A Switch between Two Intrinsically Disordered Conformational Ensembles Modulates the Active Site of a Basic-Helix-Loop-Helix Transcription Factor

Abstract

We report a conformational switch between two distinct intrinsically disordered subensembles within the active site of a transcription factor. This switch highlights an evolutionary benefit conferred by the high plasticity of intrinsically disordered domains, namely, their potential to dynamically sample a heterogeneous conformational space housing multiple states with tailored properties. We focus on proto-oncogenic basic-helix-loop-helix (bHLH)-type transcription factors, as these play key roles in cell regulation and function. Despite intense research efforts, the understanding of structure-function relations of these transcription factors remains incomplete as they feature intrinsically disordered DNA-interaction domains that are difficult to characterize, theoretically as well as experimentally. Here we characterize the structural dynamics of the intrinsically disordered region DNA-binding site of the vital MYC-associated transcription factor X (MAX). Integrating nuclear magnetic resonance (NMR) measurements, molecular dynamics (MD) simulations, and electron paramagnetic resonance (EPR) measurements, we show that, in the absence of DNA, the binding site of the free MAX₂ homodimer samples two intrinsically disordered conformational subensembles. These feature distinct structural properties: one subensemble consists of a set of highly flexible and spatially extended conformers, while the second features a set of "hinged" conformations. In this latter ensemble, the disordered N-terminal tails of MAX₂ fold back along the dimer, forming transient long-range contacts with the HLH-region and thereby exposing the DNA binding site to the solvent. The features of these divergent substates suggest two mechanisms by which protein conformational dynamics in MAX₂ might modulate DNA-complex formation: by enhanced initial recruitment of free DNA ligands, as a result of the wider conformational space sampled by the extended ensemble, and by direct exposure of the binding site and the corresponding strong electrostatic attractions presented while in the hinged conformations.

Figure 1

(a) Sketch of MAX₂’s functional elements. The DNA-binding NTD spans residues 1–16, followed by a helix–loop–helix segment and a leucine zipper. (b) Evolution of distances r(5–5) between the C^α atoms of the two N-terminal R5 residues of each MAX subunit during an MD trajectory. (c) Structural representation of hinged (left) and extended (right) subensembles found during the MD trajectory of panel b. Examples for the R5–R5 distances are indicated. The two different subensembles account for the large distance fluctuations in panel b. Note that the hinged subensemble appears more defined than the extended one. The different MAX domains are indicated on the right. (d) RMSD of the NTD only relative to the starting structure. On the left, the narrow dispersion of the hinged ensemble is indicated in green (ΔRMSD < 0.3 nm). On the right the larger RMSD fluctuations of the extended ensemble are highlighted in orange (ΔRMSD > 1.3 nm). The RMSD analysis confirms that the extended subensemble is less constrained than the hinged one.

Figure 2

(a) Residue dependence of experimental PRE signal suppression ratios V. Three distinct PRE sites are observed, indicated as S (short-range), L1 (long-range 1), and L2 (long-range 2). The spin label site R5C is indicated by the red dot. Reduced PRE values ≪1 indicate transient contacts between residue R5C and the lateral loop of the HLH (site L1) and LZ segments (site L2). (b) Calculated PRE values from the two different conformational ensembles (hinged and extended) sampled in an MD trajectory. The experimentally observed values can be reproduced within the precision of the approach that is outlined in the main text. The extended subensemble cannot account for long-range PREs, only for PREs at site S. The hinged ensemble could again be divided into two substates: one where residue R5 approaches the HLH domain and another one where it approaches the LZ domain. The former leads to reproduction of the effect at site L1 and the latter at site L2. The bottom panel displays a superposition of the theoretical PRE values from the different subensembles. The match between measured and calculated PREs shows that the MD simulation indeed captures the conformational properties of MAX₂. (c) The simulated conformational ensembles used to calculate PREs for sites S, L1, and L2. The green dot indicates the position of residue R5, which was the SDSL site in the experiment.

Figure 3

(a) Experimentally determined SL–SL distance distribution P(r) (black, error in red) obtained for the MTSL tagged R5C-R5C mutant. Superimposed are calculated distance distributions from precomputed rotamer libraries (blue for a hinged state and in red for an extended conformation). The C^α distances are indicated. The calculated distributions fit well within the experimentally determined distance range. (b) DEER form-factor (black) underlying the distance distribution in panel a and fit (red) as obtained through the Tikhonov regularization approach. (c and d) Simulated structures underlying the calculated distance distributions panel a. (e and f) Distance histograms of C^ζ–C^ζ distances for residues R5 obtained from two 200 ns long MD trajectory (panel e corresponds to Figure 1). Within the experimental precision (see main text), the simulated distances match the experimentally determined distances. (g and h) Evolution of C^α–C^α distances for residues R5 for the MD trajectory underlying the histograms in panels e and f.

Figure 4

(a) Depiction of the solvent-exposed surfaces (yellow) of the positively charged amino acids in the NTD of a hinged conformation. The RKRRDH stretch is indicated. (b) DEER-derived distance distribution P(r) for the DNA-free R5C mutant (red) and a DNA-bound R5C mutant (blue) as published earlier (see main text). The errors are shown as superimposed shading. (c) Form factors and fits underlying the data in panel a (DEER signals before background correction are reported in Supporting Information Figure S6). Evidently, the sampled range of distances does not change significantly upon DNA binding. The arrows serve as a guide to the eye.