Evolution of Thyroglobulin Loop Kinetics in EpCAM

Epithelial cell-activating molecule (EpCAM) is an important cancer biomarker and therapeutic target given its elevated expression in epithelial cancers. EpCAM is a type I transmembrane protein that forms cis-dimers along the thyroglobulin type-1A-like domain (TYD) in the extracellular region. The thyroglobulin loop (TY loop) within the TYD is structurally dynamic in the monomer state of human EpCAM, binding reversibly to a TYD site. However, it is not known if this flexibility is prevalent across different species. Here, we conduct over 17 micros of all-atom molecular dynamics simulations to study EpCAM TY loop kinetics of five different species, including human, mouse, chicken, frog, and fish. We find that the TY loop remains dynamic across evolution. In addition to the TYD binding site, we discover a second binding site for the TY loop in the C-terminal domain (CTD). Calculations of the dissociation rate constants from the simulation trajectories suggest a differential binding pattern of fish EpCAM and other organisms. Whereas fish TY loop has comparable binding for both TYD and CTD sites, the TY loops of other species preferably bind the TYD site. A hybrid construct of fish EpCAM with human TY loop restores the TYD binding preference, suggesting robust effects of the TY loop sequence on its dynamic behavior. Our findings provide insights into the structural dynamics of EpCAM and its implication in physiological functions.