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.

Heparins and heparan sulfate polysaccharides are negatively charged glycosaminoglycans and play important roles in cell-to-matrix and cell-to-cell signaling processes. Metal ion binding to heparins alters the conformation of heparins and influences their function. Various experimental techniques have been used to investigate metal ion-heparin interactions, frequently with inconsistent results. Exploiting the quadrupolar 23Na nucleus, we herein develop a 23Na NMR-based competition assay and monitor the binding of divalent Ca2+ and Mg2+ and trivalent Al3+ metal ions to sodium heparin and the consequent release of sodium ions from heparin. The 23Na spin relaxation rates and translational diffusion coefficients are utilized to quantify the metal ion-induced release of sodium ions from heparin. In the case of the Al3+ ion, the complementary approach of 27Al quadrupolar NMR is employed as a direct probe of ion binding to heparin. Our NMR results demonstrate at least two metal ion-binding sites with different affinities on heparin, potentially undergoing dynamic exchange. For the site with lower metal ion binding affinity, the order of Ca2+ > Mg2+ > Al3+ is obtained, in which even the weakly binding Al3+ ion is capable of displacing sodium ions from heparin. Overall, the multinuclear quadrupolar NMR approach employed here can monitor and quantify metal ion binding to heparin and capture different modes of metal ion-heparin binding.

Little is known on how maturation of Alzheimer’s disease-related amyloid β (Aβ) fibrils alters their stability and potentially influences their spreading in brain. Using high-pressure NMR, we show that progression...

Signal transduction by the ligated B cell antigen receptor (BCR) depends on the pre-organization of its intracellular components, such as the effector proteins SLP65 and CIN85 within phase-separated condensates. These liquid-like condensates are based on the interaction between three Src homology 3 (SH3) domains and corresponding proline-rich recognition motifs (PRM) in CIN85 and SLP65, respectively. However, detailed information on the protein conformation and how it impacts on the capability of SLP65/CIN85 condensates to orchestrate BCR signal transduction is still lacking. This study identifies a hitherto unknown intramolecular SH3:PRM interaction between the C-terminal SH3 domain (SH3C) of CIN85 and an adjacent PRM. We used high-resolution nuclear magnetic resonance (NMR) experiments to study the flexible linker region containing the PRM and determined the extent of the interaction in multidomain constructs of the protein. Moreover, we observed that the phosphorylation of a serine residue located in the immediate vicinity of the PRM regulates this intramolecular interaction. This allows for a dynamic modulation of CIN85’s valency towards SLP65, regulating the extent of liquid-liquid phase separation. B cell culture experiments further revealed that the PRM/SH3C interaction is crucial for maintaining the physiological level of SLP65/CIN85 condensate formation, activation-induced membrane recruitment of CIN85, and subsequent mobilization of Ca2+. Our findings therefore suggest that the intramolecular interaction to the adjacent disordered linker is effective in modulating CIN85’s valency bothin vitroandin vivo. This therefore constitutes a powerful way for the modulation of SLP65/CIN85 condensate formation and subsequent B cell signaling processes within the cell.