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.
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.
The presence of a regulatory site for monovalent cations that affects the conformation of the MgATP-binding pocket leading to enzyme activation has been demonstrated for ribokinases. This site is selective toward the ionic radius of the monovalent cation, accepting those larger than Na(+). Phosphofructokinase-2 (Pfk-2) from Escherichia coli is homologous to ribokinase, but unlike other ribokinase family members, presents an additional site for the nucleotide that negatively regulates its enzymatic activity. In this work, we show the effect of monovalent cations on the kinetic parameters of Pfk-2 together with its three-dimensional structure determined by x-ray diffraction in the presence of K(+) or Cs(+). Kinetic characterization of the enzyme shows that K(+) and Na(+) alter neither the kcat nor the KM values for fructose-6-P or MgATP. However, the presence of K(+) (but not Na(+)) enhances the allosteric inhibition induced by MgATP. Moreover, binding experiments show that K(+) (but not Na(+)) increases the affinity of MgATP in a saturable fashion. In agreement with the biochemical data, the crystal structure of Pfk-2 obtained in the presence of MgATP shows a cation-binding site at the conserved position predicted for the ribokinase family of proteins. This site is adjacent to the MgATP allosteric binding site and is only observed in the presence of Cs(+) or K(+). These results indicate that binding of the monovalent metal ions indirectly influences the allosteric site of Pfk-2 by increasing its affinity for MgATP with no alteration in the conformation of residues present at the catalytic site.
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