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Designed Mutations Alter the Binding Pathways of an Intrinsically Disordered Protein.

ABSTRACT: Many cellular functions, including signaling and regulation, are carried out by intrinsically disordered proteins (IDPs) binding to their targets. Experimental and computational studies have now significantly advanced our understanding of these binding processes. In particular, IDPs that become structured upon binding typically follow a dock-and-coalesce mechanism, whereby the docking of one IDP segment initiates the process, followed by on-target coalescence of remaining IDP segments. Multiple dock-and-coalesce pathways may exist, but one may dominate, by relying on electrostatic attraction and molecular flexibility for fast docking and fast coalescing, respectively. Here we critically test this mechanistic understanding by designing mutations that alter the dominant pathway. This achievement marks an important step toward precisely manipulating IDP functions.

SUBMITTER: Wu D

PROVIDER: S-EPMC6467919 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Designed Mutations Alter the Binding Pathways of an Intrinsically Disordered Protein.

Wu Di D Zhou Huan-Xiang HX

Scientific reports 20190416 1

Many cellular functions, including signaling and regulation, are carried out by intrinsically disordered proteins (IDPs) binding to their targets. Experimental and computational studies have now significantly advanced our understanding of these binding processes. In particular, IDPs that become structured upon binding typically follow a dock-and-coalesce mechanism, whereby the docking of one IDP segment initiates the process, followed by on-target coalescence of remaining IDP segments. Multiple ...[more]

PMID: 30992509

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Project description:The Wnt signalling pathway plays an important role in cell proliferation, differentiation, and fate decisions in embryonic development and the maintenance of adult tissues. The twelve armadillo (ARM) repeat-containing protein β-catenin acts as the signal transducer in this pathway. Here, we investigated the interaction between β-catenin and the intrinsically disordered transcription factor TCF7L2, comprising a very long nanomolar-affinity interface of approximately 4800 Å2 that spans ten of the twelve ARM repeats of β-catenin. First, a fluorescence reporter system for the interaction was engineered and used to determine the kinetic rate constants for the association and dissociation. The association kinetics of TCF7L2 and β-catenin were monophasic and rapid (7.3 ± 0.1 × 107 M-1·s-1), whereas dissociation was biphasic and slow (5.7 ± 0.4 × 10-4 s-1, 15.2 ± 2.8 × 10-4 s-1). This reporter system was then combined with site-directed mutagenesis to investigate the striking variability in the conformation adopted by TCF7L2 in the three different crystal structures of the TCF7L2-β-catenin complex. We found that the mutation had very little effect on the association kinetics, indicating that most interactions form after the rate-limiting barrier for association. Mutations of the N- and C-terminal subdomains of TCF7L2 that adopt relatively fixed conformations in the crystal structures had large effects on the dissociation kinetics, whereas the mutation of the labile sub-domain connecting them had negligible effect. These results point to a two-site avidity mechanism of binding with the linker region forming a "fuzzy" complex involving transient contacts that are not site-specific. Strikingly, the two mutations in the N-terminal subdomain that had the largest effects on the dissociation kinetics showed two additional phases, indicating partial flux through an alternative dissociation pathway that is inaccessible to the wild type. The results presented here provide insights into the kinetics of the molecular recognition of a long intrinsically disordered region with an elongated repeat-protein surface, a process found to involve parallel routes with sequential steps in each.

Dataset Information

Designed Mutations Alter the Binding Pathways of an Intrinsically Disordered Protein.

Publications

Designed Mutations Alter the Binding Pathways of an Intrinsically Disordered Protein.

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