Intrinsically Disordered Regions of the DNA-Binding Domain of Human FoxP1 Facilitate Domain Swapping

Integrating single-molecule spectroscopy and simulations for the study of intrinsically disordered proteins

“…Medina et al utilized MD simulations paired with smFRET and hydrogen-deuterium exchange mass spectrometry to probe the conformational heterogeneity and dynamics present within intermediate binding steps of FOXP1 [ 270 ]. This approach allowed the authors to probe low-population conformations that would be hidden if ensemble experiments were used exclusively.…”

Integrative structural dynamics probing of the conformational heterogeneity in synaptosomal-associated protein 25

“… 32 – 36 Our integrative method can leverage experimental data, prove specific regions of interest that would be limited in determining structures with reasonable accuracy, and provide complementary information to NMR experiments. Similar workflows resolved the structural dynamics in enzymes, 36 the dynamic heterogeneity in multi-domain proteins, 18 and other intrinsically disordered proteins, 37 , 38 and has been used for determining the ensemble switching mechanism of eukaryotic thiamin riboswitch. 39 Furthermore, the integrative structural modeling using DMD and label-based experiments can solve the remaining challenges in highly dynamic systems such as IDPs, IDRs, and complex supertertiary structural dynamics.…”

Human FoxP Transcription Factors as Tractable Models of the Evolution and Functional Outcomes of Three-Dimensional Domain Swapping

“…In the case of FoxP1, single-molecule Multiparameter Fluorescence Spectroscopy (smMFS) toolbox and HDXMS were employed to elucidate the molecular pathway of 3D-DS and the relationship with structural dynamics at different timescales [ 64 ]. These approaches were used considering their powerful temporal and structural resolution that are informative from fast structural motions [ 65 , 66 ] as well as their effects in order–disorder transitions taking place within the dimerization timescales ( Figure 2 ).…”

“…As an example, we show two FRET dimers, where one of them was generated by labeling of S57C in both monomers (C57(D)-C57(A)) and the other was generated through labelling of V78C in both monomers (C78(D)-C78(A)) ( Figure 2 ). These experiments demonstrated that different secondary structure elements in FoxP1 are more likely to remain folded in the 3D-DS conformation or in the intermediate ensemble, although extreme cases such as helices H 1 and H 5 are mostly in the extended ensemble, suggesting that they are primarily found as disordered elements ( Figure 2 ) [ 64 ]. Additionally, native-centric molecular dynamics simulations of FoxP1 using structure-based models that enable simulating folding and binding transitions [ 69 ] determined that small energy barriers separate the 3D-DS dimer and the intermediate ensemble, thus providing a thermodynamic explanation based on order–disorder transitions to the dimerization properties of FoxP1.…”

“…The kinetic approximation of HDXMS was important to detect a large amount of deuteron incorporation throughout the polypeptide chain (≈30–60%) within the timescales in which FoxP1 dimerization occurs, without significant differences between monomers and dimers except that the H 2– H 4 helix that is rearranged as an extended helix in the 3D-DS ensemble ( Figure 1 B) [ 64 ]. The monitoring of FoxP1 also showed that helix H 5 exhibits the largest extent of deuteron incorporation (≈50–60%), as previously deduced from smMFS.…”