Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions

Liquid–liquid phase separation of proteins underpins the formation of membraneless compartments in living cells. Elucidating the molecular driving forces underlying protein phase transitions is therefore a key objective for understanding biological function and malfunction. Here we show that cellular proteins, which form condensates at low salt concentrations, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, can reenter a phase-separated regime at high salt concentrations. By bringing together experiments and simulations, we demonstrate that this reentrant phase transition in the high-salt regime is driven by hydrophobic and non-ionic interactions, and is mechanistically distinct from the low-salt regime, where condensates are additionally stabilized by electrostatic forces. Our work thus sheds light on the cooperation of hydrophobic and non-ionic interactions as general driving forces in the condensation process, with important implications for aberrant function, druggability, and material properties of biomolecular condensates.

Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions

Many cellular proteins demix spontaneously from solution to form liquid condensates. These phase-separated systems have wide-ranging roles in health and disease. Elucidating the molecular driving forces underlying liquid–liquid phase separation (LLPS) is therefore a key objective for understanding biological function and malfunction. Here we show that proteins implicated in cellular LLPS, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, which form condensates at low salt concentrations, can reenter a phase-separated regime at high salt concentrations. By bringing together experiments and simulations, we demonstrate that phase separation in the high-salt regime is driven by hydrophobic and non-ionic interactions, and is mechanistically distinct from the low-salt regime, where condensates are additionally stabilized by electrostatic forces. Our work thus provides a new view on the cooperation of hydrophobicity and non-ionic interactions as non-specific driving forces for the condensation process, with important implications for aberrant function, druggability, and material properties of biomolecular condensates.

Structure of the ALS Mutation Target Annexin A11 Reveals a Stabilising N-Terminal Segment

The functions of the annexin family of proteins involve binding to Ca2+, lipid membranes, other proteins, and RNA, and the annexins share a common folded core structure at the C terminus. Annexin A11 (AnxA11) has a long N-terminal region, which is predicted to be disordered, binds RNA, and forms membraneless organelles involved in neuronal transport. Mutations in AnxA11 have been linked to amyotrophic lateral sclerosis (ALS). We studied the structure and stability of AnxA11 and identified a short stabilising segment in the N-terminal end of the folded core, which links domains I and IV. The crystal structure of the AnxA11 core highlights main-chain hydrogen bonding interactions formed through this bridging segment, which are likely conserved in most annexins. The structure was also used to study the currently known ALS mutations in AnxA11. Three of these mutations correspond to buried Arg residues highly conserved in the annexin family, indicating central roles in annexin folding. The structural data provide starting points for detailed structure–function studies of both full-length AnxA11 and the disease variants being identified in ALS.

Structure of the ALS Mutation Target Annexin A11 Reveals a Stabilising N-Terminal Segment

The functions of the annexin family of proteins involve binding to Ca2+, lipid membranes, other proteins, and RNA, and the annexins share a common folded core structure at the C terminus. Annexin A11 (AnxA11) has a long N-terminal region, which is predicted to be disordered, binds RNA, and forms membraneless organelles involved in neuronal transport. Mutations in AnxA11 have been linked to amyotrophic lateral sclerosis (ALS). We studied the structure and stability of AnxA11 and identified a short stabilising segment in the N-terminal end of the folded core, which links domains I and IV. Crystal structure of the AnxA11 core highlights main-chain hydrogen bonding interactions formed through this bridging segment, which are likely conserved in most annexins. The structure was also used to study the currently known ALS mutations in AnxA11. Three of these mutations correspond to buried Arg residues highly conserved in the annexin family, indicating central roles in annexin folding. The structural data provide starting points for detailed structure-function studies of both full-length AnxA11 and the disease variants being identified in ALS.