Detailed Evidence for an Unparalleled Interaction Mode between Calmodulin and Orai Proteins

Abstract

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...

Regulation of Store-Operated Ca2+ Entry by SARAF

“…The cell-type specific effect of CaM inhibition is somewhat surprising since CaM is ubiquitously expressed and highly abundant in cells. However, this inconsistency may be explained by the different ways through which CaM has been shown to affect the CRAC channel, including the binding of CaM to Orai1 [66,67] or to the SOAR of STIM1 [56], which promotes the decoupling of STIM1 from Orai1, and the interaction of CaM with the lipid-binding C-terminus of STIM1 or STIM2 [68,69], which may interfere with the targeting of STIM1 to ER-PM junctions and therefore with SCDI [63].…”

Ca2+/Calmodulin Binding to STIM1 Hydrophobic Residues Facilitates Slow Ca2+-Dependent Inactivation of the Orai1 Channel

“…Dysfunctions in Ca 2+ /CaM based regulatory mechanisms are linked to several human diseases [49][50][51][52]. The key components of the CRAC machinery, STIM and Orai channel proteins have been shown to interact with Ca 2+ /CaM [16,46,[53][54][55][56][57][58][59][60]. However, Ca 2+ /CaM, STIM1 and Orai1 interaction sites and the underlying regulatory mechanism of SCDI still remain unclear.…”

More Than Just Simple Interaction between STIM and Orai Proteins: CRAC Channel Function Enabled by a Network of Interactions with Regulatory Proteins

“…CaM was suggested to account for the observed Ca 2+ dependence of CRAC channel inactivation. A stepwise bivalent binding model of CaM to Orai1/Orai3 N-terminal regions was supported by independent studies [ 139 , 140 ]. The propensity of calmodulin to in vitro associate with N-terminal segments of Orai1, the calmodulin-binding domain (CBD Orai1 ), only if Ca 2+ is present, raised for instance suggestions that CaM was critical for fast inactivation (FCDI) to arise.…”