Conserved interdomain linker promotes phase separation of the multivalent adaptor protein Nck

Abstract

The organization of membranes, the cytosol, and the nucleus of eukaryotic cells can be controlled through phase separation of lipids, proteins, and nucleic acids. Collective interactions of multivalent molecules mediated by modular binding domains can induce gelation and phase separation in several cytosolic and membrane-associated systems. The adaptor protein Nck has three SRC-homology 3 (SH3) domains that bind multiple proline-rich segments in the actin regulatory protein neuronal Wiskott-Aldrich syndrome protein (N-WASP) and an SH2 domain that binds to multiple phosphotyrosine sites in the adhesion protein nephrin, leading to phase separation. Here, we show that the 50-residue linker between the first two SH3 domains of Nck enhances phase separation of Nck/N-WASP/nephrin assemblies. Two linear motifs within this element, as well as its overall positively charged character, are important for this effect. The linker increases the driving force for self-assembly of Nck, likely through weak interactions with the second SH3 domain, and this effect appears to promote phase separation. The linker sequence is highly conserved, suggesting that the sequence determinants of the driving forces for phase separation may be generally important to Nck functions. Our studies demonstrate that linker regions between modular domains can contribute to the driving forces for self-assembly and phase separation of multivalent proteins.phase separation | multivalency | interdomain linker | adaptor proteins | intrinsically disordered E ukaryotic cells are compartmentalized into different organelles, which have specific functions. Membrane-bound organelles, such as the endoplasmic reticulum and vacuoles, have been studied extensively, and their functions are relatively well understood. Non-membrane-bound organelles also exist in cells. These organelles include P-granules, Cajal bodies, promyelocytic leukemia (PML) bodies, paraspeckles, and the nucleolus, for example (1). Recently, many of these non-membrane-bound structures have been proposed to form via liquid-liquid demixing phase transitions of their constituent molecules (protein, DNA, and RNA) (2). For example, germ-line P-granules and the nucleolus show liquid-like properties, including droplet fusion, fission, and shearing, and they dissolve and condense in a fashion reminiscent of a phase separation process (3-5). Similarly, stress granules also condense and dissolve, sequestering the family of DYRK kinases (6). Organelles, such as the centrosome, have also been studied through the lens of phase separation (7).A molecular understanding of the physical properties that promote formation of new phases is important for understanding the nature, function, and regulation of non-membrane-bound organelles. We recently showed that multivalent proteins interact with multivalent ligands to form higher order species in aqueous solution that phase-separate and give rise to liquid droplets. The constituent proteins are highly concentrated in these droplets (∼100-fold) (8). This phenomenon ap...

Cryptic sequence features within the disordered protein p27 Kip1 regulate cell cycle signaling

“…Indeed, their existence was previously postulated (29). Their impact has been established for the C-terminal region of the core subunit of the Polycomb repressive complex 1 that compacts chromatin and inhibits chromatin remodeling (16) and in the Nck system that undergoes phase separation via interactions with polyvalent partners (30). Our analysis clearly shows that the sequence context of an IDR can modulate the functionality of a primary motif.…”

Protein Phase Separation: A New Phase in Cell Biology

“…In addition to serving as merely linkers, IDRs may also mediate ‘sticky’ interactions to promote phase transition [45]. McKnight and coworkers found that concentrated solutions of different IDRs could, over time, spontaneously form hydrogels [39], similar to existing observations made regarding FG-repeat-containing nuclear pore proteins [46].…”

Allosteric N-WASP activation by an inter-SH3 domain linker in Nck

“…In addition to activating N-WASP through GBD binding, the inter-SH3AB linker has been shown to bind intramolecularly to the SH3B domain (45) and mediate Nck homo-oligomerization and phase separation (46). The N-WASP C-helix motif is similarly multifunctional, binding in a mutually exclusive manner to G actin and the Arp2/3 complex (47) in addition to mediating autoinhibition by intramolecular GBD interactions.…”