Specificity of collybistin interaction with phosphoinositides: Impact of the individual protein domains

Collybistin, a brain-specific GEF, is required for gephyrin clustering at the postsynaptic membrane. This is important for the correct arrangement of GABAA and glycine receptors. Binding of collybistin to a membrane is initiated by a C-terminal PH-domain. In general, this domain is known to specifically bind to phosphoinositides (PIPs) in the membrane. The aim of this study was to design an in vitro assay that enables detailed studies of binding of collybistin binding to phosphoinositides. With this assay we analyzed the impact of different collybistin 2 domains on binding to a membrane. Solid-supported lipid bilayers (SLB) with different phosphoinositides mimicking the cellular postsynaptic membrane. This approach allowed to investigate specific binding of the protein isoforms using reflective interference spectroscopy (RIfS). We found out that the PH domain (CB2PH) from collybistin prefers binding to monophosphorylated PIPs with moderate binding affinity. The PH/DH-tandem domain (CB2SH3-) reveals a weaker binding affinity to different PIPs except for PI(3,5)P2. In addition the collybistin isoform containing an additional SH3 domain (CB2SH3+) showed no significant binding to PIPs. This suggested a closed/inactive protein conformation. Activation of CB2SH3+ is initiated in vivo by the postsynaptic cell adhesion protein Neuroligin-2. For our in vitro studies this was successfully mimicked by two point mutations in the protein isoform CB2SH3+/W24A-E262A. In contrast to CB2SH3+, the partly-activated mutant CB2SH3+/W24A-E262A binds to phosphoinositides in SLBs with decreasing binding affinity to PI(3)P, PI(4)P and PI(3,5)P2. This study provides new insights into the binding of collybistin to phosphoinositides in membranes. In order to draw a comprehensive picture of this protein to membrane binding process, the impact of each protein domain was investigated.2015-10-2