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

Ben M. Smith,Laura S. Itzhaki,Christopher M. Dobson,Pamela J. E. Rowling

doi:10.3390/biom11060827

Abstract

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.

Highlights

Upon Wnt stimulation, β-catenin translocates to the nucleus, where it binds the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) family of transcription factors [2] and associates with a number of other proteins to form the Wnt enhanceosome [3], leading to transcription initiation and elongation, as well as histone and chromatin modification [4,5], resulting in the transcription of genes that are of developmental importance and those involved in tissue homeostasis
In order to study the kinetics of TCF7L2–β-catenin complex formation and dissociation, we used a recombinant fragment of TCF7L2 comprising the N-terminal 54-residue βcatenin binding domain
In order to determine whether the S31C mutation and its subsequent labelling with fluorescein maleimide had a significant effect on TCF7L2 binding to βcatenin, equilibrium binding experiments were performed using Isothermal Titration Calorimetry (ITC)

Summary

Introduction

Β-catenin fulfils two important functions in vivo, the first of which is as the signal transducer in the canonical Wnt signalling pathway [1]. Biomolecules 2021, 11, 827 sequestered and targeted for proteasomal degradation by a multi-protein complex called the β-catenin destruction complex (BDC) that forms biological condensates [6]. In the BDC, β-catenin is hyperphosphorylated at its N-terminal disordered region by the combined action of GSK3β and CK1α [7], allowing it to be recognised and ubiquitinated by the E3 ubiquitin ligase β-TrCP [8]

Methods

Results

Conclusion