Increase in Domain Swapping of the DNA-Binding Domain of Human FoxP1 is Related to a Decrease in Monomer Folding Stability

Abstract

Domain swapping is a folding-upon-binding phenomenon that allows proteins to form dimers by exchanging segments of their structure with another equivalent subunit. Recently, solved structures of the DNA-binding domain of the P subfamily of forkhead box transcription factors, FoxP, showed that these proteins can also form swapped dimers. Biologically, mutations of the DNA-binding domain of these proteins are linked to diverse diseases in humans. Moreover, FoxP1 and FoxP2 can reach monomer-dimer equilibrium in solution after hours of incubation, suggesting a low kinetic barrier separating both species in contrast to other domain-swapping proteins where this process takes days. Using wild type FoxP1 (WT) as a model of domain swapping, we analyzed the temperature and protein concentration effects on dimer dissociation, obtaining the free energy change and enthalpy of the process, indicating that the dimerization is an enthalpy-driven process. To understand how FoxP1 could swap, we performed equilibrium unfolding experiments with the dimer, showing that it follows a three-state mechanism, where the first transition corresponds to dimer dissociation and occurs with little loss of secondary structure. To further corroborate the relevance of hinge region and helix H3 in the domain swapping process of FoxP1, we engineered a previously described hinge monomeric mutant of FoxP1 (A539P) and we constructed a mutant in helix H3 (R553H) related to human health diseases. The dimerization propensity of R553H is higher than WT, suggesting that helix 3 and hinge region are relevant in the dimerization. Folding stability comparison of A539P and R553H monomers with WT show that the order of stability is A539P>WT>R553H, concluding that the ability of FoxP1 to domain swap rapidly can be partially explained by its low monomer stability. Funding: FONDECYT 1130510, 11140601.