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 MAX 2 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 MAX 2 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 MAX 2 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.

How to assess the structural dynamics of transcription factors by integrating sparse NMR and EPR constraints with molecular dynamics simulations

“…b) Experimentally observed PRE effects as a function of residue position in MAX:MAX (top) compared to PRE effects extracted from MD simulations (bottom). The match between both data sets is good, such that the conformational ensemble sampled in the MD simulations could be verified by the experimental observations (adapted from reference [61] with permission of the publisher.) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)…”

“…Recently, approaches based on the site-specific introduction of paramagnetic labels could characterize the DNA-binding sites of (partially) disordered TFs [61] , [62] , [63] , [64] , [65] . These approaches employed the so-called paramagnetic relaxation enhancement (PRE) technique [66] .…”

“…Recently, putting the combination of PRE-based relaxometry and MD simulations to use, the intrinsically disordered DNA-binding interface of the important transcription factor MAX (MYC-associated factor X) has been investigated [61] , [63] , [77] . It could be shown that its conformational space is not merely void of long-lived structural elements.…”

“…1 depicts the extended and hinged states. The figure also displays how PRE-NMR revealed the involved long-range contacts between the DNA-binding site and other domains of the MAX:MAX homodimer and how MD-simulations could predict these effects [61] . The combination of both data sets thus enabled the validation of the simulated conformational ensemble.…”

Exploring Energy Landscapes of Intrinsically Disordered Proteins: Insights into Functional Mechanisms

“…Thus, it becomes more compact and more negatively charged in the HIPK1-PAGE4 version. Figure 7 shows R g in the interval [26,76] (N = 25). The data indicates that the region in which HIPK1-PAGE4 has the highest density of states (region A), corresponds to the lowest R g , i.e.…”