The exchanged EF-hands in calmodulin and troponin C chimeras impair the Ca2+-induced hydrophobicity and alter the interaction with Orai1: a spectroscopic, thermodynamic and kinetic study

Abstract

BackgroundCalmodulin (CaM) plays an important role in Ca2+-dependent signal transduction. Ca2+ binding to CaM triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition. CaM regulates a Ca2+-dependent inactivation process in store-operated Ca2+ entry, by interacting Orai1. To understand the relationship between Ca2+-induced hydrophobicity and CaM/Orai interaction, chimera proteins constructed by exchanging EF-hands of CaM with those of Troponin C (TnC) are used as an informative probe to better understand the functionality of each EF-hand.ResultsANS was used to assess the context of the induced hydrophobic surface on CaM and chimeras upon Ca2+ binding. The exchanged EF-hands from TnC to CaM resulted in reduced hydrophobicity compared with wild-type CaM. ANS lifetime measurements indicated that there are two types of ANS molecules with rather distinct fluorescence lifetimes, each specifically corresponding to one lobe of CaM or chimeras. Thermodynamic studies indicated the interaction between CaM and a 24-residue peptide corresponding to the CaM-binding domain of Orail1 (Orai-CMBD) is a 1:2 CaM/Orai-CMBD binding, in which each peptide binding yields a similar enthalpy change (ΔH = −5.02 ± 0.13 kcal/mol) and binding affinity (Ka = 8.92 ± 1.03 × 105 M−1). With the exchanged EF1 and EF2, the resulting chimeras noted as CaM(1TnC) and CaM(2TnC), displayed a two sequential binding mode with a one-order weaker binding affinity and lower ΔH than that of CaM, while CaM(3TnC) and CaM(4TnC) had similar binding thermodynamics as CaM. The dissociation rate constant for CaM/Orai-CMBD was determined to be 1.41 ± 0.08 s−1 by rapid kinetics. Stern-Volmer plots of Orai-CMBD Trp76 indicated that the residue is located in a very hydrophobic environment but becomes more solvent accessible when EF1 and EF2 were exchanged.ConclusionsUsing ANS dye to assess induced hydrophobicity showed that exchanging EFs for all Ca2+-bound chimeras impaired ANS fluorescence and/or binding affinity, consistent with general concepts about the inadequacy of hydrophobic exposure for chimeras. However, such ANS responses exhibited no correlation with the ability to interact with Orai-CMBD. Here, the model of 1:2 binding stoichiometry of CaM/Orai-CMBD established in solution supports the already published crystal structure.

Deuterostome Genomics: Lineage-Specific Protein Expansions That Enabled Chordate Muscle Evolution

“…The first is that the complex, consisting of two troponin components, troponin I and T, functions independently. The second is that calmodulin ( Jensen et al. 2015 ) mediates regulation as an alternative to troponin C, given the sequence similarity between those two genes.…”

Calmodulin binding to the dehydrogenase domain of NADPH oxidase 5 alters its oligomeric state

“…When the binding kinetics of CaM/CMBD complex were measured, we failed to detect any meaningful kinetic traces using a stopped-flow fluorescence device as used previously for the CaM/Orai system [ 21 ]. Therefore, our results implied that its association kinetics are much faster than the dissociation rate.…”

“…To study the CaM-DH interaction by fluorescence, we prepared Dansyl-labeled CaM (D-CaM) [ 21 ] and determined its binding to all Nox5's DH constructs by fluorescence spectroscopy. Fig.…”

Interacción calmodulina-melatonina: efectos sobre la oxidación de la calmodulina y la actividad de la calmodulina cinasa II

“…Estos datos concuerdan con estudios previos en los que la actividad de la enzima se estimuló en cultivos organotípicos y se inhibió en ensayos de reconstitución en el tubo de ensayo. Estos datos indican que existe una regulación opuesta en la actividad de la enzima que depende del microambiente, el cual favorece de distintas formas la interacción de la MEL (molécula anfifílica (Dies et al, 2015;Yu et al, 2016)) con los residuos hidrofóbicos o hidrofílicos de la CaM (Follenius & Gerard, 1984;Jensen et al, 2015;LaPorte et al, 1980;Tanaka & Hidaka, 1980), es decir, se favorecen diversas conformaciones según el microambiente, que activan o inhiben a la CaMKII.…”