Mechanical Unfolding of an Ankyrin Repeat Protein

Ankryin repeat proteins comprise tandem arrays of a 33-residue, predominantly α-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

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

<div><p>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.</p> </div

Distribution of the external force measured along the simulations.

<p>(A) Upper panel: Distribution of the force for isolated Gank (black solid line) and Gank-S6-C complex (red solid line). The peaks of the distributions were fitted to a Gaussian (dashed lines). Lower panel: Difference between the actual distribution and the fitted Gaussian, normalized using the error on the actual distributions. Peaks in the difference (at 200 pN and 250 pN, for isolated Gank and Gank-S6-C complex, respectively) were taken as thresholds to identify unambiguously peaks in the force-extension profiles. (B) Cartoon representation of the ankyrin repeats of Gank from N- to C-terminus are colored blue, green, red, cyan, magenta, yellow and dark grey, respectively. The end-to-end vector and its repeat components are indicated with black arrows.</p

Distribution of the repeat components of the transferred work at 0.01 Å/ps pulling speed after full extension of Gank is reached (at t = 64 ns).

<p>The distribution is obtained by a kernel density estimation analysis of the work data with a Gaussian bandwidth of 50 k_BT. Data for isolated Gank and Gank-S6-C complex are shown in the upper and lower panels, respectively.</p

Unfolding times of individual ankyrin repeats of Gank at 0.01 Å/ps pulling speed.

<p>Distributions of successive repeat unfolding times (e1 corresponding to the first unfolding event) in isolated Gank (continuous lines) and Gank-S6-C complex (dashed line). Distributions are obtained via kernel density estimation analysis with Gaussian bandwidth of 1.6 ns.</p

Gank residues involved in long-lived contacts with ligand S6-C.

<p>Gank residues involved in long-lived contacts with ligand S6-C.</p

Time dependence of the binding contacts between Gank and S6-C at 0.01 Å/ps pulling speed.

<p>A. Lifetime of binding contacts with S6-C for the residues of Gank (red error bars represent 1 standard error around average). Black and blue boxes represent 1 standard error around the average unfolding time of the corresponding repeats in Gank-S6-C complex and isolated Gank simulations, respectively, as measured using RMSD. Grey and green shadowed regions indicate the beta-turn and N-terminal helix regions, respectively. (B)–(C). Front and upper views of Gank (cartoon representation), colored according to the average time each residue took to lose contact with S6-C (glass surface representation, displayed to mark the position of the binding site in the native conformation). The residues that form long-lasting contacts with S6-C are shown in licorice. The figure was prepared using VMD <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002864#pcbi.1002864-Humphrey1" target="_blank">[38]</a>.</p

Order of repeat unfolding.

<p>Entries are the number of SMD runs in which the repeat indicated unfolds first, second, third etc. The data from simulations at the two pulling speeds were pooled.</p

Force-extension profiles from representative simulations.

<p>Representative profiles are shown for isolated Gank (A)–(B) and for Gank-S6-C complex (C)–(D). The peaks were fitted to a WLC model with a persistence length of 0.38 nm (dashed lines). (E) Probability distribution of the force peaks on the extension-force plane of uncomplexed Gank (black) and Gank-S6-C complex (red). The distribution was obtained by a kernel density estimation analysis of the peak coordinates from all the sampled force-extension profiles, with a Gaussian bandwidth of 20 Å and 20 pN. The figure was prepared using Octave <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002864#pcbi.1002864-Eaton1" target="_blank">[37]</a>.</p

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.Fil: Tsytlonok, Maksym. University Of Cambridge; Estados Unidos. Hutchison/MRC Research Centre; Reino UnidoFil: Craig, Patricio Oliver. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. University Of California At San Diego; Estados UnidosFil: Sivertsson, Elin. University Of Cambridge; Reino UnidoFil: Serquera, David. Hutchison/MRC Research Centre; Reino UnidoFil: Perrett, Sarah. Chinese Academy Of Sciences; República de ChinaFil: Best, Robert B.. University Of Cambridge; Reino UnidoFil: Wolynes, Peter G.. Rice University; Estados UnidosFil: Itzhaki, Laura S.. University Of Cambridge; Reino Unid