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.
Highlights
IntroductionCa2+ binding to CaM triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition
Calmodulin (CaM) plays an important role in Ca2+-dependent signal transduction
We have used 2 mM Ca2+ in all experiments to ensure that CaM and chimera proteins are saturated with Ca2+ for a fair comparison
Summary
Ca2+ binding to CaM triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition. Results: ANS was used to assess the context of the induced hydrophobic surface on CaM and chimeras upon Ca2+ binding. Conclusions: Using 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. Such ANS responses exhibited no correlation with the ability to interact with Orai-CMBD. Calmodulin (CaM) is a small, acidic protein with 148 amino acids, which plays important roles in Ca2+-dependent signal transduction in eukaryotes. Ca2+ binding to CaM triggers a major conformational change to form an extended dumbbell-shaped structure, linked by a solvent-exposed, rigid helical structure in x-ray crystallography [2,3,4] but an un-structural linker in NMR [5], suggesting both structures may coexist in solution to facilitate target complexation
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