Neuroligin-2 dependent conformational activation of collybistin reconstituted in supported hybrid membranes

Jonas Schäfer,Lucas Förster,Ingo Mey,Theofilos Papadopoulos,Nils Brose,Claudia Steinem

doi:10.1074/jbc.ra120.015347

Abstract

The assembly of the postsynaptic transmitter sensing machinery at inhibitory nerve cell synapses requires the intimate interplay between cell adhesion proteins, scaffold and adaptor proteins, and γ-aminobutyric acid (GABA) or glycine receptors. We developed an in vitro membrane system to reconstitute this process, to identify the essential protein components, and to define their mechanism of action, with a specific focus on the mechanism by which the cytosolic C terminus of the synaptic cell adhesion protein Neuroligin-2 alters the conformation of the adaptor protein Collybistin-2 and thereby controls Collybistin-2-interactions with phosphoinositides (PtdInsPs) in the plasma membrane. Supported hybrid membranes doped with different PtdInsPs and 1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DGS-NTA(Ni)) to allow for the specific adsorption of the His6-tagged intracellular domain of Neuroligin-2 (His-cytNL2) were prepared on hydrophobically functionalized silicon dioxide substrates via vesicle spreading. Two different collybistin variants, the WT protein (CB2SH3) and a mutant that adopts an intrinsically ‘open’ and activated conformation (CB2SH3/W24A-E262A), were bound to supported membranes in the absence or presence of His-cytNL2. The corresponding binding data, obtained by reflectometric interference spectroscopy, show that the interaction of the C terminus of Neuroligin-2 with Collybistin-2 induces a conformational change in Collybistin-2 that promotes its interaction with distinct membrane PtdInsPs.

Highlights

Synaptic signaling between neurons is based on the presynaptic release and postsynaptic sensing of neurotransmitters
The spreading process of small unilamellar vesicles (SUVs) after HMDS functionalization was monitored in a time-resolved manner by reflectometric interference spectroscopy (RIfS)
Adsorption of CB2SH3/W24A-E262A led to DOTCB2 of (0.67 6 0.13) nm (n = 4). These results demonstrate that the CB2SH3/W24A-E262A mutant can interact with the phosphoinositides presumably because of an open conformation induced by the mutations

Summary

Introduction

Synaptic signaling between neurons is based on the presynaptic release and postsynaptic sensing of neurotransmitters. To follow the binding of the cytosolic domain of NL2 (HiscytNL2) via its His6-tag to the membrane, we prepared lipid monolayers composed of POPC, doped with 3 mol % of PtdInsP and 3 mol % of DGS-NTA(Ni).

Results

Conclusion