Phase Separation Studied by Nuclear Magnetic Resonance
by Daniel Sieme
Date of Examination:2023-01-17
Date of issue:2024-01-04
Advisor:Prof. Dr. Christian Griesinger
Referee:Prof. Dr. Christian Griesinger
Referee:Prof. Dr. Jürgen Wienands
Persistent Address:
http://dx.doi.org/10.53846/goediss-10275
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Abstract
English
Signal transduction by the ligated B cell 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. I 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, I 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+. My findings therefore suggest that the intramolecular interaction to the adjacent disordered linker is effective in modulating CIN85’s valency both in vitro and in vivo. This therefore constitutes a powerful way for the modulation of SLP65/CIN85 condensate formation and subsequent B cell signaling processes within the cell. In addition to the scaffold proteins CIN85 and SLP65, glycosaminoglycans like heparin play an important role in immunological processes, such as inflammation. Besides their role as anti-coagulants in modern medicine, heparins can serve as a reservoir of metal ions in vivo. By exploiting the difference in NMR spectra of heparin-bound sodium and free hydrated sodium ions, I determined the relative heparin-binding affinity of the cations Ca2+, Mg2+ and Al3+. These results indicated the presence of more than one metal ion-binding site on heparin and determined the order of binding affinity for the studied ions. More generally, it highlighted the potential of multinuclear quadrupolar NMR in quantification of metal ion binding to heparins and how to distinguish various binding modes. Obtaining this type of information is crucial for understanding the interaction of heparins with soluble and cell-surface macromolecules in vivo. This is due to metal ion-specific structural changes in heparins, tuning their binding behavior. The approach presented here can therefore aid in understanding how these differences arise from differential metal-ion interactions of heparins and could help in determining potential structural causes for clinical side-effects of applying heparin, such as heparin-induced thrombocytopenia (HIT).
Keywords: cell signaling; peptides and proteins; post-translational modifications; nuclear magnetic resonance; liquid-liquid phase separation
