Parallel and Sequential Pathways of Molecular Recognition of a Tandem-Repeat Protein and Its Intrinsically Disordered...

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 Ų 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.