2-Hydroxy Fatty Acid Enantiomers of Gb 3 Impact Shiga Toxin Binding and Membrane Organization

Schütte, Ole Mathis; Patalag, Lukas J.; Weber, Lucas M.C.; Ries, Annika; Römer, Winfried; Werz, Daniel B.; Steinem, Claudia

doi:10.1016/j.bpj.2015.05.009

Cited by 31 publications

(59 citation statements)

References 31 publications

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“…To see this figure in color, go online. of a protein layer that is determined by the height of the layer as well as the surface coverage (27,31,32,42). For ezrin bound to DOGS-Ni-NTA or PIP 2 , the change in OT as a result of protein surface coverage is reflected by the increase in DOT as a function of increasing receptor lipid content in the membrane.…”

Section: Discussionmentioning

confidence: 99%

“…RIfS is an optical technique that allows label-free investigations of protein binding to a specific receptor lipid embedded in SLBs (27,(31)(32)(33)(34)(35). The adsorption process is detected as an increase in optical thickness, OT ¼ n d, i.e., the product of the refractive index n and the physical layer thickness d. RIfS experiments were employed to analyze whether ezrin wt or ezrin T567D adsorbed to either the receptor lipid DOGS-Ni-NTA or PIP 2 results in a difference in OT.…”

Section: Analysis Of Ezrin-lipid Interactions By Rifsmentioning

confidence: 99%

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Mode of Ezrin-Membrane Interaction as a Function of PIP 2 Binding and Pseudophosphorylation

Shabardina

Kramer

Gerdes

et al. 2016

Biophysical Journal

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Ezrin, a protein of the ezrin, radixin, moesin (ERM) family, provides a regulated linkage between the plasma membrane and the cytoskeleton. The hallmark of this linkage is the activation of ezrin by phosphatidylinositol-4,5-bisphosphate (PIP2) binding and a threonine phosphorylation at position 567. To analyze the influence of these activating factors on the organization of ezrin on lipid membranes and the proposed concomitant oligomer-monomer transition, we made use of supported lipid bilayers in conjunction with atomic force microscopy and fluorescence microscopy. Bilayers doped with either PIP2 as the natural receptor lipid of ezrin or a Ni-nitrilotriacetic acid-equipped lipid to bind the proteins via their His6-tags to the lipid membrane were used to bind two different ezrin variants: ezrin wild-type and ezrin T567D mimicking the phosphorylated state. Using a combination of reflectometric interference spectroscopy, atomic force microscopy, and Förster resonance energy transfer experiments, we show that only the ezrin T567D mutant, upon binding to PIP2-containing bilayers, undergoes a remarkable conformational change, which we attribute to an opening of the conformation resulting in monomeric protein on the lipid bilayer.

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Section: Discussionmentioning

confidence: 99%

Section: Analysis Of Ezrin-lipid Interactions By Rifsmentioning

confidence: 99%

Mode of Ezrin-Membrane Interaction as a Function of PIP 2 Binding and Pseudophosphorylation

Shabardina

Kramer

Gerdes

et al. 2016

Biophysical Journal

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“…The largest protein layer thickness was found for PI(3,4,5)P 3 -doped membranes with d protein-max ϭ 2.8 Ϯ 0.3 nm. Assuming a maximum protein surface coverage of 56% according to the scaled particle theory (25) and a diameter of the PH domain of 4.2 nm as obtained from the crystal structure (12), one would expect a protein layer thickness of 2.4 nm. Comparing the theoretical and experimental values, we conclude that full protein coverage is reached in the case of PI(3,4,5)P 3 , whereas for the other PIPs, a lower surface coverage is found.…”

Section: Resultsmentioning

confidence: 99%

“…3A) (19 -21). RIfS is a well established technique to quantitatively monitor proteinreceptor interactions at solid-supported membranes (22)(23)(24)(25).…”

