Hierarchical Organization of Evolutionary Constraint in Duplicated EF-Hand Motifs

Calmodulin is a conserved [Formula: see text]-binding protein composed of four EF-hand motifs that retain structural symmetry despite functional differences. How evolutionary constraint is organized across these repeated EF-hands remains unclear. Here, we analyzed calmodulin using a protein language model (PLM) to determine whether divergence is distributed uniformly across the motifs or concentrated at residues in the [Formula: see text]-binding loops. In native embedding space, EF-hands showed greater similarity between duplicated pairs than between same-lobe pairs. Position-specific differences were strongest at loop positions, including both canonical coordinating and non-coordinating positions. These results support a hierarchical organization in which the duplicated structure remains conserved while divergence emerges through localized specialization. Our findings show that the PLM captures sequence-context information that is not fully explained by alignment-based conservation.