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.

Mechanical unfolding of an ankyrin repeat protein.

Ankryin repeat proteins comprise tandem arrays of a 33-residue, predominantly alpha-helical motif that stacks roughly linearly to produce elongated and superhelical structures. They function as scaffolds mediating a diverse range of protein-protein interactions, and some have been proposed to play a role in mechanical signal transduction processes in the cell. Here we use atomic force microscopy and molecular-dynamics simulations to investigate the natural 7-ankyrin repeat protein gankyrin. We find that gankyrin unfolds under force via multiple distinct pathways. The reactions do not proceed in a cooperative manner, nor do they always involve fully stepwise unfolding of one repeat at a time. The peeling away of half an ankyrin repeat, or one or more ankyrin repeats, occurs at low forces; however, intermediate species are formed that are resistant to high forces, and the simulations indicate that in some instances they are stabilized by nonnative interactions. The unfolding of individual ankyrin repeats generates a refolding force, a feature that may be more easily detected in these proteins than in globular proteins because the refolding of a repeat involves a short contraction distance and incurs a low entropic cost. We discuss the origins of the differences between the force- and chemical-induced unfolding pathways of ankyrin repeat proteins, as well as the differences between the mechanics of natural occurring ankyrin repeat proteins and those of designed consensus ankyin repeat and globular proteins.

Complex energy landscape of a giant repeat protein.

Here, we reveal a remarkable complexity in the unfolding of giant HEAT-repeat protein PR65/A, a molecular adaptor for the heterotrimeric PP2A phosphatases. The repeat array ruptures at multiple sites, leading to intermediate states with noncontiguous folded subdomains. There is a dominant sequence of unfolding, which reflects a nonuniform stability distribution across the repeat array and can be rationalized by theoretical models accounting for heterogeneous contact density in the folded structure. Unfolding of certain intermediates is, however, competitive, leading to parallel unfolding pathways. The low-stability, central repeats sample unfolded conformations under physiological conditions, suggesting how folding directs function: certain regions present rigid motifs for molecular recognition, whereas others have the flexibility with which to broaden the search area, as in the fly-casting mechanism. Partial unfolding of PR65/A also impacts catalysis by altering the proximity of bound catalytic subunit and substrate. Thus, the repeat array orchestrates the assembly and activity of PP2A.