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

Open AccessPublished:October 30, 2020DOI:https://doi.org/10.1074/jbc.RA120.015347
      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.
      Synaptic signaling between neurons is based on the presynaptic release and postsynaptic sensing of neurotransmitters. In the mammalian brain, inhibitory synaptic signaling relies on the neurotransmitter γ-aminobutyric acid (GABA), which is detected by specific postsynaptic GABAA receptors that operate as ligand-gated Cl-channels. The clustering of these receptors in the postsynaptic plasma membrane, in direct apposition to the presynaptic transmitter release site, ensures fast signal transduction, so that GABA release induces a hyperpolarization of the postsynaptic membrane and reduced excitability (
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      A defined protein machinery is required for GABAA receptor clustering at many GABAergic postsynaptic sites (Fig. 1A). At the core of this machinery is the cell adhesion protein Neuroligin-2 (NL2) (
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      The role of collybistin in gephyrin clustering at inhibitory synapses: facts and open questions.
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      ) and the adaptor protein Collybistin (CB). Previous studies showed that CB binds to phosphoinositides (PtdInsPs) via its pleckstrin homology (PH) domain (
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      PH-Domain-driven targeting of collybistin but not Cdc42 activation is required for synaptic gephyrin clustering.
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      • Papadopoulos T.
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      • Brose N.
      Lipid binding defects and perturbed synaptogenic activity of a Collybistin R290H mutant that causes epilepsy and intellectual disability.
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      • Mueller R.
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      • Rhee H.J.
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      • Mueller R.
      • Schultz C.
      • et al.
      Endosomal phosphatidylinositol-3-phosphate promotes gephyrin clustering and GABAergic neurotransmission at inhibitory postsynapses.
      ). On binding to PtdInsPs, CB serves as an adaptor to connect Gephyrin to the plasma membrane. This triggers Gephyrin oligomerization and the subsequent clustering of GABAA receptors (
      • Soykan T.
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      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
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      • Betz H.
      • Kirsch J.
      Collybistin, a newly identified brain-specific GEF, induces submembrane clustering of gephyrin.
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      Impaired GABAergic transmission and altered hippocampal synaptic plasticity in collybistin-deficient mice.
      ).
      Figure thumbnail gr1
      Figure 1The postulated NL2-CB2 interaction at inhibitory synapses and the design of the SHM assay. A, Schematic drawing illustrating the GABAAR clustering machinery at inhibitory postsynapses. B, Recombinant CB2SH3 proteins (WT CB2SH3 or the constitutively active CB2SH3/W24A-E262A mutant) used in this study. C, Scheme of a supported hybrid membrane composed of an HMDS monolayer and a POPC monolayer (yellow) doped with DGS-NTA(Ni) (blue) and PtdInsPs (red) on a silicon dioxide substrate.
      The adaptor protein CB, a guanine nucleotide exchange factor (GEF), is expressed in several splice variants that differ in their N- and C termini (CB1-CB3) and the presence or absence of a regulatory src homology 3 (SH3) domain (
      • Harvey K.
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      • Harvey R.J.
      The GDP-GTP exchange factor collybistin: An essential determinant of neuronal gephyrin clustering.
      ). All CB variants contain a DH (Dbl homology) domain, which has GEF activity, and a C-terminal PH domain. Multiple studies demonstrated that mutations in CB cause neuronal dysfunction in various brain diseases. For example, an arginine to histidine exchange at position 290 within the DH domain affects the intramolecular interaction within the DH-PH tandem domain and thereby reduces the affinity of the PH domain to PtdIns[3]P, which is correlated to epileptic symptoms (
      • Papadopoulos T.
      • Schemm R.
      • Grubmüller H.
      • Brose N.
      Lipid binding defects and perturbed synaptogenic activity of a Collybistin R290H mutant that causes epilepsy and intellectual disability.
      ). Similarly, an arginine to tryptophan exchange at position 338 perturbs PH domain binding to PtdInsPs and causes a form of X-linked intellectual disability (
      • Long P.
      • May M.M.
      • James V.M.
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      • Harvey R.J.
      Missense mutation R338W in ARHGEF9 in a family with X-linked intellectual disability with variable macrocephaly and macro-orchidism.
      ). These and other findings demonstrate that binding of the PH domain of CB to PtdInsPs is regulated by intramolecular interactions and of pivotal importance for the assembly of inhibitory postsynapses.
      Previous studies (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ) indicated that the most abundantly expressed, full-length, SH3-domain-containing CB isoform 2, CB2SH3, adopts a closed conformation (Fig. 1B), in which the SH3, DH, and PH domains interact intramolecularly and thus render the protein inactive, as is the case with the homologous GEFs Asaf1 and Asaf2 (
      • Hamann M.J.
      • Lubking C.M.
      • Luchini D.N.
      • Billadeau D.D.
      Asef2 functions as a Cdc42 exchange factor and is stimulated by the release of an autoinhibitory module from a concealed C-terminal activation element.
      ,
      • Mitin N.
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      • Yohe M.E.
      • Der C.J.
      • Sondek J.
      • Rossman K.L.
      Release of autoinhibition of ASEF by APC leads to CDC42 activation and tumor suppression.
      ). Disrupting this intramolecular interaction, e.g. by disabling the intramolecular binding sites in the CB2SH3/W24A-E262A mutant (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ), leads to a more open and active conformation, so that the PH domain can bind PtdInsPs. It has been hypothesized that in the biological context of the synapse the cytosolic NL2 C terminus binds the SH3 domain of CB2, thus activates it, and allows its interaction with plasma membrane PtdInsPs (
      • Poulopoulos A.
      • Aramuni G.
      • Meyer G.
      • Soykan T.
      • Hoon M.
      • Papadopoulos T.
      • Zhang M.
      • Paarmann I.
      • Fuchs C.
      • Harvey K.
      • Jedlicka P.
      • Schwarzacher S.W.
      • Betz H.
      • Harvey R.J.
      • Brose N.
      • et al.
      Neuroligin 2 drives postsynaptic assembly at perisomatic inhibitory synapses through gephyrin and collybistin.
      ). However, this type of NL2-dependent CB2 activation has so far only been inferred from data obtained with corresponding knock-out neurons or with cultured neurons that express NL2 or CB2 variants that are either unable to interact or constitutively active. Clear molecular insights into the process have been lacking.
      The present study was conducted to obtain direct molecular evidence of an NL2-mediated activation of CB2, leading to increased CB2 binding to plasma membrane PtdInsPs. To this end, we established an in vitro membrane system to reconstitute this putative key step in the development of inhibitory synapses. Specifically, we used supported hybrid membranes (SHMs) on hydrophobically functionalized silicon dioxide substrates via spreading of small unilamellar vesicles (SUVs). We showed recently that SHMs are superior to supported lipid bilayers as they provide a more homogeneous distribution of PtdInsPs (
      • Schäfer J.
      • Nehls J.
      • Schön M.
      • Mey I.
      • Steinem C.
      Leaflet-dependent distribution of PtdIns[4,5]P2 in supported model membranes.
      ). SHMs were doped with PtdInsPs serving as receptor lipids for CB2, whereas 1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DGS-NTA(Ni)) was added to specifically adsorb the intracellular domain of NL2 (His-cytNL2) via an N-terminally fused His6-tag (Fig. 1C). By means of reflectometric interference spectroscopy we were able to analyze the lipid-binding behavior of WT CB2SH3 and the intrinsically activated CB2SH3/W24A-E262A mutant (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ) in the absence or presence of His-cytNL2. Our data show that the interaction of the C terminus of NL2 with CB2 induces a conformational change in CB2 that promotes its interaction with membrane PtdInsPs.

