Multiple lipid binding sites determine the affinity of PH domains for phosphoinositide-containing membranes

Association of peripheral proteins with lipid bilayers regulates membrane signaling and dynamics. Pleckstrin homology (PH) domains bind to phosphatidylinositol phosphate (PIP) molecules in membranes. The effects of local PIP enrichment on the interaction of PH domains with membranes is unclear. Molecular dynamics simulations allow estimation of the binding energy of GRP1 PH domain to PIP3-containing membranes. The free energy of interaction of the PH domain with more than two PIP3 molecules is comparable to experimental values, suggesting that PH domain binding involves local clustering of PIP molecules within membranes. We describe a mechanism of PH binding proceeding via an encounter state to two bound states which differ in the orientation of the protein relative to the membrane, these orientations depending on the local PIP concentration. These results suggest that nanoscale clustering of PIP molecules can control the strength and orientation of PH domain interaction in a concentration-dependent manner.

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Structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain

Biochemical Journal ◽

10.1042/bcj20160791 ◽

2017 ◽

Vol 474 (4) ◽

pp. 539-556 ◽

Cited By ~ 18

Author(s):

Tao Ni ◽

Antreas C. Kalli ◽

Fiona B. Naughton ◽

Luke A. Yates ◽

Omar Naneh ◽

...

Keyword(s):

Inositol Phosphate ◽

Lipid Binding ◽

Site Directed Mutagenesis ◽

Pleckstrin Homology Domain ◽

Ph Domain ◽

Pleckstrin Homology ◽

Phosphatidylinositol Phosphate ◽

Protein Membrane ◽

Dynamics Simulations ◽

Lipid Nanodiscs

Kindlins co-activate integrins alongside talin. They possess, like talin, a FERM domain (4.1-erythrin–radixin–moiesin domain) comprising F0–F3 subdomains, but with a pleckstrin homology (PH) domain inserted in the F2 subdomain that enables membrane association. We present the crystal structure of murine kindlin-3 PH domain determined at a resolution of 2.23 Å and characterise its lipid binding using biophysical and computational approaches. Molecular dynamics simulations suggest flexibility in the PH domain loops connecting β-strands forming the putative phosphatidylinositol phosphate (PtdInsP)-binding site. Simulations with PtdInsP-containing bilayers reveal that the PH domain associates with PtdInsP molecules mainly via the positively charged surface presented by the β1–β2 loop and that it binds with somewhat higher affinity to PtdIns(3,4,5)P3 compared with PtdIns(4,5)P2. Surface plasmon resonance (SPR) with lipid headgroups immobilised and the PH domain as an analyte indicate affinities of 300 µM for PtdIns(3,4,5)P3 and 1 mM for PtdIns(4,5)P2. In contrast, SPR studies with an immobilised PH domain and lipid nanodiscs as the analyte show affinities of 0.40 µM for PtdIns(3,4,5)P3 and no affinity for PtdIns(4,5)P2 when the inositol phosphate constitutes 5% of the total lipids (∼5 molecules per nanodisc). Reducing the PtdIns(3,4,5)P3 composition to 1% abolishes nanodisc binding to the PH domain, as does site-directed mutagenesis of two lysines within the β1–β2 loop. Binding of PtdIns(3,4,5)P3 by a canonical PH domain, Grp1, is not similarly influenced by SPR experimental design. These data suggest a role for PtdIns(3,4,5)P3 clustering in the binding of some PH domains and not others, highlighting the importance of lipid mobility and clustering for the biophysical assessment of protein–membrane interactions.

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Influence of Brain Gangliosides on the Formation and Properties of Supported Lipid Bilayers for Binding Kinetics Studies

10.26434/chemrxiv.7505330 ◽

2018 ◽

Author(s):

Luke Jordan ◽

Nathan Wittenberg

Keyword(s):

Lipid Bilayers ◽

Binding Kinetics ◽

Supported Lipid Bilayers ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Supported Lipid Bilayer ◽

Head Groups ◽

Lipid Diffusion ◽

Kinetics Of ◽

Brain Gangliosides

This is a comprehensive study of the effects of the four major brain gangliosides (GM1, GD1b, GD1a, and GT1b) on the adsorption and rupture of phospholipid vesicles on SiO2 surfaces for the formation of supported lipid bilayer (SLB) membranes. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we show that gangliosides GD1a and GT1b significantly slow the SLB formation process, whereas GM1 and GD1b have smaller effects. This is likely due to the net ganglioside charge as well as the positions of acidic sugar groups on ganglioside glycan head groups. Data is included that shows calcium can accelerate the formation of ganglioside-rich SLBs. Using fluorescence recovery after photobleaching (FRAP) we also show that the presence of gangliosides significantly reduces lipid diffusion coefficients in SLBs in a concentration-dependent manner. Finally, using QCM-D and GD1a-rich SLB membranes we measure the binding kinetics of an anti-GD1a antibody that has similarities to a monoclonal antibody that is a hallmark of a variant of Guillain-Barre syndrome.

