Decoding of Small-Molecule and RNA Interactions with Intrinsically Disordered Proteins

Sammanfattning

Intrinsically disordered proteins (IDPs), which lack a stable three-dimensional structure, are involved in a wide range of important cellular functions. Due to the absence of a distinct structure and defined binding pockets, characterization of IDP binding to small molecules, nucleic acids, and other biomolecules remains a major challenge in structural biology, making it difficult to rationalize how they could be targeted therapeutically. Therefore, a detailed understanding of how IDPs engage in these interactions and characterization of binding behaviour to their partners is crucial. Here we investigated binding of small molecules to engineered proteins with disordered low-complexity (LC) domains with distinct charge properties, and to the Alzheimer’s disease associated human protein tau using native mass spectrometry. We expanded our study to RNA which has important cellular functions and identified its binding preferences to our designed LC variants using mass photometry and native mass spectrometry. We find that small molecule binding is charge independent and predominantly governed by compact conformational states, while RNA binds via positively charged amino acid side chains in the LC region. Our findings serve as a proof of concept of how protein engineering, native mass spectrometry and mass photometry can be used to resolve distinct binding modes of small molecules and biomolecules to intrinsically disordered proteins.