Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation

• Published in: PloS one Vol. 7; no. 8; p. e41922
• Main Authors: Pardoux, Romain, Sauge-Merle, Sandrine, Lemaire, David, Delangle, Pascale, Guilloreau, Luc, Adriano, Jean-Marc, Berthomieu, Catherine
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 03-08-2012; Public Library of Science (PLoS)
• Subjects: Affinity; Amino acids; Analysis; Arabidopsis - chemistry; Arabidopsis - genetics; Arabidopsis - metabolism; Arabidopsis Proteins - chemistry; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Arabidopsis thaliana; Binding sites; Biochemistry; Biochemistry, Molecular Biology; Biology; Calcium; Calcium-binding protein; Calmodulin; Calmodulin - chemistry; Calmodulin - genetics; Calmodulin - metabolism; Casein kinase II; Casein Kinase II - chemistry; Casein Kinase II - genetics; Casein Kinase II - metabolism; Catalysis; Chemistry; Chromatography; Dissociation; EF-hand; Environmental Engineering; Environmental Sciences; Fluorescence; Homogeneity; Hydrogen ions; Hydrogen-Ion Concentration; Ion-exchange chromatography; Kinases; Life Sciences; Ligands; Paramecium; Peptides; pH effects; Phosphorylation; Physical chemistry; Physiological aspects; Protein Binding; Protein Engineering; Protein kinase C; Protein kinases; Protein Structure, Tertiary; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Spectrometry; Threonine; Toxicity; Tyrosine; Uranium; Uranium - chemistry; Uranium - metabolism; Uranium - toxicity; Uranium dioxide; Vibration; France; peptides uranyl binding site phosphoryl group
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0041922

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(9)TKE(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.