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.

Keywords: adaptor proteins; interdomain linker; intrinsically disordered; multivalency; phase separation.

Fig. 1.

Multivalent interactions in adaptor proteins drive phase separation. (A, Top) Model of the interaction of the multivalent proteins p-nephrin, Nck, and N-WASP. (A, Bottom) Upon mixing 3 μM p-nephrin, 2 μM N-WASP (10% Alexa488-labeled), and 10 μM Nck, micrometer-scale droplets can be visualized by fluorescence (Left) and differential interference contrast (Right) microscopies. (B) Some constructs based on Nck used in this study.

Fig. 2.

Different di-SH3 fragments of Nck are not equivalent in their phase separation properties. Phase separation of N-WASP and Nck proteins in the presence of 7.5 μM p-nephrin is shown. Red and blue symbols indicate phase separation and no phase separation, respectively. (A–D) Data for Nck, S1-L1-S2-L3-SH2, S1-L1-S3-L3-SH2, and S2-L2-S3-L3-SH2, respectively.

Fig. 3.

L1 promotes phase separation of a di-SH3 protein through a basic N-terminal element. (A) Sequences of L1 used in experiments shown in B–G. (B–G) Phase separation experiments were performed in the presence of 7.5 μM p-nephrin. Red and blue symbols indicate phase separation and no phase separation, respectively.

Fig. 4.

L1 promotes self-association of di-SH3 proteins but does not substantially alter binding to N-WASP. (A) Isothermal titration calorimetry analysis of the binding of L1-S2-L2-S3-L3-SH2 or L1chargeshuffle-S2-L2-S3-L3-SH2 to N-WASP. A total of 1.1 mM of L1-S2-L2-S3-L3-SH2 was titrated to 100 μM N-WASP (Left) and 1.1 mM L1chargeshuffle-S2-L2-S3-L3-SH2 was titrated to 50 μM N-WASP (Right), thereby giving different molar ratios in the x axis. Heats of injection (Upper), single-site fit to integrated heats (Middle), and residuals of the fit (Lower), respectively, are shown. (B) Diffusion coefficients of L1-S2-L2-S3-L3-SH2 (brown squares) and S2-L2-S3-L3-SH2 (blue circles) at different concentrations, measured by DLS. Error bars indicate replicates from three different protein preparations. (C) Pyrene-actin assembly assays contained 2 μM actin (5% pyrene-labeled) and 50 nM Arp2/3 complex (black), plus 50 nM N-WASP (green), 50 nM N-WASP and 5 μM S2-L2-S3-L3-SH2 (red), or 50 nM N-WASP and 5 μM L1-S2-L2-S3-L3-SH2. Data for S2-L2-S3-L3-SH2 and L1-S2-L2-S3-L3-SH2 reflect three replicates.

Fig. 5.

L1 is partially disordered, and also binds the second SH3 domain. (A) Overlaid ¹H/¹⁵N TROSY spectra of 250 μM U-[¹⁵N] labeled L1-S2-L2-S3-L3-SH2 (black) and 250 μM S2-L2-S3-L3-SH2 (red). (B) Overlaid Trp indole region of ¹H/¹⁵N TROSY spectra of L1-S2-L2-S3-L3-SH2 (black), 250 μM S2-L2-S3-L3-SH2 (red), and 310 μM S2 (blue). (C) Average fold change (±SD) in intensities of 15 peaks from S2 in ¹H/¹⁵N HSQC spectra of ¹⁵N-labeled L1-S2 and S2 at the indicated concentrations. Intensities were normalized to the intensities at 50 μM.

Fig. 6.

L1 is highly conserved. Sequence alignment of the L1 region in Nck1 generated by ESPript (58). Conserved charged residues are colored blue for basic residues and red for acidic residues.