FOXP1與Myocyte Nuclear Factor的DNA Binding Domains之結構動力學與功能間的關係

Abstract

Forkhead box (FOX) proteins are a family of transcription factors that play important roles in regulating the expression of genes involved in cell growth, proliferation, differentiation, and longevity. The forkhead domain, a DNA-binding motif containing 80-100 amino acid residues, is also known as the winged-helix domain due to the butterfly-like appearance of the loops in the protein structure. To study structure-function-dynamics relationships of the forkhead domain, I used NMR spectroscopy to study the forkhead domains of FOXP1 and myocyte nuclear factor (MNF). FOXP1 belongs to the P-subfamily of forkhead transcription factors. According to size exclusion chromatography analysis, the forkhead domain of FOXP1 existed as a mixture of monomer and dimer. The dissociation constants of the forkhead domain of wild-type, C61S and C61Y mutants of FOXP1 were 27.3, 28.8, and 332.0 M, respectively. In contrast, FOXP1 A39P mutant formed only a monomer. NMR analysis also showed that FOXP1 C61S and C61Y mutants existed as a mixture. The solution structure of FOXP1 A39P/C61Y mutant was similar to the X-ray structure of the FOXP2 monomer. Comparison of backbone dynamics of FOXP1 A39P/C61Y and C61Y mutants showed that the residues preceding helix 3, the hinge region, exhibited the largest conformational exchange in FOXP1 monomer. The A39 residue of FOXP1 dimer has a lower order parameter with internal motion on the ps-ns timescale, suggesting that the dynamics of the hinge region of FOXP1 are important in the formation of the swapped dimer. The analysis also showed that the residues exhibiting the motions on the ps-ns and s-ms timescales were located at the DNA-binding surface of FOXP1, suggesting the interactions between FOXP1 and DNA may be highly dynamic. Myocyte nuclear factors (MNFs) are transcription factors that are selectively expressed in myogenic stem cells. We here reported 3D structure of the DNA-binding domain of MNF that is a member of the winged helix family. MNF exhibited high affinity DNA binding with a dissociation constant of 2.57 nM. The region preceding the DNA-recognition helix, the H2-H3 loop and the wing 1, exhibited highest chemical shift perturbation upon DNA binding. Dynamics analysis showed that the N- and C-termini and the wing 1 region were the most flexible regions. The C-terminal region exhibited highest NOE change, and most of the regions became more rigid upon DNA binding. Interestingly, the residues in H2, DNA recognition helix, and wing 1 region exhibited conformational exchange upon DNA binding. The non-DNA binding residues in the H2-H3 loop of MNF exhibited the motions on both the s/ms and ps/ns time scales, which were reduced upon DNA binding. Gel retardation analysis also showed that the mutations of the residues K35, R41, and G46 in the H2-H3 loop exhibited a 1.7-, 2.1-, and 3.5-fold decrease in binding to DNA, suggesting that they are important for DNA binding. These results showed that the binding of MNF to DNA may be mediated by the dynamics of the non-DNA interacting residues. This study provides a new insight into that how domain swapping and the DNA binding of FOX proteins may be regulated by the dynamical properties of their less conserved regions.