A New Strategy for the Analysis of Biospecific Interactions, Exemplified for Calmodulin and Two of its Targets

Calcium entry into non-excitable cells is mainly carried by store-operated channels, whereby Orai acts as the calcium channel in the plasma membrane and the stromal interaction molecule (STIM) functions as the calcium sensor in the endoplasmic reticulum (ER), as well as the activator of Orai channels. Compared with activation of Orai, much less is known about the mechanism of Ca2+-dependent inactivation (CDI) processes. It has been proposed that binding of calmodulin (CaM) to a highly conserved N-terminal segment of Orai1 is important for CDI, although its exact role remains unclear. Typically, 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 adjacent to the first transmembrane helix, but the binding mechanism is only partially characterized. Here, a new concerted strategy for the analysis of biospecific interactions using microscale thermophoresis (MST), surface plasmon resonance (SPR), and atomic force microscopy (AFM) was 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.submitted by Lukas TraxlerUniversität Linz, Dissertation, 2017OeBB(VLID)244927