Structure and dynamics at N‐ and C‐terminal regions of intrinsically disordered human c‐Myc PEST degron reveal a pH‐induced transition

We investigated the structure and Brownian rotational motion of the PEST region (201‐268) from human c‐Myc oncoprotein, whose overexpression/dysregulation is associated with various types of cancer. The 77‐residue PEST fragment revealed a large Stokes radius (~3.1 nm) and CD spectrum highlighting abundance of disordered structure. Changes in structure/dynamics at two specific sites in PEST degron were observed using time‐resolved fluorescence spectroscopy by labeling Cys 9 near N‐terminal with dansyl probe and inserting a Trp 70 near C‐terminal (PEST M1). Trp in PEST M1 at pH 3 was inaccessible to quencher, showed hindered segmental motion and slow global rotation (~30 ns) in contrast to N‐terminal where the dansyl probe was free, exposed with fast global rotation (~5 ns). Remarkably, this large monomeric structure at acidic pH was retained irrespective of ionic strength (0.03‐0.25 M) and partially so in presence of 6 M Gdn.HCl. With gradual increase in pH, a structural transition (~pH 4.8) into a more exposed and freely rotating Trp was noticeable. Interestingly, the induced structure at C‐terminal also influenced the dynamics of dansyl probe near N‐terminal, which otherwise remained unstructured at pH > 5. FRET measurements confirmed a 11 Å decrease in distance between dansyl and indole at pH 4 compared to pH 9, coinciding with enhanced ANS binding and increase in strand/helix population in both PEST fragments. The protonation of glutamate/aspartate residues in C‐terminal region of PEST is implicated in this disorder‐order transition. This may have a bearing on the role of PEST in endocytic trafficking of eukaryotic proteins.