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Interactions of peripheral proteins with model membranes as viewed by molecular dynamics simulations

Biochemical Society Transactions ◽

10.1042/bst20140144 ◽

2014 ◽

Vol 42 (5) ◽

pp. 1418-1424 ◽

Cited By ~ 28

Author(s):

Antreas C. Kalli ◽

Mark S. P. Sansom

Keyword(s):

Lipid Bilayers ◽

Molecular Mechanisms ◽

Phospholipid Composition ◽

Md Simulations ◽

Multiscale Simulation ◽

Lipid Binding ◽

Coarse Grained ◽

Peripheral Proteins ◽

Dynamics Simulations ◽

Phosphatidylinositol Phosphates

Many cellular signalling and related events are triggered by the association of peripheral proteins with anionic lipids in the cell membrane (e.g. phosphatidylinositol phosphates or PIPs). This association frequently occurs via lipid-binding modules, e.g. pleckstrin homology (PH), C2 and four-point-one, ezrin, radixin, moesin (FERM) domains, present in peripheral and cytosolic proteins. Multiscale simulation approaches that combine coarse-grained and atomistic MD simulations may now be applied with confidence to investigate the molecular mechanisms of the association of peripheral proteins with model bilayers. Comparisons with experimental data indicate that such simulations can predict specific peripheral protein–lipid interactions. We discuss the application of multiscale MD simulation and related approaches to investigate the association of peripheral proteins which contain PH, C2 or FERM-binding modules with lipid bilayers of differing phospholipid composition, including bilayers containing multiple PIP molecules.

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Oligomerization and Ca2+/calmodulin control binding of the ER Ca2+-sensors STIM1 and STIM2 to plasma membrane lipids

Bioscience Reports ◽

10.1042/bsr20130089 ◽

2013 ◽

Vol 33 (5) ◽

Cited By ~ 30

Author(s):

Rajesh Bhardwaj ◽

Hans-Michael Müller ◽

Walter Nickel ◽

Matthias Seedorf

Keyword(s):

Plasma Membrane ◽

Membrane Lipids ◽

Lipid Binding ◽

Dependent Manner ◽

Activation Threshold ◽

Concentration Dependent Manner ◽

Rich Domain ◽

Α Helix ◽

Distinct Features ◽

Ca2 Channels

Ca2+ (calcium) homoeostasis and signalling rely on physical contacts between Ca2+ sensors in the ER (endoplasmic reticulum) and Ca2+ channels in the PM (plasma membrane). STIM1 (stromal interaction molecule 1) and STIM2 Ca2+ sensors oligomerize upon Ca2+ depletion in the ER lumen, contact phosphoinositides at the PM via their cytosolic lysine (K)-rich domains, and activate Ca2+ channels. Differential sensitivities of STIM1 and STIM2 towards ER luminal Ca2+ have been studied but responses towards elevated cytosolic Ca2+ concentration and the mechanism of lipid binding remain unclear. We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PI(4,5)P2-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation. In contrast, dimerization of STIM2 K-rich domain was sufficient for lipid binding. Furthermore, the K-rich domain of STIM2, but not of STIM1, forms an amphipathic α-helix. These distinct features of the STIM2 K-rich domain cause an increased affinity for PI(4,5)P2, consistent with the lower activation threshold of STIM2 and a function as regulator of basal Ca2+ levels. Concomitant with higher affinity for PM lipids, binding of CaM (calmodulin) inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca2+ and PI(4,5)P2 concentration-dependent manner. Therefore we suggest that elevated cytosolic Ca2+ concentration down-regulates STIM2-mediated ER–PM contacts via CaM binding.

