Probing the stability and folding mechanism of FOXP2-FHD

Abstract

The forkhead box (FOX) family of transcription factors is categorised by the presence of a canonical DNA binding winged helix motif, termed the forkhead domain (FHD). Of the FOX transcription factors, the FHD of the FOXP subfamily has the unique capability to form dimers by three-dimensional domain swapped dimerisation. A definitive role for domain swapping has not been established, although it has been speculated to facilitate interchromosomal association and thus enable coregulation of distal genetic elements. The FHD of FOXP2 assembles as a domain swapped dimer upon folding; however, it exhibits an unexpectedly low propensity to do so, despite dimerisation being a proposed prerequisite for its ability to regulate transcriptional activity. This investigation aims to elucidate the propensity of FOXP2-FHD to dimerise through probing its folding mechanism and structural stability. In this pursuit, experiments on wild-type FOXP2-FHD were conducted, and contrasted against a dimer-prone mutant (Y540F) which incorporated a substitution mutation uniquely present in the obligately-dimer forming FHD of FOXP3. Low-resolution DNA binding studies by electrophoretic mobility shift assay showed both proteins to have the ability to bind DNA as both dimers and monomers. Analysis by size exclusion chromatography revealed that while the wild-type and Y540F mutant readily dissociate from a dimeric to monomeric conformation post-purification, such dissociation decreases by two orders of magnitude upon denaturation and refolding. Unexpectedly, the wild-type is less prone to dissociate into a monomer in comparison with the Y540F mutant. Equilibrium unfolding studies reveal the wild-type to fold by a two-state mechanism; in comparison, the Y540F mutant displays a yet undocumented folding pathway, whereby it unfolds by a two-state mechanism and refolds by a three-state mechanism. Dissection of the refolding data for the Y540F mutant reveals the formation of a compact, stable intermediate comprising of hydrophobically buried secondary structural elements that otherwise participate in domain swapping. Overview of the data suggests the low dimerisation propensity for the wild-type and Y540F mutant to be a consequence of the bacterial expression system utilised for overexpression. Furthermore, dimerisation for the Y540F mutant is further impeded by the increased compactness and stability of its hydrophobically buried secondary structural elements that are elsewise required for domain swapping. This work suggests the reduced dimerisation propensity of FOXP2-FHD hence described in the literature to be a product of the expression protocol utilised but, to the author’s knowledge, provides the first evidence for a disparate two-/three-state unfolding/refolding mechanism for protein folding