      Results

      Formation of supported hybrid membranes

      In a first step, we produced lipid membranes on a silicon support to investigate and quantify the binding capability of CB2 to PtdInsPs in the presence or absence of the intracellular NL2 C terminus. We used a previously published protocol (
      • Schäfer J.
      • Nehls J.
      • Schön M.
      • Mey I.
      • Steinem C.
      Leaflet-dependent distribution of PtdIns[4,5]P2 in supported model membranes.
      ) to generate lipid monolayers composed of POPC and doped with different PtdInsPs. By using silicon dioxide substrates functionalized with 1,1,1-trimethyl-N-(TMS)silanamine (HMDS), to which small unilamellar vesicles (SUVs) were fused (Fig. 1C), a possible asymmetric distribution of PtdInsPs between the two lipid leaflets is prevented (
      • Schäfer J.
      • Nehls J.
      • Schön M.
      • Mey I.
      • Steinem C.
      Leaflet-dependent distribution of PtdIns[4,5]P2 in supported model membranes.
      ). Lipid monolayers composed of POPC and doped with 3 mol % of PtdIns[3]P, PtdIns[4,5]P2, or PtdIns[3,4,5]P3 on HMDS were prepared. The three PtdInsPs were chosen based on previous studies that had highlighted the involvement of these specific PtdInsPs as CB regulators in the formation of inhibitory synapses (
      • Kalscheuer V.M.
      • Musante L.
      • Fang C.
      • Hoffmann K.
      • Fuchs C.
      • Carta E.
      • Deas E.
      • Venkateswarlu K.
      • Menzel C.
      • Ullmann R.
      • Tommerup N.
      • Dalprà L.
      • Tzschach A.
      • Selicorni A.
      • Lüscher B.
      • et al.
      A balanced chromosomal translocation disrupting ARHGEF9 is associated with epilepsy, anxiety, aggression, and mental retardation.
      ,
      • Papadopoulos T.
      • Rhee H.J.
      • Subramanian D.
      • Paraskevopoulou F.
      • Mueller R.
      • Schultz C.
      • Brose N.
      • Rhee J.-S.
      • Betz H.
      • Papadopoulos T.
      • Rhee H.J.
      • Subramanian D.
      • Paraskevopoulou F.
      • Mueller R.
      • Schultz C.
      • et al.
      Endosomal phosphatidylinositol-3-phosphate promotes gephyrin clustering and GABAergic neurotransmission at inhibitory postsynapses.
      ,
      • Kilisch M.
      • Mayer S.
      • Mitkovski M.
      • Roehse H.
      • Hentrich J.
      • Schwappach B.
      • Papadopoulos T.
      A GTPase-induced switch in phospholipid affinity of collybistin contributes to synaptic gephyrin clustering.
      ).
      The spreading process of SUVs after HMDS functionalization was monitored in a time-resolved manner by reflectometric interference spectroscopy (RIfS). A characteristic time trace of the formation of a supported hybrid membrane (SHM) is depicted in Fig. 2A. Adsorption and spreading of the SUVs results in an increase in optical thickness (ΔOT) reaching a maximum at around 20 min. After monolayer formation, the system was rinsed with buffer B to remove excess lipid material and to adjust appropriate conditions for protein binding. ΔOTSHM (Fig. 2A) is used as a quality parameter for the SHM preparation. For all three PtdInsPs doped POPC monolayers, ΔOTSHM values of 2.77-2.80 nm were obtained, in good agreement with the expectation of a lipid monolayer on top of the HMDS monolayer. The mean values of ΔOTSHM were (2.77 ± 0.11) nm for PtdIns[3]P (n = 24), (2.80 ± 0.07) nm for PtdIns[4,5]P2 (n = 31) and (2.77 ± 0.10) nm for PtdIns[3,4,5]P3 (n = 22), showing that the PtdInsP species does not influence the final monolayer thickness (Fig. 2B).
      Figure thumbnail gr2
      Figure 2Preparation of supported hybrid membranes on silicon dioxide surfaces with different PtdInsPs. A, Time resolved change in optical thickness ΔOTSHM (marked in red) upon addition of SUVs composed of POPC/PtdIns[4,5]P2 (97:3, n/n) to an HMDS functionalized silicon dioxide surface at t = 0 min. The arrow indicates the time point of rinsing with buffer B. B, Box plots of ΔOTSHM of POPC monolayers doped with 3 mol% of PtdInsP. C, Box plots of ΔOTSHM of POPC monolayers doped with 3 mol% of PtdInsP and 3 mol% of DGS-NTA(Ni). The boxes extent from upper to lower quartile whereas the whiskers represent 1st and 99th percentiles. The medians are shown as horizontal lines and the means as red squares. DGS refers to DGS-NTA(Ni).
      To follow the binding of the cytosolic domain of NL2 (His-cytNL2) via its His6-tag to the membrane, we additionally prepared lipid monolayers composed of POPC, doped with 3 mol % of PtdInsP and 3 mol % of DGS-NTA(Ni). Again, ΔOTSHM were readout from the RIfS experiments (Fig. 2C). Mean values of ΔOTSHM were (2.74 ± 0.05) nm for DGS-NTA(Ni)/PtdIns[3]P (n = 38), (2.79 ± 0.05) nm for DGS-NTA(Ni)/PtdIns[4,5]P2 (n = 45) and (2.75 ± 0.07) nm for DGS-NTA(Ni)/PtdIns[3,4,5]P3 (n = 32). These results demonstrate that the addition of DGS-NTA(Ni) to the lipid composition does not alter the monolayer quality as deduced from the measured monolayer thickness. Only in case of the SHM composed of POPC and DGS-NTA(Ni), lacking a PtdInsP, a slightly larger mean ΔOTSHM value (3.0 ± 0.1 nm, n = 19) was found.
      We next tested whether the PtdInsP lipid mobility in the monolayer is influenced by the presence of the Ni2+-loaded DGS-NTA-lipid. Sufficient lipid mobility is a prerequisite to ensure that a lateral interaction between CB2 and NL2 at the membrane interface is possible. To investigate the lateral lipid mobility, we replaced 10% of the PtdInsPs by the corresponding BODIPY®-TMR labeled phosphoinositides and performed fluorescence recovery after photobleaching (FRAP) experiments. Fig. 3A shows a typical FRAP experiment of an SHM composed of POPC/DGS-NTA(Ni)/PtdIns[4,5]P2/BODIPY®-TMR-PtdIns[4,5]P2 (94:3:2.7:0.3, n/n). From the recovery curve (Fig. 3B), the diffusion coefficient D as well as the mobile fraction γ0 for each PtdInsP in the presence or absence of DGS-NTA(Ni) were calculated (Figs. 3C, D). Although the mobile fraction remains unaffected by the presence of DGS-NTA(Ni) (in the range of γ0 = 80%), the diffusion constants were decreased in the presence of DGS-NTA(Ni) by about 50%. For PtdIns[3]P, D is reduced from (2.4 ± 0.2) μm2s−1 (n = 7) without DGS-NTA(Ni) to (1.2 ± 0.2) μm2s−1 (n = 5) in its presence. A similar trend was detected for PtdIns[4,5]P2 and PtdIns[3,4,5]P3, with a decrease in D from (1.8 ± 0.2) μm2 s−1 (n = 13) to (0.9 ± 0.1) μm2 s−1 (n = 32) and from (2.1 ± 0.1) μm2 s−1 (n = 11) to (1.0 ± 0.1) μm2 s−1 (n = 6), respectively.
      Figure thumbnail gr3
      Figure 3FRAP experiment to access the mobility of PtdInsPs. A, Time lapse series of a FRAP experiment for an SHM composed of POPC/DGS-NTA(Ni)/PtdIns[4,5]P2/BODIPY®-TMR-PtdIns[4,5]P2, 94:3:2.7:0.3, n/n) at four different time points, with the bleached area indicated by a white circle. Scale bar: 5 μm. B, FRAP recovery curve for an SHM composed of POPC/DGS-NTA(Ni)/PtdIns[4,5]P2/BODIPY®-TMR-PtdIns[4,5]P2, 94:3:2.7:0.3, n/n). Box plots of the diffusion coefficients (D) in C and the mobile fractions (γ0) in D of the three different labeled PtdInsP in the presence or absence of DGS-NTA(Ni). The boxes represent the S.E. whereas the whiskers show the S.D. The medians are shown as horizontal lines and the means as red squares. Significant differences are indicated by ** p ≤ 0.01 and ***p ≤ 0.001.
      The reduced mobility of the PtdInsPs can be explained by a possible electrostatic interaction between the negatively charged PtdInsP and the DGS-NTA(Ni). However, clustering of the PtdInsP lipids, i.e. an inhomogeneity of the fluorescence intensity, was not resolved by confocal laser scanning microscopy (see Fig. 3A at t = -2 s). These results show that even in the presence of DGS-NTA(Ni) lateral mobility of the PtdInsPs is still ensured and lipid clusters of the size that could be resolved by fluorescence microscopy are not discernible. In conclusion, these data show that membranes containing both DGS-NTA(Ni) and the different PtdInsPs are suitable for the analyses of CB2 membrane binding and its dependence on His-cytNL2.