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confidence: 99%

Specificity of Collybistin-Phosphoinositide Interactions

Ludolphs

Schneeberger

Soykan

et al. 2016

Journal of Biological Chemistry

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The regulatory protein collybistin (CB) recruits the receptorscaffolding 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 (CB2 SH3؉ ) 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" CB2 SH3؉ 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.The function of neuronal synapses and the dynamic regulation of their efficacy depend on the assembly of the postsynaptic neurotransmitter receptor apparatus. The main scaffolding protein of inhibitory glycinergic and GABAergic postsynapses in mammals is gephyrin (1, 2), whose recruitment to the postsynaptic membrane is controlled by the adaptor protein collybistin (CB) 2 (3). Loss of CB results in a strong reduction of gephyrin and GABA A receptor clusters in several regions of the forebrain, which demonstrates the essential role of CB in the assembly and maintenance of GABAergic postsynaptic structures (4). CB belongs to the Dbl family of guanine nucleotide exchange factors. In mouse, four differently spliced CB mRNAs are present (CB1 SH3ϩ , CB2 SH3Ϫ , CB2 SH3ϩ , and CB3 SH3ϩ ). All four mRNAs encode a Dbl homology (DH) and a pleckstrin homology (PH) domain. The three major variants (CB1 SH3ϩ , CB2 SH3ϩ , and CB3 SH3ϩ ) encode CBs with an additional N-terminal Src homology 3 (SH3) domain but differ in their C termini. A fourth variant (CB2 SH3Ϫ ) encodes a CB2 isoform lacking the SH3 domain ( Fig. 1) but is very rare (5) as its protein product is not detectable in mouse brain (6). Importantly, the PH domain of the different CBs is required for proper function as indicated by the fact that its deletion abolishes the plasma membrane targeting of gephyrin-CB complexes when cotransfected in HEK293 cells and causes a loss of dendritic gephyrin clusters in dissociated rat cortical neurons (5).In vitro binding studies utilizing a variety of inositol headgroups, soluble phosphoinositide analogs, and liposomes containing phosphoinositides showed that PH domains bind phosphoinositides with a broad range of selectivity and affinity (7-10). An early membrane acti...

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“…For example, a recent study was able to demonstrate that the chirality of Gb3 influences protein-membrane organization and moreover affects the propensity of tubule formation in GUVs. The authors concluded, that the protein cluster formation depends on the structure of the Gb3 fatty acid chains and area demand of unsaturated fatty acids, which, in combination, impact membrane bending[105].An additional study simulating the effects of varying Gb3 concentrations (0, 12, 25, 50 and 100 mol-%) and degree of saturation confirmed that no tubule formation occurred for saturated Gb3 species. This was owing to the formation of a rigid immobile phase, resistant to bending in view of a high energetic cost.…”

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confidence: 60%

How synthetic membrane systems contribute to the understanding of lipid-driven endocytosis

Schubert

Römer

2015

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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Synthetic membrane systems, such as giant unilamellar vesicles and solid supported lipid bilayers, have widened our understanding of biological processes occurring at or through membranes. Artificial systems are particularly suited to study the inherent properties of membranes with regard to their components and characteristics. This review critically reflects the emerging molecular mechanism of lipid-driven endocytosis and the impact of model membrane systems in elucidating the complex interplay of biomolecules within this process. Lipid receptor clustering induced by binding of several toxins, viruses and bacteria to the plasma membrane leads to local membrane bending and formation of tubular membrane invaginations. Here, lipid shape, and protein structure and valency are the essential parameters in membrane deformation. Combining observations of complex cellular processes and their reconstitution on minimal systems seems to be a promising future approach to resolve basic underlying mechanisms. This article is part of a Special Issue entitled: Mechanobiology.

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2-Hydroxy Fatty Acid Enantiomers of Gb 3 Impact Shiga Toxin Binding and Membrane Organization

Cited by 31 publications

References 31 publications

Mode of Ezrin-Membrane Interaction as a Function of PIP 2 Binding and Pseudophosphorylation

Mode of Ezrin-Membrane Interaction as a Function of PIP 2 Binding and Pseudophosphorylation

Specificity of Collybistin-Phosphoinositide Interactions

How synthetic membrane systems contribute to the understanding of lipid-driven endocytosis

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