Fast association and slow transitions in the interaction between two intrinsically disordered protein domains.
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 × 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 (∼20 and ∼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 (∼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.
Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden. Jakob.Dogan@imbim.uu.se
Jakob
J
Dogan
Tanja
T
Schmidt
Xin
X
Mu
Åke
Å
Engström
Per
P
Jemth
D050882:CREB-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D020125:Mutation, Missense; D056921:Nuclear Receptor Coactivator 3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D050882:CREB-Binding Protein; C494363:CREBBP protein, human; C538866:NCOA3 protein, human; D056921:Nuclear Receptor Coactivator 3