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.

Arrhythmia mutations in calmodulin can disrupt cooperativity of Ca2+ binding and cause misfolding

“…2000; VanScyoc & Shea, 2001; Beccia et al . 2015), such that it is rare to have a stable version with only one Ca 2+ bound to an individual lobe. However, the N98S mutation may be able to reduce this cooperativity, yielding a C‐lobe with only a single Ca 2+ bound at a physiologically relevant Ca 2+ concentration.…”

“…Ca 2+ binding to the EF‐hands within each lobe is highly cooperative (Beccia et al . 2015), and the different Ca 2+ affinities between the lobes allow CaM to mediate signalling over a range of cytosolic Ca 2+ concentrations through interactions with numerous targets, including the L‐type voltage‐gated Ca 2+ channel (Ca V 1.2) (Ben‐Johny & Yue, 2014) and cardiac ryanodine receptor (RyR2) (Van Petegem, 2012; Gong et al . 2019).…”

Conserved properties of individual Ca 2+ -binding sites in calmodulin

“…Different EF-hand-containing proteins bind Ca 2+ with different affinities, suggesting that a protein with multiple EF-hands, such as CaM, may bind Ca 2+ with a different affinity at each site (19)(20)(21)(22)(23)(24)(25)(26)(27)(28). It has therefore been suggested that CaM's four sites display different affinities and perhaps cooperativity (29,30).…”

Crystal structure of the trimeric N‐terminal domain of ciliate Euplotes octocarinatus centrin binding with calcium ions

“…Ca 2+ ions are coordinated by ligands within the loop region. Upon Ca 2+ ion binding, the EF‐hand subdomain undergoes a large conformational change and exposes more hydrophobic surface . In general, every EF‐hand subdomain contains two calcium binding sites.…”

“…Centrins are required for centriole duplication and may also play a role in severing microtubules that are caused by calcium‐mediated contraction . Centrin undergoes a large conformational change and exposes more hydrophobic surface after binding Ca 2+ ion . Centrin is also a calcium‐dependent self‐assembly protein and is found in yeast (Cdc31p), algae [ Scherffelia dubiacentrin (SdCen)], or humans (HsCen1 and HsCen2), often forming multimers in the presence of Ca 2+ ions .…”