The IQ-motif/CaM complex is coupled with the Sec7-PH tandem in IQSECs and dissociated by Ca ²⁺ . (A) Schematic diagrams showing the domain organization and amino acid sequence conservation of IQSECs from different species. Amino acid sequences of the region encompassing the extended IQ-motif of IQSECs are aligned. The predicted α-helices in the extended IQ-motif are labeled. Residues in the IQ-motif of IQSEC2 found to be mutated in ID patients are indicated. Cons., conserved; Var., variable. (B) The size-exclusion chromatography coupled with multiangle light scattering (SEC-MALS) analysis shows that the IQ-motif and CaM formed homogenous stabled complexes both in the absence and presence of Ca²⁺. (C) The SEC-MALS analysis shows that the IQ-motif/CaM complex and the Sec7-PH tandem of IQSEC3 formed stable complexes in the absence of Ca²⁺ and dissociated in the presence of Ca²⁺. (D) The ITC curve of 200 μM IQ-motif/CaM complex titrating into 20 μM Sec7-PH protein of IQSEC3 in the absence and presence of Ca²⁺. N.R., fitting is not reliable due to very weak binding. (E) Superposition plot of a small region of the ¹H, ¹⁵N HSQC spectra of the ¹⁴N_IQ∼Sec7PH/¹⁵N_Ca²⁺-CaM complexes (blue) and ¹⁵N_IQ/¹⁵N_Ca²⁺-CaM complexes (black). The full ¹H, ¹⁵N HSQC spectra are shown in Fig. S1 F. The assignments of the peaks corresponding to the ¹⁵N_Ca²⁺-CaM are labeled. (F) The ITC curve of 100 μM CaM titrating into 10 μM IQ∼Sec7PH protein of IQSEC3 in the absence and presence of Ca²⁺.