      Isolation and purification of CB2 and NL2

      We next focused on the main aim of our study i.e. to assess using the established membrane system, whether the specific interaction of SH3-domain-containing CB2 (CB2SH3) with the cytosolic C terminus of NL2 (cytNL2) leads to a conformational switch in CB2SH3 from a closed (autoinhibited) conformation to a more open (active) conformation that allows binding to PtdInsPs. To address this question, we recombinantly expressed and purified CB2SH3 and His-cytNL2. As a positive control, we additionally purified the CB2SH3/W24A-E262A mutant, which exhibits increased PtdInsP binding as compared with WT CB2SH3 (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ,
      • Ludolphs M.
      • Schneeberger D.
      • Soykan T.
      • Schäfer J.
      • Papadopoulos T.
      • Brose N.
      • Schindelin H.
      • Steinem C.
      Specificity of collybistin-phosphoinositide interactions: Impact of the individual protein domains.
      ). Fig. 4 shows the results of SDS-polyacrylamide gel electrophoreses (SDS-PAGE) and Western blotting analyses (WB) of CB2SH3 (WT or the CB2SH3/W24A-E262A mutant) and His-cytNL2. After purification of both CB2 isoforms (via intein tags on a chitin resin column), a single protein band was identified in the SDS gels (Fig. 4A, B, left panel). A CB2 specific antibody (CB Ab, epitope: aa 44-229) identified these bands as CB2 (Fig. 4 A, B, right panel). For His-cytNL2 purified on a Ni2+-nitrilotriacetic acid (NTA-(Ni2+)) agarose column and a subsequent anion exchange chromatography, a band at 17 kDa was detected in the SDS gels, in agreement with the theoretical mass of the protein (Fig. 4C).
      Figure thumbnail gr4
      Figure 4Characterization of the different proteins used in this study. A, SDS-PAGE (left) and Western blotting overlay of chemiluminescence and marker image (right) of CB2SH3. B, SDS-PAGE (left) and Western blotting overlay of chemiluminescence and marker image (right) of CB2SH3/W24A-E262A. C, SDS-PAGE of His-cytNL2. M: Marker, 1: protein sample. The vertical black lines mark the splice borders.

