Myc phosphorylation in its basic helix–loop–helix region destabilizes transient α-helical structures, disrupting Max and DNA binding

Abstract

Myelocytomatosis proto-oncogene transcription factor (Myc) is an intrinsically disordered protein with critical roles in cellular homeostasis and neoplastic transformation. It is tightly regulated in the cell, with Myc phosphorylation playing a major role. In addition to the well-described tandem phosphorylation of Thr-52 and Ser-62 in the Myc transactivation domain linked to its degradation, P21 (RAC1)-activated kinase 2 (PAK2)-mediated phosphorylation of serine and threonine residues in the C-terminal basic helix-loop-helix leucine zipper (bHLH-LZ) region regulates Myc transcriptional activity. Here we report that PAK2 preferentially phosphorylates Myc twice, at Thr-358 and Ser-373, with only a minor fraction being modified at the previously identified Thr-400 site. For transcriptional activity, Myc binds E-box DNA elements, requiring its heterodimerization with Myc-associated factor X (Max) via the bHLH-LZ regions. Using isothermal calorimetry (ITC), we found that Myc phosphorylation destabilizes this ternary protein-DNA complex by decreasing Myc's affinity for Max by 2 orders of magnitude, suggesting a major effect of phosphorylation on this complex. Phosphomimetic substitutions revealed that Ser-373 dominates the effect on Myc-Max heterodimerization. Moreover, a T400D substitution disrupted Myc's affinity for Max. ITC, NMR, and CD analyses of several Myc variants suggested that the effect of phosphorylation on the Myc-Max interaction is caused by secondary structure disruption during heterodimerization rather than by a change in the structurally disordered state of Myc or by phosphorylation-induced electrostatic repulsion in the heterodimer. Our findings provide critical insights into the effects of PAK2-catalyzed phosphorylation of Myc on its interactions with Max and DNA.

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

“…Instead, it contains a heterogeneous ensemble of states that enables rapid switching between extended and 'hinged' conformations. These findings complement long-standing research programs that aim at structure-elucidation of the DNA-binding sites of basic-helix-loop-helix-type transcription factors [78] , [79] , [80] , [81] , [82] .…”

Mapping Hidden Residual Structure within the Myc bHLH-LZ Domain Using Chemical Denaturant Titration

“…3) corresponds to the position within the leucine zipper that is important in 5 ensuring specificity for the interaction with Max, and mutation of residues E417, R423 and 6 R424 results in significant homodimerisation (Soucek et al, 1998). The long-range tertiary 7 contact defined by the PREs (between the 360-380 and 400-410 regions) coincides with the 8 phosphorylation sites S373 and T400 (Macek et al, 2018), suggesting that phosphorylation 9 perturbs this conformational ensemble, leading to its role in the mechanism of gene regulation. 10…”

Quo Vadis Biomolecular NMR Spectroscopy?

“…Many examples of such stabilizing and destabilizing phosphorylation effects in classically folded proteins are known [144]. The emergence of IDPs has helped to extend this concept to regions of residual secondary structure, where phosphorylation appears to exert even greater effects [93,108,145]. Given that pre-structured motifs usually function as molecular recognition elements in IDP–ligand interactions [146], signaling and phosphorylation-dependent changes in these structural propensities influence binding energies and affinities in pronounced ways, especially in binding-induced disorder-to-order and order-to-disorder transitions [147].…”