Three-Dimensional Domain Swapping Changes the Folding Mechanism of the Forkhead Domain of FoxP1

Abstract

The forkhead family of transcription factors (Fox) controls gene transcription during key processes such as regulation of metabolism, embryogenesis, and immunity. Structurally, Fox proteins feature a conserved DNA-binding domain known as forkhead. Interestingly, solved forkhead structures of members from the P subfamily (FoxP) show that they can oligomerize by three-dimensional domain swapping, whereby structural elements are exchanged between adjacent subunits, leading to an intertwined dimer. Recent evidence has largely stressed the biological relevance of domain swapping in FoxP, as several disease-causing mutations have been related to impairment of this process. Here, we explore the equilibrium folding and binding mechanism of the forkhead domain of wild-type FoxP1, and of two mutants that hinder DNA-binding (R53H) and domain swapping (A39P), using size-exclusion chromatography, circular dichroism, and hydrogen-deuterium exchange mass spectrometry. Our results show that domain swapping of FoxP1 occurs at micromolar protein concentrations within hours of incubation and is energetically favored, in contrast to classical domain-swapping proteins. Also, DNA-binding mutations do not significantly affect domain swapping. Remarkably, equilibrium unfolding of dimeric FoxP1 follows a three-state N2 ↔ 2I ↔ 2U folding mechanism in which dimer dissociation into a monomeric intermediate precedes protein unfolding, in contrast to the typical two-state model described for most domain-swapping proteins, whereas the A39P mutant follows a two-state N ↔ U folding mechanism consistent with the second transition observed for dimeric FoxP1. Also, the free-energy change of the N ↔ U in A39P FoxP1 is ∼2 kcal⋅mol(-1) larger than the I ↔ U transition of both wild-type and R53H FoxP1. Finally, hydrogen-deuterium exchange mass spectrometry reveals that the intermediate strongly resembles the native state. Our results suggest that domain swapping in FoxP1 is at least partially linked to monomer folding stability and follows an unusual three-state folding mechanism, which might proceed via transient structural changes rather than requiring complete protein unfolding as do most domain-swapping proteins.

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

“…In these experiments, highly flexible regions become fully deuterated in a few minutes, whereas well-packed regions such as the hydrophobic core exhibit a low extent of exchange. The advantages of this strategy is that it can be applied to proteins of any size (Balasubramaniam and Komives, 2013 ), under varying temperature (Ramírez-Sarmiento et al, 2013 ), and solvent conditions (Medina et al, 2016 ) and in the absence and presence of ligands (Chalmers et al, 2011 ). Comparative analysis of deuterium incorporations of local regions of a psychrophilic and a thermophilic alcohol dehydrogenase led to strengthen the notion that only those functional regions related to substrate binding exhibit greater flexibility in the cold-active enzyme than in the warm-adapted homolog, suggesting that local flexibility can be uncoupled from thermal stability (Liang et al, 2004 ).…”

Dissecting FOXP2 Oligomerization and DNA Binding

“…P5 featured the A539P substitution in the FHD which reportedly disrupts the ability to form domain‐swapped dimers . Since P4 and P5 behave the same, domain swapping is not relevant for our concentration regime …”

The protonation state of an evolutionarily conserved histidine modulates domain swapping stability of FoxP1

“…Most Fox members have been described to fold into monomers in vitro regardless of the presence of their cognate DNA. However, only the forkhead domain from the P subfamily (FoxP) has been characterized to form domain-swapped dimers in solution both in absence and presence of DNA 8–11 , despite the high sequence identity (~70–80%) with other Fox members. These intriguing aspects motivated the analysis of the structural and functional properties of FoxP proteins and the molecular basis of their unique behaviour.…”