A Preformed Binding Interface in the Unbound Ensemble of an Intrinsically Disordered Protein: Evidence from Molecular Simulations
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. While many proteins have a specific ‘native’ conformation, so-called intrinsically disordered proteins (IDPs) adopt many different conformations in rapid succession—a characteristic that may be advantageous for rapid binding and promiscuous association. However, this characteristic also makes it very hard to make experimental measurements over times that are short enough to see changes of conformation. In this work, we use the results of a large-scale molecular simulation to explore conformations of NCBD, which is an IDP that adopts specific conformations when it binds either of two other proteins (ACTR and IRF-3). Our results point to the following hypothesis: to help NCBD bind ACTR, those parts of NCBD that make contact with it in the bound conformation are biased towards that conformation, even in the absence of ACTR. Other parts of NCBD tend to avoid the ACTR-bound conformation, to help ensure that NCBD is disordered when unbound.