Structural investigation of the small heat shock protein HSPB1 in the presence of a phase-separated client.

Abstract

Many of the proteins found in pathological protein fibrils and aggregates also exhibit tendencies for liquid-liquid phase separation (LLPS) both in vitro and in cells. The transition between the LLPS state and the aggregate state can be modulated by protein chaperones that can block the liquid-to-solid transition and help maintain the LLPS state. Previously, we have characterized the structural features exhibited by FUS LC, a protein that has been demonstrated to undergo LLPS and form fibrils, as it transitions from the LLPS state to the fibril state. Here, we build on this work and examine the α-crystallin chaperone HSPB1 as it interacts with FUS LC in the LLPS state, and compare the structural features of HSPB1 without a client protein to its structural features in the presence of a phase-separated client protein. The heterogeneous nature of the chaperone and client system and the wide range of dynamics adopted by the component proteins present unique challenges for structural interrogation. We therefore integrate solid-state magic angle spinning NMR spectroscopy, coarse-grained molecular dynamics simulations, and cryo-EM to build a model for the dynamics adopted by HSPB1 as it interacts with client proteins, with a specific focus on the role of the N-terminal domain of HSPB1 in client recognition and chaperone function.