Abstract
Intrinsically disordered proteins play an important role in cellular signalling, mediated by their interactions with other biomolecules. A key question concerns the nature of their binding mechanism, and whether the bound structure is induced only by proximity to the binding partner. This is difficult to answer through experiment alone because of the very heterogeneous nature of the unbound ensemble, and the probable rapid interconversion of the various unbound structures. Here we report the most extensive set of simulations on NCBD to date: we use large-scale replica exchange molecular dynamics to explore the unbound state. An important feature of the study is the use of an atomistic force field that has been parametrised against experimental data for weakly structured peptides, together with an accurate explicit water model. Neither the force field nor the starting conformations are biased towards a particular structure. The regions of NCBD that have high helical propensity in the simulations correspond closely to helices in the ‘core’ unbound conformation determined by NMR, although no single member of the simulated unbound ensemble closely resembles the core conformation, or either of the two known bound conformations. We have validated the results against NMR spectroscopy and SAXS measurements, obtaining reasonable agreement. The two helices which most stabilise the binding of NCBD with ACTR are formed readily; the third helix, which is less important for binding but is involved in most of the intraprotein contacts of NCBD in the bound conformation, is formed more rarely, and tends not to coexist with the other helices. These results support a mechanism by which NCBD gains the advantages of disorder, while forming binding-competent structures in the unbound state. We obtain support for this mechanism from coarse-grained simulations of NCBD with, and without, its binding partner.
Highlights
We must consider all the conformations that a protein populates, if we want to understand completely its function and behaviour
NCBD as an intrinsically disordered protein, and NCBD-ACTR binding, we look at the contacts that are present in the bound structure 1KBH
The simulation results successfully reproduce the essential characteristics of unbound NCBD: it is found to be a molten globule with residual a-helical structure
Summary
We must consider all the conformations that a protein populates, if we want to understand completely its function and behaviour. Disordered proteins (IDPs) [2,3,4,5] do not form stable structures in isolation under physiological conditions. Instead, they sample multiple conformations, often while retaining some residual structure [6]. Protein disorder has a number of possible advantages: for example, a larger effective binding surface may increase binding rates (via ‘flycasting’ [12] or ‘non-native steering’ [13]), while greater conformational flexibility might help a protein to bind multiple ligands [2,14,15]. It has been proposed that disorder can aid allosteric coupling [16], enable ultrasensitivity [17] or, in contrast, help to stabilise a system against perturbations in its environment [18], low temperatures [19] or desiccation [20]
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