      CB2 binding to phosphoinositides

      Based on the established SHMs, we investigated the binding of CB2 to the different PtdInsPs in the absence (Fig. 5) and in the presence (Fig. 7) of bound cytNL2, whereas all other experimental conditions remain the same. Characteristic time traces of ΔOT upon SHM formation and CB2 addition (ΔOTCB2¸ definition see Fig. 5B) to a POPC/PtdIns[3,4,5]P3/DGS-NTA(Ni) (94:3:3, n/n) SHM in absence of cytNL2 are shown in Fig. 5A (CB2SH3) and B (CB2SH3/W24A-E262A). A protein concentration of 1 μm was chosen for these experiments to obtain sufficiently high protein coverage without wasting too much protein (Fig. S1A, B). Different lipid compositions were used to elucidate the binding properties of WT CB2SH3 and the CB2SH3/W24A-E262A mutant, which also allowed us to assess their nonspecific binding behavior. SHMs doped with only DGS-NTA(Ni) or with one of the three phosphoinositides were used or both receptor lipids were reconstituted simultaneously.
      Figure thumbnail gr5
      Figure 5Results of RIfS experiments to analyze the binding of CB2SH3 and CB2SH3/W24A-E262A in the absence of cytNL2. Change in optical thickness (ΔOT) versus time during the adsorption of A, CB2SH3 and B, CB2SH3/W24A-E262A. The SHMs were composed of POPC/PtdIns[3,4,5]P3/DGS-NTA(Ni) (94:3:3, n/n). Monolayer formation was initiated by the addition of SUVs (I). After rinsing with buffer B (II), CB2 was added (III, 1 μm). The difference in OT (marked in red) is defined as ΔOTCB2. C, Box plots of ΔOTCB2 upon binding of CB2SH3 (white) and CB2SH3/W24A-E262A (gray) to SHMs doped with 3 mol % of the different receptor lipids. The boxes represent the S.E. whereas the whiskers show the S.D. The medians are shown as horizontal lines and the means as central squares. Significant differences are indicated by * p ≤ 0.05 and **p ≤ 0.01. DGS is DGS-NTA(Ni).
      Figure thumbnail gr7
      Figure 7Results of RIfS experiments to analyze the binding of CB2SH3 and CB2SH3/W24A-E262A in the presence of cytNL2. Change in optical thickness (ΔOT) versus time during the adsorption of A, CB2SH3 and B, CB2SH3/W24A-E262A. The SHMs were composed of POPC/PtdIns[3,4,5]P3/DGS-NTA(Ni) (94:3:3, n/n). Monolayer formation was initiated by the addition of SUVs (I). After rinsing with buffer B (II), His-cytNL2 was added (III, 1.36 μm). After a second rinsing with buffer B (IV), CB2 was added (V, 1 μm). The difference in OT (marked in red) is defined as ΔOTCB2*. C, Box plot of ΔOTCB2* upon binding of CB2SH3 (white) and CB2SH3/W24A-E262A (gray) to SHMs doped with 3 mol % of the different receptor lipids after previous adsorption of His-cytNL2. The boxes represent the S.E. whereas the whiskers show the S.D. The medians are shown as horizontal lines and the means as central squares. Significant differences are indicated by * p ≤ 0.05. DGS is DGS-NTA(Ni).
      ΔOTCB2 for the different lipid compositions are depicted in Fig. 5C for CB2SH3 and CB2SH3/W24A-E262A. In case of CB2SH3, a small increase in ΔOT of about 0.1 nm was observed independently of the chosen lipid composition [DGS-NTA(Ni) or PtdInsP or a combination thereof] except for the POPC/PtdIns[4,5]P2/DGS-NTA(Ni) (94:3:3, n/n) membrane composition. The small ΔOTCB2 values of about 0.1 nm indicate that there is no specific interaction between CB2SH3 and the lipids analyzed. They can in part be ascribed to changes in the refractive index n caused by the addition of protein to the aqueous solution. On SHMs composed of POPC/PtdIns[4,5]P2/DGS-NTA(Ni) (94:3:3, n/n) a slightly larger ΔOTCB2 of (0.24 ± 0.05) nm (n = 4) was found indicating an increased amount of adsorbed CB2SH3. We can only speculate that the electrostatics on the surface is slightly different owing to an interaction of DGS-NTA(Ni) with PtdIns[4,5]P2. This can alter the position of the PtdIns[4,5]P headgroup protruding more from the membrane surface (
      • Li Z.
      • Venable R.M.
      • Rogers L.A.
      • Murray D.
      • Pastor R.W.
      Molecular dynamics simulations of PIP2 and PIP3 in lipid bilayers: determination of ring orientation, and the effects of surface roughness on a Poisson-Boltzmann description.
      ), and thus induces electrostatically driven interactions. For CB2SH3/W24A-E262A significantly increased binding to the phosphoinositides was observed. Although there is a small nonspecific binding on DGS-NTA(Ni)-doped SHMs with ΔOTCB2 = (0.20 ± 0.09) nm (n = 3), CB2SH3/W24A-E262A addition resulted in larger ΔOTCB2, well distinguishable from the base line level, when only phosphoinositides were present. For PtdIns[3]P and PtdIns[4,5]P2 containing SHMs, ΔOTCB2 was determined to be (0.28 ± 0.05) nm (n = 7) and (0.37 ± 0.07) nm (n = 7), respectively. The overall binding affinity given as a change in optical thickness at 1 μm protein concentration was largest for PtdIns[3,4,5]P3. Adsorption of CB2SH3/W24A-E262A led to ΔOTCB2 of (0.67 ± 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. In contrast, the WT CB2SH3 apparently remains in a closed, inactive conformation, rendering the protein incapable of interacting with PtdInsPs.
      The adsorption of CB2SH3/W24A-E262A in presence of DGS-NTA(Ni) resulted in ΔOTCB2 of (0.35 ± 0.04) nm (PtdIns[3]P, n = 8), (0.57 ± 0.05) nm (PtdIns[4,5]P2, n = 7) and (0.43 ± 0.07) nm (PtdIns[3,4,5]P3, n = 9) showing that DGS-NTA(Ni) does not significantly influence the binding of the active mutant.

      Binding of His-cytNL2 to the SHMs

      To induce the postulated conformational change in CB2SH3 upon interaction with the cytosolic part of NL2, the two proteins need to interact with each other at the membrane interface. Thus, we investigated whether His-cytNL2 can be specifically bound via DGS-NTA(Ni) to the membrane. The specific adsorption of His-cytNL2 to DGS-NTA(Ni) doped membranes was measured by RIfS (Fig. S1C). For a concentration of 1.36 μm a specific binding with a mean change in OT ΔOTNL2 = (0.69 ± 0.09) nm (n = 4) was observed (Fig. 6). To show the specificity of binding via the His6-tag to the DGS-NTA(Ni) lipid, the N-terminal His6-tag was cleaved off with the TEV (tobacco etch virus) protease (Fig. S2). After cleavage, only a small change of ΔOTNL2 = (0.10 ± 0.04) nm (n = 4) was observed.
      Figure thumbnail gr6
      Figure 6Binding of cytNL2 to different membrane compositions. Box plot of the change in optical thickness (ΔOTNL2) upon addition of cytNL2 (1.36 μm) with and without His6-tag (His-cytNL2: white; cytNL2: gray). The boxes represent the S.E. whereas the whiskers show the S.D. The medians are shown as horizontal lines and the means as central squares. DGS is DGS-NTA(Ni).
      We also investigated whether nonspecific binding of cytNL2 occurs on phosphoinositide-doped membranes. Although cytNL2 does not show any additional amount of binding, slightly larger ΔOTNL2 were observed for His-cytNL2, which might be a result of the positively charged His6-tag interacting with the negatively charged PtdInsP lipids.

