title: Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation.
abstract: 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.
authors.affiliation: CEA, DSV IBEB, Laboratoire des Interactions Protéine-Métal, Saint-Paul-lez-Durance, France.
authors.forename: Romain
authors.initials: R
authors.lastname: Pardoux
authors.forename: Sandrine
authors.initials: S
authors.lastname: Sauge-Merle
authors.forename: David
authors.initials: D
authors.lastname: Lemaire
authors.forename: Pascale
authors.initials: P
authors.lastname: Delangle
authors.forename: Luc
authors.initials: L
authors.lastname: Guilloreau
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Adriano
authors.forename: Catherine
authors.initials: C
authors.lastname: Berthomieu
mesh_terms: D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D047390:Casein Kinase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010766:Phosphorylation; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014501:Uranium / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity
chemical_list: D029681:Arabidopsis Proteins; D002147:Calmodulin; D011994:Recombinant Proteins; D014501:Uranium; D047390:Casein Kinase II