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cADP-ribose activates reconstituted ryanodine receptors from coronary arterial smooth muscle

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.280.1.h208 ◽

2001 ◽

Vol 280 (1) ◽

pp. H208-H215 ◽

Cited By ~ 41

Author(s):

Pin-Lan Li ◽

Wang-Xian Tang ◽

Hector H. Valdivia ◽

Ai-Ping Zou ◽

William B. Campbell

Keyword(s):

Smooth Muscle ◽

Lipid Bilayers ◽

Ryanodine Receptors ◽

Fold Increase ◽

Ruthenium Red ◽

Dependent Manner ◽

High Concentration ◽

Open Probability ◽

Arterial Smooth Muscle ◽

Concentration Dependent Manner

The present study was designed to test the hypothesis that cADP-ribose (cADPR) increases Ca2+release through activation of ryanodine receptors (RYR) on the sarcoplasmic reticulum (SR) in coronary arterial smooth muscle cells (CASMCs). We reconstituted RYR from the SR of CASMCs into planar lipid bilayers and examined the effect of cADPR on the activity of these Ca2+ release channels. In a symmetrical cesium methanesulfonate configuration, a 245 pS Cs+ current was recorded. This current was characterized by the formation of a subconductance and increase in the open probability (NPo) of the channels in the presence of ryanodine (0.01–1 μM) and imperatoxin A (100 nM). A high concentration of ryanodine (50 μM) and ruthenium red (40–80 μM) substantially inhibited the activity of RYR/Ca2+ release channels. Caffeine (0.5–5 mM) markedly increased the NPo of these Ca2+release channels of the SR, but d- myo-inositol 1,4,5-trisphospate and heparin were without effect. Cyclic ADPR significantly increased the NPo of these Ca2+release channels of SR in a concentration-dependent manner. Addition of cADPR (0.01 μM) into the cis bath solution produced a 2.9-fold increase in the NPo of these RYR/Ca2+release channels. An eightfold increase in the NPo of the RYR/Ca2+ release channels (0.0056 ± 0.001 vs. 0.048 ± 0.017) was observed at a concentration of cADPR of 1 μM. The effect of cADPR was completely abolished by ryanodine (50 μM). In the presence of cADPR, Ca2+-induced activation of these channels was markedly enhanced. These results provide evidence that cADPR activates RYR/Ca2+ release channels on the SR of CASMCs. It is concluded that cADPR stimulates Ca2+ release through the activation of RYRs on the SR of these smooth mucle cells.

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Influence of Brain Gangliosides on the Formation and Properties of Supported Lipid Bilayers for Binding Kinetics Studies

10.26434/chemrxiv.7505330.v1 ◽

2018 ◽

Author(s):

Luke Jordan ◽

Nathan Wittenberg

Keyword(s):

Lipid Bilayers ◽

Binding Kinetics ◽

Supported Lipid Bilayers ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Supported Lipid Bilayer ◽

Head Groups ◽

Lipid Diffusion ◽

Kinetics Of ◽

Brain Gangliosides

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Gramicidin increases lipid flip-flop in symmetric and asymmetric lipid vesicles

10.1101/383463 ◽

2018 ◽

Cited By ~ 1

Author(s):

M. Doktorova ◽

F. A. Heberle ◽

D. Marquardt ◽

R. Rusinova ◽

L. Sanford ◽

...

Keyword(s):

Effective Means ◽

Transmembrane Proteins ◽

Lipid Vesicles ◽

Melting Transition ◽

Dependent Manner ◽

Scanning Calorimetry ◽

Concentration Dependent Manner ◽

Flip Flop ◽

Lipid Distribution ◽

Dynamics Simulations

ABSTRACTUnlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Non-enzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels which have well-defined structures and known function, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small-angle X-ray scattering, circular dichroism and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time suggestive of scrambling activity, and the effect of the channel on the transbilayer lipid distribution in both symmetric POPC and asymmetric POPC/DMPC vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the amount of membrane deformation in the samples. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.

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Molecular mechanism for NLRP6 inflammasome assembly and activation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1817221116 ◽

2019 ◽

Vol 116 (6) ◽

pp. 2052-2057 ◽

Cited By ~ 33

Author(s):

Chen Shen ◽

Alvin Lu ◽

Wen Jun Xie ◽

Jianbin Ruan ◽

Roberto Negro ◽

...

Keyword(s):

Molecular Mechanism ◽

Conformational Changes ◽

Protein Complexes ◽

Multiple Roles ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Pyrin Domain ◽

Dynamics Simulations ◽

Inflammatory Caspases ◽

Lrr Domain

Inflammasomes are large protein complexes that trigger host defense in cells by activating inflammatory caspases for cytokine maturation and pyroptosis. NLRP6 is a sensor protein in the nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)-containing (NLR) inflammasome family that has been shown to play multiple roles in regulating inflammation and host defenses. Despite the significance of the NLRP6 inflammasome, little is known about the molecular mechanism behind its assembly and activation. Here we present cryo-EM and crystal structures of NLRP6 pyrin domain (PYD). We show that NLRP6 PYD alone is able to self-assemble into filamentous structures accompanied by large conformational changes and can recruit the ASC adaptor using PYD–PYD interactions. Using molecular dynamics simulations, we identify the surface that the NLRP6 PYD filament uses to recruit ASC PYD. We further find that full-length NLRP6 assembles in a concentration-dependent manner into wider filaments with a PYD core surrounded by the NBD and the LRR domain. These findings provide a structural understanding of inflammasome assembly by NLRP6 and other members of the NLR family.

