Abstract
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 μs 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.
Highlights
To characterize the thyroglobulin loop (TY loop) dynamics, we calculated the contact ratio of the TY loop with Epithelial cell-activating molecule (EpCAM) based on the number of structures that a contact is present to the total of 12,500 molecular dynamics (MD) structures (Figure 1A)
We define a contact as the distance between the geometric center of all heavy atoms of the TY loop and each EpCAM residue using cutoff distances of 6.5, 8.0, and 12.0 Å
To characterize the TY loop dynamics, we calculated the contact ratio of the TY loop with EpCAM based on the number of structures that a contact is present to the total 3of of 10
Summary
Epithelial cell-activating molecule (EpCAM, CD326) is a cell surface protein that is a prominent cancer biomarker and therapeutic target due to its overexpression on epithelial tumors [1,2]. The extracellular region of EpCAM consists of three domains: an. N-terminal domain (NTD), a thyroglobulin type-1A-like domain (TYD), and a C-terminal domain (CTD) which connects to a transmembrane helix [3,4]. EpCAM exists as a dimer, in which the identical monomers associate along the interface of their TYDs, leaving the oft-antibody targeted NTD surface exposed [3]. EpCAM function remains unknown; numerous interaction partners have implied several roles, including cell proliferation and differentiation [5,6] as well as various signaling pathways including regulated intramembrane proteolysis [7,8]
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