Detailed Evidence for an Unparalleled Interaction Mode between Calmodulin and Orai Proteins
Sprache des Titels:
Calmodulin (CaM) binds most of its targets by
wrapping around an amphipathic a-helix. The N-terminus of
Orai proteins contains a conserved CaM-binding segment but
the binding mechanism has been only partially characterized.
Here, microscale thermophoresis (MST), surface plasmon
resonance (SPR), and atomic force microscopy (AFM) were
employed to study the binding equilibria, the kinetics, and the
single-molecule interaction forces involved in the binding of
CaM to the conserved helical segments of Orai1 and Orai3.
The results consistently indicated stepwise 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.