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The Exchanged EF‐Hands in Calmodulin and Troponin C Alter the Interaction with Orai1
Author(s) -
Jensen Drake,
Thomas McKenzie,
Schafer Kyle,
Wei ChinChuan
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.881.4
Subject(s) - chemistry , calmodulin , biophysics , stereochemistry , orai1 , kinetics , dissociation constant , fluorescence , peptide , biochemistry , stim1 , receptor , biology , membrane , physics , quantum mechanics , enzyme
CaM regulates a Ca 2+ ‐dependent inactivation process in store‐operated Ca 2+ entry, by interacting Orai1. To understand the relationship between Ca 2+ ‐induced hydrophobicity and CaM/Orai interaction, chimera proteins constructed by exchanging the four EF‐hands of CaM with those of Troponin C (TnC) were employed. Using an ANS fluorescence probe, it was shown that the exchanged EF‐hands resulted in reduced hydrophobicity compared with wild‐type CaM. ANS lifetime measurements indicated that two types of ANS molecules with rather distinct fluorescence lifetimes exist, each specifically corresponding to one lobe of CaM or chimera. However, such ANS responses exhibited no correlation with the ability to interact with Orai‐CMBD. 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 (K a 8.92 ± 1.03 x 10 5 M ‐1 ). With the exchanged EF1 and EF2, chimeras displayed a two sequential binding mode with a one‐order weaker binding affinity and lower ΔH than that of CaM, while the exchanged EF3 and EF4 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 are exchanged. In conclusion, exchanging EF‐hands has provided insights on the unique 1:2 CaM/Orai‐CMBD open binding conformation, as demonstrated by the existing crystal structure.