Fast Association and Slow Transitions in the Interaction between Two Intrinsically Disordered Protein Domains
2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 41, 34316-34324 p.Article in journal (Refereed) Published
Proteins that contain long disordered regions are prevalent in the proteome and frequently associated with diseases. However, the mechanisms by which such intrinsically disordered proteins (IDPs) recognize their targets are not well understood. Here, we report the first experimental investigation of the interaction kinetics of the nuclear co-activator binding domain of CREB-binding protein and the activation domain from the p160 transcriptional co-activator for thyroid hormone and retinoid receptors. Both protein domains are intrinsically disordered in the free state and synergistically fold upon binding each other. Using the stopped-flow technique, we found that the binding reaction is fast, with an association rate constant of 3 x 10(7) M-1 s(-1) at 277 K. Mutation of a conserved buried intermolecular salt bridge showed that electrostatics govern the rapid association. Furthermore, upon mutation of the salt bridge or at high salt concentration, an additional kinetic phase was detected (similar to 20 and similar to 40 s(-1), respectively, at 277 K), suggesting that the salt bridge may steer formation of the productive bimolecular complex in an intramolecular step. Finally, we directly measured slow kinetics for the IDP domains (similar to 1 s(-1) at 277 K) related to conformational transitions upon binding. Together, the experiments demonstrate that the interaction involves several steps and accumulation of intermediate states. Our data are consistent with an induced fit mechanism, in agreement with previous simulations. We propose that the slow transitions may be a consequence of the multipartner interactions of IDPs.
Place, publisher, year, edition, pages
2012. Vol. 287, no 41, 34316-34324 p.
Coactivator Binding Domain, Conformational Selection, Unstructured Proteins, Complex, Cbp, Recruitment, Recognition, Mechanism, State
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:uu:diva-185193DOI: 10.1074/jbc.M112.399436ISI: 000309654200035OAI: oai:DiVA.org:uu-185193DiVA: diva2:571444