Identifying Unfolding Intermediates of FN-III10 by Steered Molecular Dynamics

Experimental studies have indicated that FN-III modules undergo reversible unfolding as a mechanism of elasticity. The unfolding of FN-III modules, including the cell-binding FN-III10 module, has further been suggested to be functionally relevant by exposing buried cryptic sites or modulating cell binding. While steered molecular dynamics (SMD) simulations have provided one tool to investigate this process, computational requirements so far have limited detailed analysis to the early stages of unfolding. Here, we use an extended periodic box to probe the unfolding of FN-III10 for extensions longer than 60 Å. Up to three plateaus, corresponding to three metastable intermediates, were observed in the extension-time profile from SMD stretching of FN-III10. The first and second plateaus correspond to a twisted and an aligned state prior to unraveling FN-III10 β-strands. The third plateau, at an extension of ∼100 Å, follows unraveling of FN-III10 A and B-strands and precedes breaking of inter-strand hydrogen bonds between F and G-strands. The simulations revealed three forced unfolding pathways of FN-III10, one of which is preferentially selected under physiological conditions. Implications for fibronectin fibrillogenesis are discussed.