Calmodulin (CaM) binds most of its targets by wrapping around an amphipathic a-helix. The N-terminus of Orai proteins contains ac onserved CaM-binding segment but the binding mechanismh as been only partially characterized. Here,m icroscale thermophoresis (MST), surface plasmon resonance (SPR), and atomic force microscopy( AFM) were employed to study the binding equilibria, the kinetics,a nd 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.T he analogous experiments with smooth muscle myosin light chain kinase (smMLCK) showed only the expected 1:1b inding,c onfirming the validity of our methods.
Orai1andOrai3areCa2+ channels in the plasma membrane of non-excitable cells which are activated by Ca 2+ depletion of the endoplasmic reticulum (ER);areduction of [Ca 2+ ]inthe ER causes the stromal interaction molecule (STIM) in the ER membrane to oligomerize,w hereupon it binds to Orai and activates its channel function. [1][2][3][4] Much less is known about the Ca 2+ -dependent inactivation (CDI) of Orai which is important in the regulation of the intracellular Ca 2+ level. In anumber of studies,calmodulin (CaM) was found to bind to ahighly conserved N-terminal segment of Orai1/3, and to act as an egative regulator of channel function, [5][6][7] although this was lately questioned.[8] Thec rystal structure of the complex between CaM and the isolated CaM-binding segment of Orai1 (aa69-88) revealed an unusual extended conformation of CaM with only its C-terminal lobe binding one Orai1 peptide.[9] Parallel pulldown and isothermal titration calorimetry (ITC) experiments suggested stepwise binding of two Orai1 69-88 peptides with different affinities,o ne on the Cterminal lobe of CaM (K d = 1.1 mm)a nd one on the Nterminal lobe (K d = 4.6 mm). Motivated by these findings,weaimed at acomprehensive characterization of this interaction mechanism, for the following reasons:( i) the measured K d values (1.1 and 4.6 mm)a re much higher than those usually reported for Ca 2+ -induced CaM binding (typically 10 À7 to 10 À11 m) [10] and were probably influenced by the high calmodulin concentration used in ITC,( ii)the transitions between monovalent and bivalent bond formation have not yet been addressed, and, most prominently,( iii)quantitative information about the kinetics of stepwise association and dissociation is still missing. Here,t hese questions were addressed by complementary in vitro methods.E quilibrium measurements by MST yielded the distinct affinities of both binding steps at equilibrium, SPR provided information about the kinetic rate constants,a nd AFM revealed the interaction forces of monovalent and bivalent binding,aswell as the...