Journal
BIOMOLECULES
Volume 12, Issue 11, Pages -Publisher
MDPI
DOI: 10.3390/biom12111703
Keywords
uranium; protein; cooperativity; metal binding competition; thermodynamics; spectroscopy
Categories
Funding
- CEA
- ECCOREV Research Federation
- Programme Investissement Avenir DEMETERRES
Ask authors/readers for more resources
Uranyl-protein interactions play a significant role in uranyl trafficking or toxicity to cells. This study investigates the thermodynamic properties of uranyl interaction with different binding sites on calmodulin (CaM) and the whole CaM N-terminal domain. The binding affinity and thermodynamic contributions vary between different sites, and phosphorylation enhances the uranyl-binding affinity and cooperativity. A structural role of phosphorylation in affinity modulation is suggested by the obtained structural model.
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available