title: 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.
authors.affiliation: Department of Biophysics, UT Southwestern Medical Center, Dallas, TX 75390; Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Sudeep
authors.initials: S
authors.lastname: Banjade
authors.affiliation: Department of Biophysics, UT Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wu
authors.affiliation: Department of Biomedical Engineering and Center for Biological Systems Engineering, Washington University in St. Louis, St. Louis, MO 63130.
authors.forename: Anuradha
authors.initials: A
authors.lastname: Mittal
authors.affiliation: Department of Biophysics, UT Southwestern Medical Center, Dallas, TX 75390; Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX 75390;
authors.forename: William B
authors.initials: WB
authors.lastname: Peeples
authors.affiliation: Department of Biomedical Engineering and Center for Biological Systems Engineering, Washington University in St. Louis, St. Louis, MO 63130.
authors.forename: Rohit V
authors.initials: RV
authors.lastname: Pappu
authors.affiliation: Department of Biophysics, UT Southwestern Medical Center, Dallas, TX 75390; Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX 75390; michael.rosen@utsouthwestern.edu.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Rosen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017124:Conserved Sequence; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C078332:Nck protein; D015513:Oncogene Proteins; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D011392:Proline
keywords: adaptor proteins; interdomain linker; intrinsically disordered; multivalency; phase separation