A Disorder-Induced Domino-Like Destabilization Mechanism Governs the Folding and Functional Dynamics of the Repeat Protein IκBα

Abstract

The stability of the repeat protein IκBα, a transcriptional inhibitor in mammalian cells, is critical in the functioning of the NF-κB signaling module implicated in an array of cellular processes, including cell growth, disease, immunity and apoptosis. Structurally, IκBα is complex, with both ordered and disordered regions, thus posing a challenge to the available computational protocols to model its conformational behavior. Here, we introduce a simple procedure to model disorder in systems that undergo binding-induced folding that involves modulation of the contact map guided by equilibrium experimental observables in combination with an Ising-like Wako-Saitô-Muñoz-Eaton model. This one-step procedure alone is able to reproduce a variety of experimental observables, including ensemble thermodynamics (scanning calorimetry, pre-transitions, m-values) and kinetics (roll-over in chevron plot, intermediates and their identity), and is consistent with hydrogen-deuterium exchange measurements. We further capture the intricate distance-dynamics between the domains as measured by single-molecule FRET by combining the model predictions with simple polymer physics arguments. Our results reveal a unique mechanism at work in IκBα folding, wherein disorder in one domain initiates a domino-like effect partially destabilizing neighboring domains, thus highlighting the effect of symmetry-breaking at the level of primary sequences. The offshoot is a multi-state and a dynamic conformational landscape that is populated by increasingly partially folded ensembles upon destabilization. Our results provide, in a straightforward fashion, a rationale to the promiscuous binding and short intracellular half-life of IκBα evolutionarily engineered into it through repeats with variable stabilities and expand the functional repertoire of disordered regions in proteins.

Single-Molecule FRET Reveals Hidden Complexity in a Protein Energy Landscape

“…The absence of sequence-distant contacts likely affords repeat proteins extreme flexibility and particular molecular recognition capabilities ( Ferreiro et al., 2005; Sivanandan and Naganathan, 2013 ), consistent with the proposal that they are a distinct class midway between globular structured proteins and intrinsically disordered proteins ( Forwood et al., 2010 ). An example is IκBα, the two C-terminal ankyrin repeats of which possess features of intrinsic disorder ( Lamboy et al., 2011, 2013 ).…”

Mapping the Topography of a Protein Energy Landscape

“…The folding and function of repeat proteins have been studied by both experiment and simulation [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] , and they have been found to possess certain features that distinguish them from the more commonly studied globular proteins and that arise from the symmetry inherent in their structures and the absence of long-range interactions. In particular, the modularity of repeat proteins leads to relatively easy dissection of their biophysical properties and consequently they are highly amenable to redesign -of their thermodynamic stability, folding mechanisms and molecular recognition.…”

Cooperativity and modularity in protein folding

“…We should note that there are combinatorially many ways of segment creation, growth, and coalescence, and the statistical weight of these different pathways is evaluated with the WSME model to explain the distribution of folding pathways observed in the ensemble of protein molecules. The WSME model quantitatively explains free-energy landscapes, pathways, φ-values, and kinetic rates of the folding of various proteins [14][15][16][17][18][19][20][21][22][23]. In Fig.…”

“…However, such invalidity was due to the particular approximation used in the calculation and the problem disappeared when the exact solution of the model was used. Since then, the Wako-Saitô-Muñoz-Eaton (WSME) model has been widely applied in calculating pathways [14][15][16][17][18][19][20][21][22][23] and kinetics [14,19,[23][24][25] of folding as well as in explaining mechanical unfolding [26,27], amyloidosis [28], and allosteric transitions and functions [29][30][31][32]. In this review, we discuss the physics behind the WSME model and its applications to folding and other intriguing biophysical problems.…”