Abstract
Intrinsically disordered proteins (IDPs) are common in eukaryotes. However, relatively few experimental studies have addressed the nature of the rate-limiting transition state for the coupled binding and folding reactions involving IDPs. By using site-directed mutagenesis in combination with kinetics measurements we have here characterized the transition state for binding between the globular TAZ1 domain of CREB binding protein and the intrinsically disordered C-terminal activation domain of Hif-1α (Hif-1α CAD). A total of 17 Hif-1α CAD point-mutations were generated and a Φ-value binding analysis was carried out. We found that native hydrophobic binding interactions are not formed at the transition state. We also investigated the effect the biologically important Hif-1α CAD Asn-803 hydroxylation has on the binding kinetics, and found that the whole destabilization effect due the hydroxylation is within the dissociation rate constant. Thus, the rate-limiting transition state is “disordered-like”, with native hydrophobic binding contacts being formed cooperatively after the rate-limiting barrier, which is clearly shown by linear free energy relationships. The same behavior was observed in a previously characterized TAZ1/IDP interaction, which may suggest common features for the rate-limiting transition state for TAZ1/IDP interactions.
Highlights
It has been recognized for some time that intrinsically disordered proteins (IDPs) and regions (IDRs), constitute a substantial fraction of the eukaryotic proteome[1,2,3,4,5]
The analysis of the binding kinetics for transcriptional adaptor zinc-binding 1 (TAZ1)/Hif-1α CAD mutant variants reveal that native hydrophobic binding contact formations at the rate-limiting transition state are absent, since all of the calculated Φ-values are
Even though the topology of both Hif-1α CAD8 and TAD-STAT220, which wrap around TAZ1, is rather different compared to the single α-helix conformation that represents the bound Intrinsically disordered proteins (IDPs) in other binding reactions[17,21,22], the end-result of these studies are, with the exception of a few studies[22], quite similar, i.e. the rate-limiting transition state for binding is rather unstructured, in agreement with MD simulations studies[23,24]
Summary
It has been recognized for some time that intrinsically disordered proteins (IDPs) and regions (IDRs), constitute a substantial fraction of the eukaryotic proteome[1,2,3,4,5] Their frequent involvement in various biological processes have during the last decade sparked a significant interest on the role of disorder for protein function[6,7]. Most of these studies have been performed on IDPs and their complexes under equilibrium conditions, and while such studies are crucial for a better understanding of the relationship between disorder and function, experimental kinetic studies are imperative in order to delineate the binding mechanisms and the nature of the transition state for binding, but such studies are much less prevalent. Similar results were obtained in the previously characterized TAZ1/TAD-STAT2 interaction[12], and may suggest a potential common mechanism for interactions involving TAZ1 and IDPs
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