein kinase A ternary complexes shed new light on its activity.

Kovalevsky Andrey Y AY Johnson Hanna H Hanson B Leif BL Waltman Mary Jo MJ Fisher S Zoe SZ Taylor Susan S Langan Paul P

Acta crystallographica. Section D, Biological crystallography 20120615 Pt 7

Post-translational protein phosphorylation by protein kinase A (PKA) is a ubiquitous signalling mechanism which regulates many cellular processes. A low-temperature X-ray structure of the ternary complex of the PKA catalytic subunit (PKAc) with ATP and a 20-residue peptidic inhibitor (IP20) at the physiological Mg(2+) concentration of ∼0.5 mM (LT PKA-MgATP-IP20) revealed a single metal ion in the active site. The lack of a second metal in LT PKA-MgATP-IP20 renders the β- and γ-phosphoryl groups ...[more]

PMID: 22751671

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Project description:Purpose: We aimed to provide a basis for the adaptive mechanism and a rich resource for the discovery and identification of novel genes involved in the cold stress response in Solenopsis japonica. Retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: Solenopsis japonica was reared at lab condition, and incubated at 2 different temperature for 24h (9, and 25℃) under dark conditions. RNA was extracted using Trizol reagent and Illumina sequencing was performed at Macrogen. Illumina short reads were quality-filtered and Illumina-based de novo transcriptome assembly was performed. Differential Gene Expression Analysis was studied for different tempearture conditions. Results: Totally 89,657 unigenes was obtained which overall 43,375 were annotated with gene descriptions, gene ontology terms, and metabolic pathways. 86 GO functional sub-groups and 25 EggNOG terms resulted. DEGs with FC≥2 and FC≤-2, P-value<0.05 were screened and were compared at two different temperatures. We found 138 DEG down regulated and 271 DEG up regulated when the S. japonica incubated at 9 ℃ and compared with 25℃ treated group. Comparing transcriptome profiles for differential gene expression resulted various DE proteins and genes, including cytochrome P450, NADH dehydrogenase subunit 1, cuticle protein and HSP which have previously been reported to be involved in cold and high temperature resistance. GO analysis revealed that antioxidant activity up-regulated under high temperature stress. Conclusions: we compared the transcriptomes of S. japonica under normal room temperature and low-temperature using RNA-Seq technology based on the high-throughput sequencing. Comparative transcriptome analysis identified many genes and a large number of changes were discovered in metabolic pathway through the GO and KEGG enrichment analysis. Our data will facilitate further molecular investigations and genomic research. Many low-temperature significantly up-regulated genes were first identified in this study. These newly found genes may be important and necessary to S. japonica overwintering and its behavior for adaptation in new environment.

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Low- and room-temperature X-ray structures of protein kinase A ternary complexes shed new light on its activity.

Publications

Low- and room-temperature X-ray structures of protein kinase A ternary complexes shed new light on its activity.

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