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

Romain Pardoux (1, 2, 3, 4) , Sandrine Sauge-Merle (3, 4, 1, 2, 5) , David Lemaire (3, 4, 1, 2) , Pascale Delangle (2) , Luc Guilloreau (3, 4, 1, 2) , Jean-Marc Adriano (3, 4, 1, 2) , C. Berthomieu (3, 4, 1, 2, 5)

1 CNRS - Centre National de la Recherche Scientifique
2 DRF (CEA) - Direction de Recherche Fondamentale (CEA)
3 AMU - Aix Marseille Université
4 BVME - Biologie végétale et microbiologie environnementale - UMR7265
5 IPM - Interactions Protéine Métal

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 T9TKE12 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 Kd = 2566 nM to Kd = 561 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (Kd = 0.2560.06 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 nas(P-O) and ns(PO) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in nas(UO2)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.

Mots clés: peptides uranyl binding site phosphoryl group

Domaines: Biochimie [q-bio.BM]; Ingénierie de l'environnement

Romain Pardoux, Sandrine Sauge-Merle, David Lemaire, Pascale Delangle, Luc Guilloreau, et al.. Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation. PLoS ONE, 2012, 7 (8), pp.e41922. ⟨10.1371/journal.pone.0041922⟩. ⟨hal-01402784⟩