Forkhead box P (FoxP) proteins are unique transcription factors that spatiotemporally regulate gene expression by tethering two chromosome loci together via functional domain-swapped dimers formed through their DNA-binding domains. Further, the differential kinetics on this dimerization mechanism underlie an intricate gene regulation network at physiological conditions. Nonetheless, poor understanding of the structural dynamics and steps of the association process impedes to link the functional domain swapping to human-associated diseases. Here, we have characterized the DNA-binding domain of human FoxP1 by integrating single-molecule Förster Resonance Energy Transfer (FRET) and Hydrogen-Deuterium Exchange coupled with Mass Spectrometry (HDXMS) data with Molecular Dynamics (MD) simulations. Our results confirm the formation of a previously postulated domain-swapped (DS) FoxP1 dimer in solution and reveal the presence of highly populated, heterogeneous, and locally disordered dimeric intermediates along the dimer dissociation pathway. The unique features of FoxP1 provide a glimpse of how intrinsically disordered regions can facilitate domain swapping oligomerization and other tightly regulated association mechanisms relevant in biological processes.

Forkhead box P (FoxP) proteins are members of the versatile Fox transcription factors, which control the timing and expression of multiple genes for eukaryotic cell homeostasis. Compared to other Fox proteins, they can form domain-swapped dimers through their DNA-binding –forkhead– domains, enabling spatial reorganization of distant chromosome elements by tethering two DNA molecules together. Yet, domain swapping stability and DNA binding affinity varies between different FoxP proteins. Experimental evidence suggests that the protonation state of a histidine residue conserved in all Fox proteins is responsible for pH-dependent modulation of these interactions. Here, we explore the consequences of the protonation state of another histidine (H59), only conserved within FoxM/O/P subfamilies, on folding and dimerization of the forkhead domain of human FoxP1. Dimer dissociation kinetics and equilibrium unfolding experiments demonstrate that protonation of H59 leads to destabilization of the domain-swapped dimer due to an increase in free energy difference between the monomeric and transition states. This pH–dependence is abolished when H59 is mutated to alanine. Furthermore, anisotropy measurements and molecular dynamics evidence that H59 has a direct impact in the local stability of helix H3. Altogether, our results highlight the relevance of H59 in domain swapping and folding stability of FoxP1.

Oxygen supply implies higher production cost and reduction of maximum theoretical yields. Thus, generation of fermentation products is more cost-effective. Aiming to find a key piece for the production of (poly)-3-hydroxybutyrate (PHB) as a fermentation product, here we characterize an acetoacetyl-CoA reductase, isolated from a Candidatus Accumulibacter phosphatis-enriched mixed culture, showing a (k_cat^NADH/K_M^NADH)/(k_cat^NADPH/K_M^NADPH)>500. Further kinetic analyses indicate that, at physiological concentrations, this enzyme clearly prefers NADH, presenting the strongest NADH preference so far observed among the acetoacetyl-CoA reductases. Structural and kinetic analyses indicate that residues between E37 and P41 have an important role for the observed NADH preference. Moreover, an operon was assembled combining the phaCA genes from Cupriavidus necator and the gene encoding for this NADH-preferring acetoacetyl-CoA reductase. Escherichia coli cells expressing that assembled operon showed continuous accumulation of PHB under oxygen limiting conditions and PHB titer increased when decreasing the specific oxygen consumption rate. Taken together, these results show that it is possible to generate PHB as a fermentation product in E. coli, opening opportunities for further protein/metabolic engineering strategies envisioning a more efficient anaerobic production of PHB.