FC0096
Adaptor protein Nck  -  Nck SH3

Biological function
Nck plays a role in diverse cellular processes, including axon guidance, cell movement, cell–cell fusion, stress responses, and maintenance of cell–cell adhesions, which are achieved via the polymerization of actin through Nck/neuronal Wiskott-Aldrich syndrome protein (N-WASP)/Arp2/3 complex pathways. Nck phase-separates upon interaction with proline-rich motifs (PRMs) of N-WASP, which occurs at lower concentrations when pTyr-containing phosphorylated nephrin (p-nephrin) is present. Phase separation is dependent on both the number of pTyr motifs in p-nephrin and SH3 domains in Nck.

Domain organization/sequence features
Nck is composed of an SRC homology 2 (SH2) domain, which binds phosphotyrosine (pTyr) in pYDEV sequence motifs and three SRC-homology 3 (SH3) domains, which interact with a variety of PRMs. The SH2 and SH3 modules (marked as S1, S2, S3) are connected by linkers (L1, L2, L3) ranging from 24 to 50 residues.

Structural evidence
NMR analysis of Nck2 has shown that the linkers are mostly disordered, although a portion of the linker between the first and second SH3 domains interacts weakly with the latter domain. The spectrum of L1-S2-L2- S3-L3-SH2 and L1-S2 shows a series of intense, poorly dispersed resonances, representing disordered, dynamic residues. Peaks corresponding to the C-terminal acidic portion of L1 have high and relatively uniform intensity, whereas peaks in the N terminus are appreciably weaker. Five of the N-terminal 11 residues could not be assigned due to exchange broadening; the first Lys residue of the KVKRK motif also could not be assigned. These data indicate variable, weak-affinity interactions, which was estimated to have Kd value of 1.3 mM based on ¹H/¹⁵N HSQC spectra.

Biochemical evidence
The linker between the first and second SH3 domains of Nck (L1) affects the phase separation behavior of p-nephrin/Nck/N- WASP oligomers. The S2-L2- S3-L3-SH2 does not phase-separate even up to a concentration of 250 μM plus 250 μM N- WASP, but adding L1 lowers the phase boundary to 30 μM (for both partners). The L1 has 10 basic residues lie within its N- terminal half, and five acidic residues lie within its C-terminal half. Deletion of the first N-terminal 17 residues, or only the KVKRK motif, or even mutation of three Lys in this region, inhibited phase-separation (boundary above 250 μM concentration plus 250 μM N-WASP). In contrast, increasing the overall positive charge of L1 or charge density in the N terminal region enhances phase separation. These trends were similar in the presence of p-nephrin. L1 does not impact the affinity for N- WASP, instead it promotes self-assembly of Nck based on dynamic light scattering measurements, also supported by NMR data (see above).

Structure/Mechanism
The linker between the first and second SH3 domain of Nck consists of a basic N-terminal element and an acidic C-terminal element. The basic element promotes phase separation of Nck assemblies through two short linear motifs (e.g. KVKRK) via their weak interactions with the second SH3 domain. The overall positively charged character of the linker enables to interact with the negatively charged promixal second SH3 domain. All these interactions act synergistically to promote collective interactions and crosslinking, ultimately leading to phase separation.

Mechanism category
tethering

Significance
Fuzzy, weak interactions between the linker and the SH3 domain of Nck contribute to increasing valency and combinatorial crosslinking within the Nck/N-WASP/nephrin assembly and promote phase separation.