Solution NMR Investigation of Proline-Rich Domains Reveals Mechanism of Modulation of Signal Transduction

Abstract

Proline-rich domains are abundant in eukaryotic proteome and play an essential role in signal transduction by acting as docking sites for a multitude of signaling molecules. This is partly due to the restrictive backbone mobility afforded by the cyclical pyrrolidine ring of proline, which imposes unique conformational constraints and provides a distinct entropic advantage upon binding to its signaling partners. Despite their importance, proline-rich domains are often unexplored by structural biophysical techniques, including X-ray crystallography and NMR, due to the problems associated with their expression and purification in E. coli and due to their disordered nature. Moreover, as prolines lack the amide protons, they are not amenable to conventional 1H-detected NMR methods. Here, we present our latest results related to one such proline-rich domain - 168 amino acids, containing ∼30% prolines - belonging to a human protein ALIX, a multifaceted adaptor protein involved in apoptosis, endocytic membrane trafficking, cell adhesion, viral budding and tumor growth. We demonstrate new strategies to successfully express this domain in E. coli (∼ 40 mg protein / L of bacterial culture), and new NMR methods to carry out complete backbone resonance assignments of this domain. Biophysical investigation of interactions with its binding partners as well as Src-kinase mediated post-translational modifications using modern NMR methods reveal the mechanistic role of prolines in modulation of signal transduction.