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

Itzhaki LS (2013) Effects of Ligand Binding on the Mechanical Properties of Ankyrin Repeat Protein Gankyrin. PLoS Comput Biol 9(1): e1002864. doi:10.1371/journal.pcbi.1002864 Effects of Ligand Binding on the Mechanical Properties of Ankyrin Repeat Protein Gankyrin Giovanni Settanni 0 David Serquera 0 Piotr E. Marszalek 0 Emanuele Paci 0 Laura S. Itzhaki 0 Shi-Jie Chen, University of Missouri, United States of America 0 1 Physics Department, Johannes Gutenberg University , Mainz, Germany , 2 MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Cambridge, United Kingdom, 3 Department of Mechanical Engineering and Materials Science, Duke University , Durham , North Carolina, United States of America, 4 School of Molecular and Cellular Biology, University of Leeds , Leeds , United Kingdom , 5 University of Cambridge Department of Chemistry , Cambridge , United Kingdom 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. - Funding: This research was funded by the Center for Computational Science of the University of Mainz and by the Max-Planck Graduate Center with the University of Mainz (GS), NIH grant R01-GM079563 (PEM), Medical Research Council of the UK (DS and LSI, including grant G1002329), Medical Research Foundation (LSI) and the Cambridge Gates Trust (DS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Tandem repeat proteins, also known as solenoid proteins, are a special class of proteins comprising tandem arrays of small structural motifs (2040 residues) that pack in a roughly linear fashion to produce elongated, superhelical architectures, thereby presenting extended surfaces that act as scaffolds for molecular recognition. Examples include ankyrin, tetratricopeptide, HEAT and leucine rich repeats. Their structures are characterized by short-range interactions between residues either within a repeat or in adjacent repeats. As such they contrast with globular proteins, which are stabilized by many sequence-distant interactions that frequently result in complex topologies, and with polyproteins like titin, in which independently folded domains are covalently linked in tandem arrays but without significant non-covalent interactions between the individual domains. It is thought that the lack of sequence-distant contacts affords repeat proteins a high degree of flexibility and elasticity [1,2], and atomic force microscopy (AFM) studies have identified certain unique properties that underlie this spring-like mechanical behavior [35,6]. However, the relationship between spring and scaffold functions of repeat proteins is not understood and requires a determination of the mechanics of these proteins upon ligand binding. Gankyrin is an oncoprotein that is overexpressed in hepatocellular carcinomas [7]. It belongs to the ankyrin repeat family of proteins, which are involved in numerous protein-protein interactions and which have been postulated to be the spring elements in mechanotransduction [8]. Each ankyrin repeat forms a b-turn followed by two antiparallel a-helices and a loop. Gankyrin binds the S6 ATPase subunit of the 19S regulatory particle of the 26S proteasome and it enhances the degradation of the tumour suppressors pRb and p53. The interaction of Gankyrin with S6 Cterminal domain (S6-C) is typical of repeat protein molecular recognition in that the whole length of Gankyrin is used to create an extended surface for binding [9] (Figure 1). All but the Cterminal ankyrin repeat of Gankyrin (repeat seven) make contacts with S6-C. The interaction involves complementary charged residues on the two proteins that form several positively and negatively charged patches along the elongated interface. The latter comprises residues from the b-turns and the N-terminal helices of repeats 16 of Gankyrin. An important process in mechanotransduction is mechanoswitching. For example, force has the potential to partially unfold proteins, shutting off or triggering biochemical reactions by disrupting binding motifs or exposing cryptic binding sites. Force modulates a proteins free energy surface; a small force does not necessarily abolish the native minimum but may cause the breakage of non-covalent bonds or, conversely, activate catch bonds that bind more tightly with force [10]. Likewise, binding can affect the mechanical response of proteins to external stress, as 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 Nterminus 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 mechanoswitching mechanism (...truncated)