      Effect of membrane bound cytNL2 on CB2 activation

      With the information that cytNL2 is specifically bound to DGS-NTA(Ni), we now compare the binding of CB2SH3 to the different PtdInsPs in the absence (Fig. 5) with that in the presence (Fig. 7) of cytNL2 under otherwise exact same conditions. The results allow us to address the question whether WT CB2SH3 is capable of binding to PtdInsP-doped membranes if cytNL2 is present at the membrane interface. For a control, we also analyzed the binding behavior of the mutant CB2SH3/W24A-E262A, which is expected to bind to phosphoinositides even without any activation via cytNL2. After His-cytNL2 was bound to the SHMs doped with DGS-NTA(Ni) and one of the three PtdInsPs (III in Fig. 7A and B), either CB2SH3 or CB2SH3/W24A-E262A was added (V in Fig. 7A and B), and the change in optical thickness (ΔOTCB2*, definition see Fig. 7A) was monitored by RIfS. In contrast to the result shown in Fig. 5A, where CB2SH3 was added to the same membrane but in the absence of cytNL2, and where no change in OT was observed, a clear change in OT was found for CB2SH3 in the presence of cytNL2 (Fig. 7A).
      Fig. 7C summarizes the results for the three different PtdInsPs, which can be directly compared with the ΔOTCB2 values obtained in the absence of cytNL2 (Fig. 5C, lanes PtdInsPs & DGS). The ΔOTCB2* values for CB2SH3 adsorption to PtdIns[3]P and PtdIns[4,5]P2 were determined to be (0.16 ± 0.02) nm (n = 9) and (0.26 ± 0.02) nm (n = 7), respectively. Compared with the ΔOTCB2 values (in absence of cytNL2) for CB2SH3 adsorption to PtdIns[3]P ((0.09 ± 0.07) nm) and PtdIns[4,5]P2 ((0.24 ± 0.05) nm), there appears to be no significant difference in the amount of bound CB2SH3 if cytNL2 is present. However, in case of PtdIns[3,4,5]P3 ΔOTCB2* was significantly (p < 0.01) increased to (0.25 ± 0.04) nm (n = 9) in presence of cytNL2, as compared with ΔOTCB2 = (0.04 ± 0.02) nm (n = 4) in its absence. This result indicates an activation of the CB2SH3 WT rendering the protein capable of selectively interacting with PtdIns[3,4,5]P3.
      However, there is another aspect that needs to be considered. A significantly (p < 0.001) reduced amount of the CB2SH3/W24A-E262A mutant protein binds to the membrane surface in presence of cytNL2 (Fig. 7C) compared with that found in its absence (Fig. 5C, lanes PtdInsPs & DGS). ΔOTCB2* was determined to be (0.12 ± 0.02) nm (n = 8) and (0.17 ± 0.02) nm (n = 5) for PtdIns[3]P and PtdIns[4,5]P2 containing SHMs, respectively (Fig. 7C). For comparison ΔOTCB2 = (0.35 ± 0.04) nm (PtdIns[3]P) and (0.57 ± 0.05) nm (PtdIns[4,5]P2) (Fig. 5C) was measured. Only in case of PtdIns[3,4,5]P3 doped membranes the overall amount of adsorbed CB2SH3/W24A-E262A was not significantly influenced in the presence of cytNL2 with ΔOTCB2* = (0.37 ± 0.04) nm (n = 8) compared with ΔOTCB2 = (0.43 ± 0.07) nm (n = 9) in its absence (compare Fig. 5C and Fig. 7C). These results indicate that the natively unfolded cytNL2 occludes some of the PtdInsPs, possibly by ionic interactions. This inaccessibility of binding sites for CB2 leads to a reduction in binding capability of the CB2SH3/W24A-E262A mutant. Taken the reduced ΔOTCB2* values into account, one can calculate the fraction of remaining available binding sites (ABS) in the presence of cytNL2, defined as:
      ABS=ΔOTCB2mutant/ΔOTCB2mutant,


      resulting in 34% (PtdIns[3]P), 30% (PtdIns[4,5]P2) and 86% (PtdIns[3,4,5]P3). Thus, a direct comparison of CB2 WT adsorption in presence and absence of cytNL2 appears not to be reasonable as cytNL2's potential to compromise the CB2-PtdInsP interaction has to be considered. Despite this fact, the trend that the amount of bound CB2SH3 in the presence of cytNL2 increases becomes obvious, if we compare the obtained ΔOTCB2 and ΔOTCB2* values of CB2SH3 related to the corresponding values for CB2SH3/W24A-E262A that are measured under identical conditions and are set to 100%. This calculation leads to an increase of the relative amount of adsorbed CB2SH3 from 25% to 133% (PtdIns[3]P), from 42% to 153% (PtdIns[4,5]P2) and from 9% to 68% (PtdIns[3,4,5]P3).

