Detailed Evidence for an Unparalleled Interaction Mode between Calmodulin and Orai Proteins
Calmodulin (CaM) binds most of its targets by wrappingaround an amphipathic ?-helix. The N-terminus of Orai proteins contains a conserved CaM-binding segment but the binding mechanism is only partially characterized. Here, microscale thermophoresis (MST), surface plasmon resonance (SPR), andatomic force microscopy (AFM) were employed to study the binding equilibria, the kinetics, and the single-molecular interaction forces involved in the binding of CaM to the conserved helical segments of Orai1 and Orai3. The results consistently indicated step-wise binding of two separate target peptides to the two lobes of CaM. An unparalleled high affinity was found when two Orai peptides were dimerized or immobilized at high lateral density, thereby mimicking the close proximity of the N-termini in native Orai oligomers. The analogous experiments with smooth muscle myosin light chain kinase (smMLCK) showed only the expected 1:1 binding, confirming the validity of our methods.
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