Exploring the dynamics and interactions of the N-myc transactivation domain through solution NMR

Abstract

The myc family of proteins (c-, N- and L-myc) are transcription factors (TFs) responsible for maintaining the proliferative program in cells. They consist of a C-terminal domain that mediates heterodimerisation with Max and DNA binding, and an N-terminal disordered region culminating in the transactivation domain (TAD). The TAD participates in many protein-protein interactions, notably with kinases that promote stability (Aurora-A) or degradation (ERK1, GSK3) via the ubiquitin-proteasome system. Structural characterization of the TAD of N-myc, is very limited, with the exception of a crystal structure of Aurora-A bound to a helical region of N-myc. We probed the structure, dynamics and interactions of N-myc TAD using nuclear magnetic resonance (NMR) spectroscopy following its complete backbone assignment enabled by a truncation approach. Chemical shift analysis revealed that N-myc has two regions with clear helical propensity: one region within Trp77-Glu86 and the second between Ala122-Glu132. These regions also have more restricted ps-ns motions than the rest of the TAD, and, along with another known interaction site (myc box I), have comparatively high transverse (R2) 15N relaxation rates, indicative of slower timescale dynamics and/or chemical exchange. Collectively these features suggest differential propensities for structure and interaction, either internal or with binding partners, across the TAD. Solution studies on the interaction between N-myc and Aurora-A revealed a previously uncharacterised binding site. The specificity and kinetics of sequential phosphorylation of N-myc by ERK1 and GSK3 were characterised using NMR and showed no significant structural changes through the rest of the TAD. When doubly phosphorylated on residues Ser62 and Thr58, N-myc formed a robust interaction with the Fbxw7-Skp1 complex. Our study provides foundational insights into N-myc TAD dynamics and a backbone assignment that will underpin future work on the structure, dynamics, interactions and regulatory post-translational modifications of this key oncoprotein.