      Discussion

      The present study provides direct molecular evidence of an NL2-mediated activation of CB2, leading to increased CB2 binding to plasma membrane PtdInsPs.
      Previous studies led to the hypothesis that the cytosolic NL2 C terminus binds the SH3 domain of CB2 in the biological context of the postsynapse, thereby inducing a more open conformation, which allows CB to interact with PtdInsPs located at the postsynaptic membrane (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ,
      • Poulopoulos A.
      • Aramuni G.
      • Meyer G.
      • Soykan T.
      • Hoon M.
      • Papadopoulos T.
      • Zhang M.
      • Paarmann I.
      • Fuchs C.
      • Harvey K.
      • Jedlicka P.
      • Schwarzacher S.W.
      • Betz H.
      • Harvey R.J.
      • Brose N.
      • et al.
      Neuroligin 2 drives postsynaptic assembly at perisomatic inhibitory synapses through gephyrin and collybistin.
      ). By using SHMs, which provide a homogeneous distribution of PtdInsPs (
      • Schäfer J.
      • Nehls J.
      • Schön M.
      • Mey I.
      • Steinem C.
      Leaflet-dependent distribution of PtdIns[4,5]P2 in supported model membranes.
      ), we were able to establish an in vitro molecular model that allowed us to investigate directly how the interaction of CB2 with the cytosolic part of NL2 influences its ability to bind to different PtdInsPs. Our results show that, without the membrane-associated part of NL2, the WT protein CB2SH3 does not bind to any of the three PtdInsPs tested. This agrees with previous studies indicating that CB2SH3 adopts a closed, autoinhibited conformation, in which the PH domain is not accessible for PtdInsP binding (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ,
      • Ludolphs M.
      • Schneeberger D.
      • Soykan T.
      • Schäfer J.
      • Papadopoulos T.
      • Brose N.
      • Schindelin H.
      • Steinem C.
      Specificity of collybistin-phosphoinositide interactions: Impact of the individual protein domains.
      ). As previously shown (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ), the mutant protein CB2SH3/W24A-E262A, in which intramolecular interactions between the C-terminal PH and N-terminal SH3 domains of CB are weakened, adopts a more open conformation, which allowed the protein to interact with all three PtdInsPs used in the present study. We have chosen PtdIns[3]P as one of the receptor lipids based on previous studies highlighting the high affinity of CB2 to this PtdInsP (
      • Kalscheuer V.M.
      • Musante L.
      • Fang C.
      • Hoffmann K.
      • Fuchs C.
      • Carta E.
      • Deas E.
      • Venkateswarlu K.
      • Menzel C.
      • Ullmann R.
      • Tommerup N.
      • Dalprà L.
      • Tzschach A.
      • Selicorni A.
      • Lüscher B.
      • et al.
      A balanced chromosomal translocation disrupting ARHGEF9 is associated with epilepsy, anxiety, aggression, and mental retardation.
      ,
      • Ludolphs M.
      • Schneeberger D.
      • Soykan T.
      • Schäfer J.
      • Papadopoulos T.
      • Brose N.
      • Schindelin H.
      • Steinem C.
      Specificity of collybistin-phosphoinositide interactions: Impact of the individual protein domains.
      ). The other two PtdInsPs are the most abundant ones in the post synaptic plasma membrane (
      • Dickson E.J.
      Recent advances in understanding phosphoinositide signaling in the nervous system.
      ,
      • Volpatti J.R.
      • Al-Maawali A.
      • Smith L.
      • Al-Hashim A.
      • Brill J.A.
      • Dowling J.J.
      The expanding spectrum of neurological disorders of phosphoinositide metabolism.
      ) and present during postsynapse formation.
      Whereas the interaction of CB with PtdIns[3]P allows the accumulation of CB and CB-associated proteins on early-endosomal membranes (
      • Papadopoulos T.
      • Rhee H.J.
      • Subramanian D.
      • Paraskevopoulou F.
      • Mueller R.
      • Schultz C.
      • Brose N.
      • Rhee J.-S.
      • Betz H.
      • Papadopoulos T.
      • Rhee H.J.
      • Subramanian D.
      • Paraskevopoulou F.
      • Mueller R.
      • Schultz C.
      • et al.
      Endosomal phosphatidylinositol-3-phosphate promotes gephyrin clustering and GABAergic neurotransmission at inhibitory postsynapses.
      ), the small GTPase TC10 binds to the PH domain of CB and induces a phospholipid affinity switch in CB, which allows CB to specifically interact with PtdInsP-species present at the plasma membrane, such as PtdIns[4,5]P2 and PtdIns[3,4,5]P3 (
      • Kilisch M.
      • Mayer S.
      • Mitkovski M.
      • Roehse H.
      • Hentrich J.
      • Schwappach B.
      • Papadopoulos T.
      A GTPase-induced switch in phospholipid affinity of collybistin contributes to synaptic gephyrin clustering.
      ,
      • Mayer S.
      • Kumar R.
      • Jaiswal M.
      • Soykan T.
      • Ahmadian M.R.
      • Brose N.
      • Betz H.
      • Rhee J.-S.
      • Papadopoulos T.
      Collybistin activation by GTP-TC10 enhances postsynaptic gephyrin clustering and hippocampal GABAergic neurotransmission.
      ). Similarly to the interaction of NL2 with CB2SH3, the TC10-CB2SH3 interaction was previously suggested to interfere with intramolecular interactions between the different domains of CB2SH3, leading to a transition toward an open state of CB, which allows the PH domain to specifically bind to PtdInsPs located at the plasma membrane (
      • Mayer S.
      • Kumar R.
      • Jaiswal M.
      • Soykan T.
      • Ahmadian M.R.
      • Brose N.
      • Betz H.
      • Rhee J.-S.
      • Papadopoulos T.
      Collybistin activation by GTP-TC10 enhances postsynaptic gephyrin clustering and hippocampal GABAergic neurotransmission.
      ).
