Molecular mechanisms of Ded1p phase separation

Abstract

Liquid-liquid phase separation (LLPS) describes a mechanism that results in the clustering of biomacromolecules, such as nucleic acids and proteins, into large microscopically visible foci. On a molecular level, LLPS is the result of a not fully characterized, redundant network of weak protein-protein and protein-RNA interactions. It is now widely understood that LLPS plays an important role in the formation of many membraneless organelles. In this work, I used nuclear magnetic resonance (NMR) spectroscopy and biochemical assays to elucidate the intermolecular interactions that govern the LLPS of the yeast DEAD-box RNA helicase Definition of Essential Domain 1 (Ded1p), which is a main component of stress granules (SG). The Ded1p protein consists of a highly conserved helicase core of two folded RecA domains that constitute the active site and additional N- and C-terminal disordered extensions. Based on an in vitro LLPS assay, I established that LLPS of Ded1p is mainly mediated by its C-terminal extension. Using paramagnetic relaxation enhancement (PRE) and spin relaxation experiments, the molecular grammar that governs the driving force of LLPS was identified for the C-terminal region. Tryptophan and arginine residues were shown to be highly potent stickers that dynamically drive condensation of the C‑terminal intrinsically disordered region (IDR) of Ded1p. My data also uncover a direct relationship between the mobility of individual amino acids and the importance of these residues for LLPS. Importantly, I established that these findings for the isolated IDR can be transferred to the LLPS behavior of the full-length protein. In addition, I identified interactions between the N- and C-terminal extensions and the folded helicase core that also contribute to LLPS. In a second line of work, I used NMR titration experiments to study the interaction between the RecA core domains and their substrates adenosine triphosphate (ATP) and RNA. Based on five different DEAD-box helicases, I established a conserved energetic coupling between ATP and RNA binding, which is mediated by a conformational rearrangement of the RecA core domains. In the apo state, the RecA domains tumble independently, however, in the presence of both substrates, a transition to a rigid closed conformation takes place. The formation of this closed helicase core state is required for helicase unwinding activity in all tested DEAD-box RNA helicases. Taken together, this work highlights the importance of IDRs for Ded1p LLPS and increases our understanding of the molecular mechanisms of DEAD-box RNA helicases.