Native Top-Down Mass Spectrometry and Ion Mobility Spectrometry of the Interaction of Tau Protein with a Molecular Tweezer Assembly Modulator

Abstract

Native top-down mass spectrometry (MS) and ion mobility spectrometry were applied to characterize the interaction of a molecular tweezer assembly modulator, CLR01, with tau, a protein believed to be involved in a number of neurodegenerative disorders, including Alzheimer’s disease. The tweezer CLR01 has been shown to inhibit aggregation of amyloidogenic polypeptides without toxic side effects. ESI-MS spectra for different forms of tau protein (full-length, fragments, phosphorylated, etc) in the presence of CLR01 indicate a primary binding stoichiometry of 1:1. The relatively high charging of the protein measured from nondenaturing solutions is typical of intrinsically disordered proteins, such as tau. Top-down mass spectrometry using electron capture dissociation (ECD) is a tool to determine not only the sites of post-translational modifications, but also the binding site(s) of non-covalent interacting ligands to biomolecules. The intact protein and the protein-modulator complex were further subjected to ECD-MS to obtain sequence information, map phosphorylation sites, and pinpoint the sites of inhibitor binding. ESI-MS data for intact tau proteins indicate that top-down MS is amenable to the study of various tau isoforms and their post-translational modifications (PTMs). The ECD-MS data point to a CLR01 binding site in the microtubule-binding region of tau, spanning residues K294-K331, which includes a six-residue nucleating segment PHF6 (VQIVYK) implicated in aggregation. Furthermore, ion mobility experiments on the tau fragment reveal a shift towards a more compact structure in the presence of CLR01. The mass spectrometry study suggests a picture for the molecular mechanism for the modulation of protein-protein interactions in tau by CLR01.