title: Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity.
abstract: Calmodulin (CaM) is an essential Ca(II)-dependent regulator of cell physiology. To understand its interaction with Ca(II) at a molecular level, it is essential to examine Ca(II) binding at each site of the protein, even if it is challenging to estimate the site-specific binding properties of the interdependent CaM-binding sites. In this study, we evaluated the site-specific Ca(II)-binding affinity of sites I and II of the N-terminal domain by combining site-directed mutagenesis and spectrofluorimetry. The mutations had very low impact on the protein structure and stability. We used these binding constants to evaluate the inter-site cooperativity energy and compared it with its lower limit value usually reported in the literature. We found that site I affinity for Ca(II) was 1.5 times that of site II and that cooperativity induced an approximately tenfold higher affinity for the second Ca(II)-binding event, as compared to the first one. We further showed that insertion of a tryptophan at position 7 of site II binding loop significantly increased site II affinity for Ca(II) and the intra-domain cooperativity. ΔH and ΔS parameters were studied by isothermal titration calorimetry for Ca(II) binding to site I, site II and to the entire N-terminal domain. They showed that calcium binding is mainly entropy driven for the first and second binding events. These findings provide molecular information on the structure-affinity relationship of the individual sites of the CaM N-terminal domain and new perspectives for the optimization of metal ion binding by mutating the EF-hand loops sequences.
authors.affiliation: CEA, DSV, IBEB, Laboratoire des Interactions Protéine Métal, Saint-Paul-Lez-Durance, France.
authors.forename: Maria Rosa
authors.initials: MR
authors.lastname: Beccia
authors.forename: Sandrine
authors.initials: S
authors.lastname: Sauge-Merle
authors.forename: David
authors.initials: D
authors.lastname: Lemaire
authors.forename: Nicolas
authors.initials: N
authors.lastname: Brémond
authors.forename: Romain
authors.initials: R
authors.lastname: Pardoux
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Blangy
authors.forename: Philippe
authors.initials: P
authors.lastname: Guilbaud
authors.forename: Catherine
authors.initials: C
authors.lastname: Berthomieu
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
chemical_list: D002147:Calmodulin; D002118:Calcium