Inter-Site Cooperativity of Calmodulin N-Terminal Domain and Phosphorylation Synergistically Improve the Affinity and Selectivity for Uranyl

Maria Rosa Beccia (1) , Sandrine Sauge-Merle (1) , Nicolas Brémond (1) , David Lemaire (1) , Pierre Henri (2, 3) , Christine Battesti (1) , Philippe Guilbaud (4) , Serge Crouzy (5) , Catherine Berthomieu (1)

1 BIAM - Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB)
2 LPC2E - Laboratoire de Physique et Chimie de l'Environnement et de l'Espace
3 Observatoire de la Côte d'Azur
4 DMRC - Département de recherche sur les procédés pour la mine et le recyclage du combustible
5 COMX - Chimie computationnelle, modélisation et rayons X

Uranyl-protein interactions participate in uranyl trafficking or toxicity to cells. In addition to their qualitative identification, thermodynamic data are needed to predict predominant mechanisms that they mediate in vivo. We previously showed that uranyl can substitute calcium at the canonical EF-hand binding motif of calmodulin (CaM) site I. Here, we investigate thermodynamic properties of uranyl interaction with site II and with the whole CaM N-terminal domain by spectrofluorimetry and ITC. Site II has an affinity for uranyl about 10 times lower than site I. Uranyl binding at site I is exothermic with a large enthalpic contribution, while for site II, the enthalpic contribution to the Gibbs free energy of binding is about 10 times lower than the entropic term. For the N-terminal domain, macroscopic binding constants for uranyl are two to three orders of magnitude higher than for calcium. A positive cooperative process driven by entropy increases the second uranyl-binding event as compared with the first one, with Delta Delta G = -2.0 +/- 0.4 kJ mol(-1), vs. Delta Delta G = -6.1 +/- 0.1 kJ mol(-1) for calcium. Site I phosphorylation largely increases both site I and site II affinity for uranyl and uranyl-binding cooperativity. Combining site I phosphorylation and site II Thr7Trp mutation leads to picomolar dissociation constants Kd(1) = 1.7 +/- 0.3 pM and Kd(2) = 196 +/- 21 pM at pH 7. A structural model obtained by MD simulations suggests a structural role of site I phosphorylation in the affinity modulation.

Keywords: uranium; protein; cooperativity; metal binding competition; thermodynamics; spectroscopy

Domains: Toxicology; Biochemistry, Molecular Biology

Maria Rosa Beccia, Sandrine Sauge-Merle, Nicolas Brémond, David Lemaire, Pierre Henri, et al.. Inter-Site Cooperativity of Calmodulin N-Terminal Domain and Phosphorylation Synergistically Improve the Affinity and Selectivity for Uranyl. Biomolecules, 2022, 12 (11), pp.1703. ⟨10.3390/biom12111703⟩. ⟨hal-04028779⟩