pKID Binds to KIX via an Unstructured Transition State with Nonnative Interactions

Abstract

Understanding the detailed mechanism of interaction of intrinsically disordered proteins with their partners is crucial to comprehend their functions in signaling and transcription. Through its interaction with KIX, the disordered pKID region of CREB protein is central in the transcription of cAMP responsive genes, including those involved in long-term memory. Numerous simulation studies have investigated these interactions. Combined with experimental results, these can provide valuable and comprehensive understanding of the mechanisms involved. Here, we probe the transition state of this interaction experimentally through analyzing the kinetic effect of mutating both interface and solvent exposed residues in pKID. We show that very few specific interactions between pKID and KIX are required in the initial binding process. Only a small number of weak interactions are formed at the transition state, including nonnative interactions, and most of the folding occurs after the initial binding event. These properties are consistent with computational results and also the majority of experimental studies of intrinsically disordered protein coupled folding and binding in other protein systems, suggesting that these may be common features.

Diffusion-limited association of disordered protein by non-native electrostatic interactions

“…Sugase et al have found that the association rate can be enhanced by non-native hydrophobic interactions for a much slower binding system (binding of the disordered phosphorylated kinase inducible activation domain (pKID) and the KIX domain of CBP) using NMR relaxation dispersion experiments 33 . ϕ -Value analysis has also shown the involvement of non-native interactions for the same system 49 . The enhancement of the association by non-native interactions have also been found for various systems in simulations 23 , 24 , 52 .…”

“…In this case, the formation of the native interactions will be hardly affected by the ionic strength of the solution. Therefore, the electrostatic interactions must be involved in the formation of the TC and these interactions are non-native, although non-native hydrophobic interactions can also contribute 33 , 49 . The long lifetime of TC indicates that once non-native contacts are formed between charged side chains, two molecules would stick together so that TAD rearranges and folds on the surface of NCBD without dissociation (small k − in Fig.…”

“…Therefore, the formation of a stable TC via non-native interactions may be a general mechanism for IDP binding. Although non-native hydrophobic interactions can contribute 33 , 49 , we would like to point out that non-native electrostatic interactions should prevail since IDPs usually possess a large number of charged residues 53 while their binding interfaces are hydrophobic 54 (e.g., amphipathic helix formation).…”

Templated folding of intrinsically disordered proteins

“…Several IDPs were found to conform to robust two-state kinetics, displaying single exponential time courses, as well as a linear dependence on reactant concentrations under pseudo-first-order conditions, another hallmark of two-state behaviour (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51). Note that apparent two-state does not imply that there are no intermediates, but that any intermediates are high-energy states, which rapidly convert to the equilibrium bound state or back to the free state.…”

“…The Φ value analysis represents the only experimental method available to describe the structure of a transition state and it has been employed extensively to describe the mechanism of folding of globular proteins (62,63), and lately the coupled binding and folding of IDPs (36)(37)(38)40,48,50,59,60,(64)(65)(66)(67)(68)(69)(70)(71)(72)(73). Interestingly, it has been generally observed that Φ values tend to be between zero and one, with very few cases of unusual values (i.e.…”