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      Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides: Effect of Phosphorylation

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          To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T 9TKE 12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K d = 25±6 nM to K d = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (K d = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν as(P-O) and ν s(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν as(UO 2) 2+ vibration (from 923 cm −1 to 908 cm −1) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH.

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          Most cited references 39

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          One-thousand-and-one substrates of protein kinase CK2?

          CK2 (formerly termed "casein kinase 2") is a ubiquitous, highly pleiotropic and constitutively active Ser/Thr protein kinase whose implication in neoplasia, cell survival, and virus infection is supported by an increasing number of arguments. Here an updated inventory of 307 CK2 protein substrates is presented. More than one-third of these are implicated in gene expression and protein synthesis as being either transcriptional factors (60) or effectors of DNA/RNA structure (50) or translational elements. Also numerous are signaling proteins and proteins of viral origin or essential to virus life cycle. In comparison, only a minority of CK2 targets (a dozen or so) are classical metabolic enzymes. An analysis of 308 sites phosphorylated by CK2 highlights the paramount relevance of negatively charged side chains that are (by far) predominant over any other residues at positions n+3 (the most crucial one), n+1, and n+2. Based on this signature, it is predictable that proteins phosphorylated by CK2 are much more numerous than those identified to date, and it is possible that CK2 alone contributes to the generation of the eukaryotic phosphoproteome more so than any other individual protein kinase. The possibility that CK2 phosphosites play some global role, e.g., by destabilizing alpha helices, counteracting caspase cleavage, and generating adhesive motifs, will be discussed.
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            Calmodulin: a prototypical calcium sensor.

            Calmodulin is the best studied and prototypical example of the E-F-hand family of Ca2+-sensing proteins. Changes in intracellular Ca2+ concentration regulate calmodulin in three distinct ways. First, at the cellular level, by directing its subcellular distribution. Second, at the molecular level, by promoting different modes of association with many target proteins. Third, by directing a variety of conformational states in calmodulin that result in target-specific activation. The calmodulin-dependent regulation of protein kinases illustrates the potential mechanisms by which Ca2+-sensing proteins can recognize and generate affinity and specificity for effectors in a Ca2+-dependent manner.
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              Protein kinase CK2: a challenge to canons.

               Lorenzo Pinna (2002)
              CK2 is an extremely conserved pleiotropic protein kinase with a growing list of more than 300 substrates, the majority of which are proteins implicated in signal transduction, gene expression and other nuclear functions. The CK2 phosphoacceptor sites are specified by multiple acidic residues, with the one at position +3 relative to the target residue being of crucial relevance. The CK2 holoenzyme is composed of two catalytic subunits (alphaalpha, alpha'alpha' or alphaalpha'), which are essential for cell viability, and a dimer of two non-catalytic beta subunits, whose precise function is still poorly understood. Although the beta subunits deeply affect many properties of CK2, both the isolated catalytic subunits and the holoenzyme are constitutively active, which is probably responsible for the oncogenic potential of CK2. Given the structure of the holoenzyme, the beta subunits could undergo reversible dissociation under physiological conditions and play a role as anchoring elements and/or as a docking platform for protein substrates and effectors. These unusual features are likely to be instrumental in the involvement of CK2 in a number of key biological functions, notably RNA synthesis, Wnt signaling, ubiquitination and cell survival.

                Author and article information

                [1 ]CEA, DSV IBEB, Laboratoire des Interactions Protéine-Métal, Saint-Paul-lez-Durance, France
                [2 ]CNRS, UMR Biologie Végétale et Microbiologie Environnementale, Saint-Paul-lez-Durance, France
                [3 ]Université d’Aix-Marseille, Saint-Paul-lez-Durance, France
                [4 ]CEA, INAC, Service de Chimie Inorganique et Biologique (UMR_E 3 CEA UJF), Grenoble, France
                [5 ]CEA, DSV IBEB, Laboratoire de Bioénergétique et Biotechnologie des Bactéries et Microalgues, Saint Paul-lez-Durance, France
                Medical School of Hannover, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: RP SSM DL CB. Performed the experiments: RP SSM DL PD LG JMA. Analyzed the data: RP SSM DL PD CB. Contributed reagents/materials/analysis tools: RP SSM DL PD JMA CB. Wrote the paper: RP SSM DL PD CB.

                Role: Editor
                PLoS One
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                3 August 2012
                : 7
                : 8

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Pages: 10
                This work was funded in part by the “Toxicologie” program of the Commissariat à l’énergie atomique et aux énergies alternatives (CEA) (CalUMo). RP acknowledges a PhD grant from the CEA international program IRTELIS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article
                Recombinant Proteins
                Bioinorganic Chemistry
                Biomacromolecule-Ligand Interactions
                Protein Chemistry
                Biological Systems Engineering
                Environmental Biotechnology
                Toxic Agents
                Analytical Chemistry
                Chemical Biology
                Environmental Chemistry
                Heavy Metals
                Inorganic Chemistry



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