Effects of Ligand Binding on the Mechanical Properties of Ankyrin Repeat Protein Gankyrin

Ankyrin repeat proteins are elastic materials that unfold and refold sequentially, repeat by repeat, under force. Herein we use atomistic molecular dynamics to compare the mechanical properties of the 7-ankyrin-repeat oncoprotein Gankyrin in isolation and in complex with its binding partner S6-C. We show that the bound S6-C greatly increases the resistance of Gankyrin to mechanical stress. The effect is specific to those repeats of Gankyrin directly in contact with S6-C, and the mechanical ‘hot spots’ of the interaction map to the same repeats as the thermodynamic hot spots. A consequence of stepwise nature of unfolding and the localized nature of ligand binding is that it impacts on all aspects of the protein's mechanical behavior, including the order of repeat unfolding, the diversity of unfolding pathways accessed, the nature of partially unfolded intermediates, the forces required and the work transferred to the system to unfold the whole protein and its parts. Stepwise unfolding thus provides the means to buffer repeat proteins and their binding partners from mechanical stress in the cell. Our results illustrate how ligand binding can control the mechanical response of proteins. The data also point to a cellular mechano-switching mechanism whereby binding between two partner macromolecules is regulated by mechanical stress. Author Summary: Here we use molecular dynamics simulation to compare the mechanical properties of the 7-ankyrin-repeat oncoprotein Gankyrin in isolation and in complex with binding partner S6-C. Tandem repeat proteins like Gankyrin comprise tandem arrays of small structural motifs that pack linearly to produce elongated architectures. They are elastic, mechanically weak molecules and they unfold and refold repeat by repeat under force. We show that S6-C binding greatly increases the resistance of Gankyrin to mechanical stress. The enhanced mechanical stability is specific to those ankyrin repeats in contact with S6-C, and the localized nature of the effect results in fundamental changes in the way the protein responds to force. Thus, the forced unfolding of isolated Gankryin involves a diverse set of pathways with a preference for a C- to N-terminus unfolding mechanism whereas this diversity is reduced upon complex formation with the central repeats, which are those most tightly bound to the ligand, tending to unfold last. Our study shows how stepwise unfolding can buffer repeat proteins and their binding partners from mechanical stress in the cell. It also points to a mechano-switching mechanism whereby binding between two partner macromolecules is regulated by mechanical stress.