      In order to induce the postulated conformational change in CB2SH3 upon interaction with the cytosolic part of NL2, the two proteins have to interact with each other at the membrane interface. To bind the cytosolic part of NL2 to the membrane, we exploited a His6-tag-DGS-NTA(Ni) strategy. Even though two oppositely charged lipids were inserted into the POPC matrix, a homogeneous SHM was produced, with laterally mobile lipids. However, the diffusion coefficients D of the BODIPY®-TMR PtdInsPs were reduced by about 50% compared with those without DGS-NTA(Ni). These diffusion coefficients are still in the range of those found in cellular plasma membranes of fibroblasts and epithelial cells with an average of D = (0.8 ± 0.2) μm2/s (
      • Golebiewska U.
      • Nyako M.
      • Woturski W.
      • Zaitseva I.
      • McLaughlin S.
      Diffusion coefficient of fluorescent phosphatidylinositol 4,5-bisphosphate in the plasma membrane of cells.
      ) and hence appear to be sufficient to allow for lateral protein-protein interactions at the membrane interface.
      Our comparative analysis of the binding of CB2SH3 (WT and the W24A-E262A mutant) to PtdInsPs in the presence or absence of cytNL2 clearly indicates that the cytosolic C terminus of NL2 alters the conformation of CB2 and thereby controls CB2-interactions with PtdInsPs at the plasma membrane. In the absence of cytNL2, only the “open conformation-mutant”, CB2SH3/W24A-E262A, efficiently interacted with PtdInsPs. In contrast, in the presence of cytNL2, similar amounts of WT CB2SH3 and the CB2SH3/W24A-E262A mutant bound to PtdInsPs. However, there is an overall decrease in the amount of CB2SH3/W24A-E262A bound to the three PtdInsPs in the presence of cytNL2, as compared with that in the absence of membrane anchored cytNL2. One cannot rule out that the W24A-E262A double-mutation of CBSH3 alters the specificity for the different PtdInsPs compared with the WT protein, as shown previously for CB and other proteins (
      • Lemmon M.A.
      • Ferguson K.M.
      • O'Brien R.
      • Sigler P.B.
      • Schlessinger J.
      Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain.
      ,
      • Chiou T.-T.
      • Long P.
      • Schumann-Gillett A.
      • Kanamarlapudi V.
      • Haas S.A.
      • Harvey K.
      • O'Mara M.L.
      • De Blas A.L.
      • Kalscheuer V.M.
      • Harvey R.J.
      Mutation p.R356Q in the collybistin phosphoinositide binding site is associated with mild intellectual disability.
      ). Moreover, in agreement with this hypothesis, a previous study indicated that a single (R290H) mutation in the DH domain of CB, which leads to epilepsy and intellectual disability in humans, alters the strength of intramolecular interactions between the DH and the PH domains of CB, thereby leading to a reduced PtdIns[3]P-binding of CB (
      • Papadopoulos T.
      • Schemm R.
      • Grubmüller H.
      • Brose N.
      Lipid binding defects and perturbed synaptogenic activity of a Collybistin R290H mutant that causes epilepsy and intellectual disability.
      ). Thus, whereas the interaction of WT CBSH3 with endogenous activator-proteins, such as the cell-adhesion protein NL2 studied here or the small Rho-like GTPase TC10 (
      • Kilisch M.
      • Mayer S.
      • Mitkovski M.
      • Roehse H.
      • Hentrich J.
      • Schwappach B.
      • Papadopoulos T.
      A GTPase-induced switch in phospholipid affinity of collybistin contributes to synaptic gephyrin clustering.
      ), leads to a fine-tuned increase of the interaction of CB2 with certain PtdInsPs enriched at the plasma membrane, the “open-conformation mutant” CB2SH3/W24A-E262A might lead to a more general and less specific increase of CB binding to a broader range of PtdInsPs.
      As regards WT CB2SH3, an interesting finding of our study is that for PtdIns[3,4,5]P3 the ΔOTCB2* (in the presence of cytNL2) was significantly increased, as compared with the ΔOTCB2 (in the absence of cytNL2). For PtdIns[3]P and PtdIns[4,5]P2, a similar trend toward increased ΔOTCB2* was observed but did not reach significance, as compared with ΔOTCB2. However, for both, WT CB2SH3 and the W24A-E262A mutant, it appears that the PtdInsP-accessibility is reduced upon His-cytNL2 binding to the membrane, resulting in an overall decrease in the amount of bound CB2 proteins. If this is assumed, we can relate the ΔOTCB2 values obtained in the absence of cytNL2 to the changes in optical thickness for CB2SH3 in presence of cytNL2 and calculate an increase in the amount of bound CB2SH3 for all three PtdInsPs with a maximum increase for PtdIns[3,4,5]P3. This agrees with a previous study, indicating that endogenous CB2 activators can induce a switch in the conformation of CB2, which allows enhanced interaction with plasma membrane-PtdInsPs (
      • Kilisch M.
      • Mayer S.
      • Mitkovski M.
      • Roehse H.
      • Hentrich J.
      • Schwappach B.
      • Papadopoulos T.
      A GTPase-induced switch in phospholipid affinity of collybistin contributes to synaptic gephyrin clustering.
      ). Thus, we conclude that in the presence of the cytosolic domain of NL2 a switch from a closed to an open conformation of CB2 is induced, which enables CB2 to properly anchor at nascent inhibitory postsynapses enriched in PtdIns[4,5]P2 and PtdIns[3,4,5]P3. It is likely that this conformational switch is induced by the interaction of the N-terminal SH3 domain of CB with poly-proline sequences in cytNL2 (
      • Papadopoulos T.
      • Soykan T.
      The role of collybistin in gephyrin clustering at inhibitory synapses: facts and open questions.
      ,
      • Hoon M.
      • Soykan T.
      • Falkenburger B.
      • Hammer M.
      • Patrizi A.
      • Schmidt K.-F.
      • Sassoe-Pognetto M.
      • Lowel S.
      • Moser T.
      • Taschenberger H.
      • Brose N.
      • Varoqueaux F.
      Neuroligin-4 is localized to glycinergic postsynapses and regulates inhibition in the retina.
      ). As we only adsorbed the water-soluble cytosolic domain of NL2, we moreover conclude that a dimerization of NL2 via the transmembrane domains, as it was observed for NL2 in vivo (
      • Poulopoulos A.
      • Soykan T.
      • Tuffy L.P.
      • Hammer M.
      • Varoqueaux F.
      • Brose N.
      Homodimerization and isoform-specific heterodimerization of neuroligins.
      ), is not required for CB2 activation.