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Redefining the specificity of phosphoinositide-binding by human PH domain-containing proteins

Nature Communications ◽

10.1038/s41467-021-24639-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Nilmani Singh ◽

Adriana Reyes-Ordoñez ◽

Michael A. Compagnone ◽

Jesus F. Moreno ◽

Benjamin J. Leslie ◽

...

Keyword(s):

Single Molecule ◽

Learning Algorithm ◽

Specific Interaction ◽

Lipid Vesicles ◽

Lipid Binding ◽

Ph Domain ◽

Structural Requirements ◽

Pulldown Assay ◽

Ph Domains ◽

Functional Relevance

AbstractPleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins are unclear. Here we employ a single-molecule pulldown assay to study interactions of lipid vesicles with full-length proteins in mammalian whole cell lysates. Of 67 human PH domain-containing proteins initially examined, 36 (54%) are found to have affinity for PIPs with various specificity, the majority of which have not been reported before. Further investigation of ARHGEF3 reveals distinct structural requirements for its binding to PI(4,5)P2 and PI(3,5)P2, and functional relevance of its PI(4,5)P2 binding. We generate a recursive-learning algorithm based on the assay results to analyze the sequences of 242 human PH domains, predicting that 49% of them bind PIPs. Twenty predicted binders and 11 predicted non-binders are assayed, yielding results highly consistent with the prediction. Taken together, our findings reveal unexpected lipid-binding specificity of PH domain-containing proteins.

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Lipid raft components cholesterol and sphingomyelin increase H+/OH− permeability of phosphatidylcholine membranes

Biochemical Journal ◽

10.1042/bj20051620 ◽

2006 ◽

Vol 398 (3) ◽

pp. 485-495 ◽

Cited By ~ 27

Author(s):

Rebekah H. Gensure ◽

Mark L. Zeidel ◽

Warren G. Hill

Keyword(s):

Lipid Bilayers ◽

Lipid Raft ◽

Water Permeability ◽

Fold Increase ◽

Proton Flux ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Crude Lipid ◽

Lipid Order ◽

Lipid Environment

H+/OH− permeation through lipid bilayers occurs at anomalously high rates and the determinants of proton flux through membranes are poorly understood. Since all life depends on proton gradients, it is important to develop a greater understanding of proton leak phenomena. We have used stopped-flow fluorimetry to probe the influence of two lipid raft components, chol (cholesterol) and SM (sphingomyelin), on H+/OH− and water permeability. Increasing the concentrations of both lipids in POPC (palmitoyl-2-oleoyl phosphatidylcholine) liposomes decreased water permeability in a concentration-dependent manner, an effect that correlated with increased lipid order. Surprisingly, proton flux was increased by increasing the concentration of chol and SM. The chol effect was complex with molar concentrations of 17.9, 33 and 45.7% giving 2.8-fold (P<0.01), 2.2-fold (P<0.001) and 5.1-fold (P<0.001) increases in H+/OH− permeability from a baseline of 2.4×10−2 cm/s. SM at 10 mole% effected a 2.8-fold increase (P<0.01), whereas 20 and 30 mole% enhanced permeability by 3.6-fold (P<0.05) and 4.1-fold respectively (P<0.05). Supplementing membranes containing chol with SM did not enhance H+/OH− permeability. Of interest was the finding that chol addition to soya-bean lipids decreased H+/OH− permeability, consistent with an earlier report [Ira and Krishnamoorthy (2001) J. Phys. Chem. B 105, 1484–1488]. We speculate that the presence of proton carriers in crude lipid extracts might contribute to this result. We conclude that (i) chol and SM specifically and independently increase rates of proton permeation in POPC bilayers, (ii) domains enriched in these lipids or domain interfaces may represent regions with high H+/OH− conductivity, (iii) H+/OH− fluxes are not governed by lipid order and (iv) chol can inhibit or promote H+/OH− permeability depending on the total lipid environment. Theories of proton permeation are discussed in the light of these results.

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