The transition state structure for coupled binding and folding of disordered protein domains

Abstract

Intrinsically disordered proteins are abundant in the eukaryotic proteome, and they are implicated in a range of different diseases. However, there is a paucity of experimental data on molecular details of the coupled binding and folding of such proteins. Two interacting and relatively well studied disordered protein domains are the activation domain from the p160 transcriptional co-activator ACTR and the nuclear co-activator binding domain (NCBD) of CREB binding protein. We have analyzed the transition state for their coupled binding and folding by protein engineering and kinetic experiments (Φ-value analysis) and found that it involves weak native interactions between the N-terminal helices of ACTR and NCBD, but is otherwise "disordered-like". Most native hydrophobic interactions in the interface between the two domains form later, after the rate-limiting barrier for association. Linear free energy relationships suggest a cooperative formation of native interactions, reminiscent of the nucleation-condensation mechanism in protein folding.

Discriminating binding mechanisms of an intrinsically disordered protein via a multi-state coarse-grained model

“…Our finding of an induced-fit mechanism for NCBD binding to ACTR is confirmed by recent experimental evidence from a protein engineering analysis of the binding transition states, which show that, apart from weak native-like interactions, the transition state is largely disordered, 33,34 with -values for binding in the range 0-0.2. We have also calculated -values for binding of NCBD to ACTR based on the simulations, using a simple contact based analysis, i.e., we define the value of residue i as (i) = (q ‡ (i) − q U (i))/(q F (i) − q U (i)), where q ‡ (i), q U (i) and q F (i) are the fraction of its native contacts formed by residue i in the binding transition state, unfolded state and folded state, respectively.…”

“…40 We have previously 38 used large scale atomistic simulations to investigate structure formation in the unbound state of NCBD, and others have used a multi-scale approach to study unbound NCBD. 41 Here, we study the binding mechanism of NCBD to its partners; this complements recent experimental studies, focussed on the association of NCBD to ACTR, [33][34][35] as we discuss below. Although ACTR also undergoes a disorder to order transition upon binding, we have chosen to restrict our attention here to the mechanism as seen from the point of view of NCBD.…”

“…The most powerful methods are single molecule FRET experiments, which can provide information on the overall dimensions 25 and dynamics 26 of unbound IDPs, and NMR experiments which give more localised and higher resolution structural data 27,28 and also mechanistic insights. 29 Kinetic experiments can yield information on binding mechanism, [30][31][32][33][34][35] in particular when used in conjunction with a protein engineering approach. In general, however, it is challenging to resolve mechanistic scenarios from experiment alone.…”

A Frustrated Binding Interface for Intrinsically Disordered Proteins

“…Protein Expression and Purification-Human NCBD and ACTR were expressed and purified as described previously (7,8).…”

“…We have previously used ACTR/NCBD as a model system to investigate the mechanisms of coupled binding and folding (7)(8)(9). The association between NCBD and ACTR is fast, with a transition state that contains only a few native hydrophobic interactions, followed by a cooperative formation of native contacts after the rate-limiting barrier, resembling the nucleation condensation mechanism in protein folding (10).…”