      Experimental Procedures

      Materials

      C16 derivatives and BODIPY®-TMR labeled derivatives of PtdInsPs (PtdIns[3]P, PtdIns[4,5]P2, and PtdIns[3,4,5]P3) were obtained as from Echelon Biosciences (Salt Lake City, UT). 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and 1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DGS-NTA(Ni)) were purchased from Avanti Polar Lipids (Alabaster, Alabama). BL21(DE3) competent Escherichia coli cells were purchased from Invitrogen whereas BL21(DE3) Rosetta competent E. coli cells were from VWR International (Darmstadt, Germany). Chitin resin was obtained from New England Biolabs (Ipswich, MA) and NTA(Ni2+) agarose (Protino™) from Macherey-Nagel (Düren, Germany). Antibodies specific for CB2 (1: 1000, Cat. No. 261 011) and NL2 (1: 1000, Cat. No. 129 213) were purchased from Synaptic Systems (Göttingen, Germany) whereas the His-tag antibody (1: 1000, ab18184) was obtained from Abcam (Cambridge, UK). Silicon wafers were purchased from Silicon Materials (Kaufering, Germany). 1,1,1-Trimethyl-N-(TMS)silanamine (HMDS) was purchased from VWR International (Darmstadt, Germany).

      Protein purification

      Proteins were recombinantly expressed in E. coli following previously described protocols (
      • Soykan T.
      • Schneeberger D.
      • Tria G.
      • Buechner C.
      • Bader N.
      • Svergun D.
      • Tessmer I.
      • Poulopoulos A.
      • Papadopoulos T.
      • Varoqueaux F.
      • Schindelin H.
      • Brose N.
      A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.
      ,
      • Koltzscher M.
      • Neumann C.
      • König S.
      • Gerke V.
      Ca 2+-dependent binding and activation of dormant ezrin by dimeric S100P.
      ). Briefly, His-cytNL2 was obtained from transformed E. coli BL21(DE3) Rosetta cells containing the bacterial expression vector pETM11 (EMBL, Heidelberg, Germany). The plasmid was kindly provided by the group of Hermann Schindelin (Rudolf-Virchow-Zentrum, Würzburg, Germany). It encodes the intracellular domain of NL2 with an N-terminally fused histidine tag. First, the cells were grown to an OD600 = 0.8 in kanamycin (50 μg·ml−1) containing LB medium. Protein expression was induced by addition of 0.5 mm isopropyl-β-d-thiogalactopyranoside (IPTG). After incubation for ≥15 h at 15 °C, the cells were harvested by centrifugation (4,000 × g, 20 min, 4 °C), and the pellet was resuspended in lysis buffer A (500 mm NaCl, 100 mm HEPES, 10%(v/v) glycerol, 6 mm benzamidine, 5 mm mercaptoethanol, 2 mm PMSF, protease inhibitor mixture (cOmplete; Roche Diagnostics, Basel, Switzerland, pH 8.0). Lysis was completed using a microfluidizer (1 kbar, three cycles, ice cooled, LM10 processor, Microfluidics, Westwood, MA). Afterward, the bacterial lysate was clarified by centrifugation (57,500 × g, 45 min, 4 °C). The supernatant was then applied to a Ni2+-nitrilotriacetic acid (NTA-(Ni2+)) agarose column and incubated for 30 min. Then the column was washed with 20 CV lysis buffer A and wash buffer A (500 mm NaCl, 100 mm HEPES, 10%(w/v) glycerol, 5 mm mercaptoethanol, pH 8.0) each. His-cytNL2 was eluted using buffer A (250 mm NaCl, 250 mm imidazole, 500 mm HEPES, 5 mm mercaptoethanol, pH 8.0) after incubation for 30 min. The protein solution was then dialyzed against imidazole-free Äkta-buffer (50 mm NaCl, 25 mm HEPES, 1 mm EDTA, pH 8.0) and applied to an anion-exchange chromatography column (Mono Q 5/50 GL, GE Healthcare, Uppsala, Sweden). The protein was eluted with an ion gradient created by use of Äkta-buffer containing 2 m NaCl. The purified protein was dialyzed against buffer B (100 mm NaCl, 25 mm HEPES, pH 8.0) and stored at 4 °C until use after its concentration was determined via Bradford test.
      CB2SH3 and CB2SH3/W24A-E262A were obtained from transformed E. coli BL21(DE3) cells. Plasmids based on the IMPACT system vector pTXB1 encoding both full-length forms of CB2 with C-terminal intein tags were transformed into E. coli cells and cells were grown to an OD600 of 0.9–1.0 at 37 °C in LB-Miller medium containing ampicillin (100 μg·ml−1). Protein expression was induced by addition of 0.5 mm IPTG. After overnight incubation at 15 °C, cells were harvested by centrifugation (4,000 × g, 20 min, 4 °C). The cell pellets were resuspended in lysis buffer B (250 mm NaCl, 20 mm HEPES, 2 mm EDTA, 10%(w/v) glycerol, pH 8.0), and cell lysis was performed using a microfluidizer (1 kbar, three cycles, ice cooled; LM10 processor, Microfluidics, Westwood, MA, USA). After centrifugation (70,000 × g, 30 min, 4 °C), the supernatant was applied to the equilibrated chitin resin column for 1 h. The resin was rinsed with 500 ml of wash buffer B (1 m NaCl, 20 mm HEPES, 2 mm EDTA, pH 8.0), and protein cleavage was induced by incubation with 50 mm DTT in buffer C (250 mm NaCl, 20 mm HEPES, 2 mm EDTA, pH 8.0) for > 24 h. Finally, the protein was eluted with buffer C containing 5 mm DTT. Concentration and dialysis to buffer B (100 mm NaCl, 25 mm HEPES, pH 8.0) was performed by ultrafiltration using spin concentrators (Sartorius, Göttingen, Germany). Protein concentrations were determined by UV/Vis spectroscopy using extinction coefficients of ε280(CB2SH3) = 98,945 m−1cm−1 and ε280(CB2SH3/W24A-E262A) = 93,445 m−1cm−1.
      All proteins were analyzed by SDS-PAGE and Western Blots using a Gel-Imager (Azure c300, azure biosystems, Dublin, USA) for documentation.

      Substrate preparation

      Silicon substrates with a SiO2 layer thickness of 5 μm were cleaned two times with detergent solution followed by rinsing with ultrapure water in an ultrasonic bath for 15 min each. Afterward, the substrates were treated with O2-plasma for 30 s and then exposed to hexamethyldisilazane (HMDS) as previously described in detail (
      • Schäfer J.
      • Nehls J.
      • Schön M.
      • Mey I.
      • Steinem C.
      Leaflet-dependent distribution of PtdIns[4,5]P2 in supported model membranes.
      ).

      Vesicle preparation

      A stock solution of POPC was prepared in chloroform at a concentration of 10 mg·ml−1. Lyophilized PtdInsPs were dissolved in mixtures of chloroform/methanol/water to concentrations of 1 mg·ml−1. Lipid stock solutions (0.4–0.8 mg of total lipid material) were mixed in a test tube preloaded with 100 μl chloroform at the desired molar ratio. Fluorophores were added as indicated. Organic solvent was evaporated with a gentle stream of nitrogen at 25 °C. To remove residual solvent, the lipid film was dried under vacuum for 3 h at the corresponding temperature. Lipid films were stored at 4 °C until use. A lipid film was rehydrated by adding 0.5–1.2 ml of spreading buffer (20 mm citrate, 50 mm KCl, 0.1 mm NaN3, pH 4.8) and incubated for 30 min. Multilamellar vesicles (MLVs) were obtained by vortexing for 3 × 30 s at 5 min intervals. The MLV suspension was transferred to an Eppendorf cup and sonicated for 30 min using an ultrasonic homogenizer (Sonopuls HD2070, resonator cup; Bandelin, Berlin, Germany) to obtain small unilamellar vesicles (SUVs).

      Reflectometric interference spectroscopy (RIfS)

      RIfS is a label-free, noninvasive technique determining the optical thickness (n × d) of a thin layer by measuring white light interference. This interference is caused by partial reflection at interfaces whose distance is within the coherence length of white light (
      • Hänel C.
      • Gauglitz G.
      Comparison of reflectometric interference spectroscopy with other instruments for label-free optical detection.
      ). RIfS was employed to monitor the formation of SHMs and subsequent protein adsorption to receptor lipid containing membranes in a label-free and time-resolved manner. The experimental setup is described in detail elsewhere (
      • Krick R.
      • Busse R.A.
      • Scacioc A.
      • Stephan M.
      • Janshoff A.
      • Thumm M.
      • Kühnel K.
      Structural and functional characterization of the two phosphoinositide binding sites of PROPPINs, a β-propeller protein family.
      ). Briefly, a Flame-S-UV/Vis spectrometer (Ocean Optics, Dunedin, FL, USA) was used to record interference spectra at intervals of 2 s. Data were evaluated applying a MATLAB (The MathWorks, Natick, MA, USA) tool following the work of Krick et al. (
      • Krick R.
      • Busse R.A.
      • Scacioc A.
      • Stephan M.
      • Janshoff A.
      • Thumm M.
      • Kühnel K.
      Structural and functional characterization of the two phosphoinositide binding sites of PROPPINs, a β-propeller protein family.
      ).

      Confocal laser scanning microscopy (CLSM)

      CLSM images were taken with a confocal laser scanning microscope LSM 880 (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) equipped with a 40× objective (W Plan-Apochromat, NA = 1.0, Zeiss). BODIPY®-TMR-PtdInsPs were monitored at 520 − 650 nm after excitation at 488 nm.

      Fluorescence recovery after photobleaching (FRAP)

      Fluorescence intensity in a region of interest (ROI) of a model membrane doped with one of the BODIPY®-TMR-PtdInsPs (λbleach = 488 nm) was bleached by a short laser pulse, and the time-dependent fluorescence recovery was recorded with a frame rate of 3-4 frames·s−1. The diffusion coefficients and mobile fractions were calculated using a Hankel transformation (
      • Jönsson P.
      • Jonsson M.P.
      • Tegenfeldt J.O.
      • Höök F.
      A method improving the accuracy of fluorescence recovery after photobleaching analysis.
      ).

      Data availability

      All data are contained within the article.

      Acknowledgments

      We thank Jutta Gerber-Nolte for technical assistance.

      Supplementary Material

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