title: 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: BACKGROUND
  Calmodulin (CaM) plays an important role in Ca(2+)-dependent signal transduction. Ca(2+) binding to CaM triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition. 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 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.
  
  
  RESULTS
  ANS was used to assess the context of the induced hydrophobic surface on CaM and chimeras upon Ca(2+) 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 (K(a) = 8.92 ± 1.03 × 10(5) 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.
  
  
  CONCLUSIONS
  Using ANS dye to assess induced hydrophobicity showed that exchanging EFs for all Ca(2+)-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.
authors.affiliation: Department of Chemistry, Southern Illinois University Edwardsville, Edwardsville, IL, 62026, USA. djensen@siue.edu.
authors.forename: Drake
authors.initials: D
authors.lastname: Jensen
authors.affiliation: Department of Chemistry, Southern Illinois University Edwardsville, Edwardsville, IL, 62026, USA. nreynol82@gmail.com.
authors.forename: Nicole
authors.initials: N
authors.lastname: Reynolds
authors.affiliation: Department of Pathobiology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, 44195, USA. yyny@yahoo.com.
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Yang
authors.affiliation: Department of Chemistry, Southern Illinois University Edwardsville, Edwardsville, IL, 62026, USA. sshakya@siue.edu.
authors.forename: Shubha
authors.initials: S
authors.lastname: Shakya
authors.affiliation: Department of Chemistry, Kent State University at Tuscarawas, New Philadelphia, OH, 44663, USA. zwang3@kent.edu.
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Wang
authors.affiliation: Department of Pathobiology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, 44195, USA. stuehrd@ccf.org.
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr
authors.affiliation: Department of Chemistry, Southern Illinois University Edwardsville, Edwardsville, IL, 62026, USA. cwei@siue.edu.
authors.forename: Chin-Chuan
authors.initials: CC
authors.lastname: Wei
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D020832:EF Hand Motifs; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D012997:Solvents / Q000737:chemistry; D013816:Thermodynamics; D019209:Troponin C / Q000737:chemistry / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D012997:Solvents; D019209:Troponin C; D002118:Calcium