The Wnt signalling pathway plays a crucial role in the regulation of cell proliferation, differentiation, and tissue homeostasis. Its dysregulation is closely associated with the development of numerous cancers. The central mediator of the canonical Wnt pathway is β catenin, which functions as a transcriptional co-activator regulating the expression of target genes. Among its binding partners is FHL2 (four and a half LIM domain protein 2). Despite the biological importance of the β-catenin:FHL2 complex, the molecular mechanisms underlying their interaction remain poorly understood.
FHL2 is one of four structurally related members of the FHL protein family (FHL1, FHL3, and FHL5), and their comparison provides insight into the structural and functional determinants that govern the specificity of β-catenin binding. We prepared recombinant protein constructs of FHL family members. Expression and purification protocols were established or optimized using a bacterial expression system. Particular attention was given to FHL5, which exhibited markedly low solubility; therefore, various expression and lysis conditions were tested, including pH adjustment, modulation of Zn²⁺ ion concentration, and the use of the pRARE2 plasmid. After obtaining sufficient amounts of purified proteins, their interactions with β catenin were analysed using isothermal titration calorimetry (ITC) and size exclusion chromatography coupled with static light scattering (SEC-SLS), enabling the assessment of binding affinity and complex stability. The experimental analyses were complemented by bioinformatic approaches such as sequence comparison, solubility prediction, electrostatic property analysis, and comparison of predicted three-dimensional models of LIM domains.
Sufficient quantities of purified FHL1 and FHL3 were obtained for interaction analyses, whereas the yield of soluble FHL5 remained limited and represented the main experimental constraint. Interaction studies demonstrated that the FHL3:β-catenin complex forms a weak and transient interaction with an estimated dissociation constant in the 10 µM range or weaker. No specific binding was detected for FHL1. Due to the limited availability of FHL5, its interaction with β-catenin could not be evaluated. The results showed that FHL proteins differ in their expression, solubility and interaction behaviour, while bioinformatic analyses demonstrated that LIM domains are structurally conserved but differ mainly in the surface properties. We found that LIM domains cluster according to their positional identity rather than protein origin, supporting the modular evolution of these proteins. Differences in the isoelectric points of individual domains and full-length proteins further indicate distinct electrostatic profiles that may contribute to differences in interactions with β-catenin.
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