Specificity of Collybistin-Phosphoinositide Interactions: IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS

Michaela Ludolphs,Daniela Schneeberger,Tolga Soykan,Jonas Schäfer,Theofilos Papadopoulos,Nils Brose,Hermann Schindelin,Claudia Steinem

doi:10.1074/jbc.m115.673400

Abstract

The regulatory protein collybistin (CB) recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2SH3+) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally "opens" CB2SH3+ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering.

Highlights

The regulatory protein collybistin (CB) recruits the receptorscaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells
We showed that small unilamellar vesicles (SUVs) composed of POPC doped with PI[4,5]P2 do not spread to a continuous bilayer on silicon/silicon dioxide substrates at pH 7.4 if the PI[4,5]P2 concentration is larger than 4 mol % [23]
It allowed for a quantitative analysis of the binding properties of CB2 in a label-free manner by means of reflectometric interference spectroscopy

Summary

IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS*

Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2SH3؉) adopts a closed and autoinhibited conformation that largely prevents membrane binding This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally “opens” CB2SH3؉ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. The question arises as to whether the phosphoinositide specificity of CB observed with overlay assays properly reflects the lipid binding specificity of CB in intact phospholipid bilayers That this is a critical issue is ence spectroscopy; SH3, Src homology 3; PI[3,4,5]P3, phosphatidylinositol 3,4,5-trisphosphate; PI[3]P, phosphatidylinositol 3-monophosphate; PI[3,4]P2, phosphatidylinositol 3,4-bisphosphate; PI[4,5]P2, phosphatidylinositol 4,5-bisphosphate; PI[4]P, phosphatidylinositol 4-monophosphate; PI[3,5]P2, phosphatidylinositol 3,5-bisphosphate; SUV, small unilamellar vesicle. RIfS is a well established technique to quantitatively monitor proteinreceptor interactions at solid-supported membranes [22,23,24,25]

Experimental Procedures

Results

Random coil

Discussion