Conserved IQ motifs, divergent apo‐calmodulin recognition in UBE3B and UBE3C

Calmodulin (CaM) regulates diverse signaling proteins through recognition of short linear motifs, including isoleucine‐glutamine (IQ) motifs that commonly bind Ca 2+ ‐free CaM (apo‐CaM). The homologous to E6AP carboxyl terminus (HECT) E3 ubiquitin ligases ubiquitin protein ligase E3B (UBE3B) and ubiquitin protein ligase E3C (UBE3C) are closely related enzymes that both contain N ‐terminal IQ motifs, yet whether these motifs encode conserved modes of apo‐CaM recognition has remained unclear. Here, we combined isothermal titration calorimetry, nuclear magnetic resonance (NMR) spectroscopy, and computational modeling to compare apo‐CaM binding by the UBE3B and UBE3C IQ motifs. The UBE3B IQ motif bound apo‐CaM with high affinity and preferentially engaged the CaM C ‐lobe, whereas the UBE3C IQ motif interacted more weakly under the same conditions. NMR titrations revealed distinct molecular recognition modes—UBE3B IQ binding occurred in slow exchange and produced a localized C ‐lobe interaction surface, while UBE3C IQ binding occurred in fast exchange and generated smaller, more distributed chemical shift perturbations. Structural models and molecular dynamics simulations provide a framework for interpreting these divergent apo‐CaM engagement modes despite conservation of canonical IQ motif features. Phylogenetic analysis further suggests that the UBE3B and UBE3C IQ motifs have followed divergent evolutionary trajectories within the broader IQ motif family. Together, these findings show that conserved IQ motifs in related HECT ligases can encode different apo‐CaM binding modes, providing a molecular basis for divergent CaM‐mediated regulation of UBE3B and UBE3C.