Quantitative description of the phase separation behavior of the multivalent SLP65-CIN85 complex

Abstract

Biomolecular condensates play a major role in cell compartmentalization, besides membrane-enclosed organelles. The multivalent SLP65 and CIN85 proteins are downstream B cell receptor (BCR)-signaling effectors, required for a proper immune response. Both proteins phase separate together with vesicles to form pre-signaling clusters. Within this tripartite system, six PRMs of SLP65 interact promiscuously with three SH3 domains of the CIN85 monomer, establishing 18 individual SH3-PRM interactions whose individual dissociation constants we determined. Based on these 18 dissociation constants, we measured the phase separation properties of the natural SLP65/CIN85 system as well as designer constructs that emphasize the strongest SH3/PRM interactions. By modelling these various SLP65/CIN85 constructs with the program LASSI (LAttice simulation engine for Sticker and Spacer Interactions) we reproduced the observed phase separation properties. In addition, LASSI revealed a deviation in the experimental measurement, which was independently identified as a previously unknown intramolecular interaction. Thus, thermodynamic properties of the individual PRM/SH3 interactions allow to model the phase separation behavior of the SLP65/CIN85 system faithfully.

Autoinhibition in the Signal Transducer CIN85 Modulates B Cell Activation

“…30 In past studies on similar systems, the introduction of R/A mutations was found to be effective in perturbing this type of interaction. 15,17,23,31 We therefore used CIN85-PRM1 mutant peptide binding to the SH3C domain to determine the role of both arginines (R227 and R229) in this interaction. The K D increased 4-fold for the R227A and 12-fold for the R229A mutant, with complete abolition of binding only after mutating both residues (see Figure 2B, Table 1, and Figure S3).…”

“…The recognition of PRM by SH3 domains typically depends on the presence of positively charged residues, such as arginines at the motif’s N- or C-terminus, which often form cation−π interactions with conserved tryptophan residues in the SH3 binding interface . In past studies on similar systems, the introduction of R/A mutations was found to be effective in perturbing this type of interaction. ,,, We therefore used CIN85-PRM1 mutant peptide binding to the SH3C domain to determine the role of both arginines (R227 and R229) in this interaction. The K D increased 4-fold for the R227A and 12-fold for the R229A mutant, with complete abolition of binding only after mutating both residues (see Figure B, Table , and Figure S3).…”

“…Because the promiscuous interactions of CIN85 SH3 domains with SLP65 PRMs drive the LLPS in conjunction with small vesicles, modulating the valency of the CIN85 protein should also have an effect on the propensity for phase separation. Indeed, a study conducted in parallel by our group has investigated the phase separation behavior of CIN85 and SLP65 by in vitro droplet reconstitution assays and lattice-based computational modeling . One main finding of that study was that the threshold concentration for LLPS was lowered for the CIN85 1–333 -R227A/R229A mutant in which the interaction between SH3C and CIN85-PRM1 was abolished (see Figure S8 in Maier et al).…”

“…Indeed, a study conducted in parallel by our group has investigated the phase separation behavior of CIN85 and SLP65 by in vitro droplet reconstitution assays and lattice-based computational modeling . One main finding of that study was that the threshold concentration for LLPS was lowered for the CIN85 1–333 -R227A/R229A mutant in which the interaction between SH3C and CIN85-PRM1 was abolished (see Figure S8 in Maier et al). This can be understood quantitatively only if one assumes the presence of an autoinhibitory interaction, preventing one of the SH3 domains from interacting with SLP65 PRMs.…”