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.
Highlights
Disordered proteins are abundant in the eukaryotic proteome, and they are implicated in a range of different diseases
Protein engineering together with detailed kinetic analyses allowed us to determine a structural model of the rate-limiting transition state in the coupled binding and folding involving the Intrinsically disordered proteins (IDPs), ACTR, and the molten globule, nuclear co-activator binding domain (NCBD) (Fig. 3)
Native interactions in the binding interface are formed and consolidated subsequent to the rate-limiting barrier for association. This is in agreement with previous studies on the kinetic binding mechanism of IDPs and disordered regions using NMR27, MD28 or stopped-flow spectroscopy[17,29,30,31,32], in which data suggest that native interactions form late on the reaction pathway
Summary
Disordered proteins are abundant in the eukaryotic proteome, and they are implicated in a range of different diseases. Two disordered domains that participate in the formation of a multicomponent protein assembly that is involved in the activation and regulation of gene expression[12,13] are the activation domain from the p160 transcriptional co-activator for thyroid hormone and retinoid receptors (ACTR) and the nuclear co-activator binding domain (NCBD) of CREB binding protein (CBP). The bimolecular complex of the two domains displays a welldefined tertiary structure[8] (Fig. 1) and is a classical example of coupled binding and folding of IDPs. To shed light on the molecular details of the coupled binding and folding reaction of ACTR and NCBD we have here used protein engineering in conjunction with kinetic experiments (W value analysis20,21) to determine the structure of the transition state in terms of formation of native hydrophobic tertiary contacts in the binding interface. A nucleus with partial formation of native hydrophobic interactions was found to be present between the N-terminal helices of both IDP domains
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