Cholesterol Increases Lipid Binding Rate and Changes Binding Behavior of Bacillus thuringiensis Cytolytic Protein

Tharad, Sudarat; Üzülmez, Öykü; Promdonkoy, Boonhiang; Toca-Herrera, José L.

doi:10.3390/ijms19123819

Cited by 14 publications

(11 citation statements)

References 37 publications

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“…The planar structure of cholesterol promotes orientational order in the liquid disordered phase of the bilayer, leading to reduced lateral diffusion and increased hydrophobic thickness ( Figure 7A ; Crane and Tamm, 2004 ; de Meyer and Smit, 2009 ; Filippov et al, 2003 ; Hung et al, 2007 ). For instance, as much as 20% increase in thickness can be expected for a POPC bilayer when the cholesterol concentration is varied from 0% to 30% ( Mouritsen and Bagatolli, 2016 ; Tharad et al, 2018 ).…”

Section: Resultsmentioning

confidence: 99%

Allosteric modulation of the adenosine A2A receptor by cholesterol

Huang

Almurad

Pejana

et al. 2022

eLife

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Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A2A receptor (A2AR) in reconstituted phospholipid nanodiscs. This model system conveniently excludes possible effects arising from cholesterol-induced phase separation or receptor oligomerization and focuses on the question of allostery. GTP hydrolysis assays show that cholesterol weakly enhances the basal signaling of A2AR while decreasing the agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy shows that this enhancement arises from an increase in the receptor’s active state population and a G-protein-bound precoupled state. 19F NMR of fluorinated cholesterol analogs reveals transient interactions with A2AR, indicating a lack of high-affinity binding or direct allosteric modulation. The combined results suggest that the observed allosteric effects are largely indirect and originate from cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.

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Section: Resultsmentioning

confidence: 99%

Allosteric modulation of the adenosine A2A receptor by cholesterol

Huang

Almurad

Pejana

et al. 2022

eLife

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“…For GM1 liposomes, it was assumed that ∼57% lipids (and GM1) are in the outer leaflet, based on their diameter and the assumed thickness of the bilayer (5 nm). 39 Similar to the NDs, the K a,app measured for the liposomes decreases with the increasing %GM1 [from 1.1 × 10 6 M −1 (1%) to 1.4 × 10 5 M −1 (30%)]. The GM1 glycomicelles exhibit the lowest K a,app (4.1 × 10 4 M −1 ).…”

Section: Materials and Methods Proteins Styrene Maleic Acidmentioning

confidence: 62%

“…The K a,app for the 1% GM1 ND (1.6 × 10 6 M –1 ) is of similar magnitude; the values for the higher % GM1 NDs are ∼10 5 M –1 . For GM1 liposomes, it was assumed that ∼57% lipids (and GM1) are in the outer leaflet, based on their diameter and the assumed thickness of the bilayer (5 nm) . Similar to the NDs, the K a,app measured for the liposomes decreases with the increasing %GM1 [from 1.1 × 10 6 M –1 (1%) to 1.4 × 10 5 M –1 (30%)].…”

Section: Resultsmentioning

confidence: 99%

How Choice of Model Membrane Affects Protein–Glycosphingolipid Interactions: Insights from Native Mass Spectrometry

et al. 2022

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Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) are involved in numerous physiological and pathophysiological processes. Many model membrane systems are available for studying GBP–GSL interactions, but a systematic investigation has not been carried out on how the nature of the model membrane affects binding. In this work, we use electrospray ionization mass spectrometry (ESI-MS), both direct and competitive assays, to measure the binding of cholera toxin B subunit homopentamer (CTB5) to GM1 ganglioside in liposomes, bilayer islands [styrene maleic acid lipid particles (SMALPs), nanodiscs (NDs), and picodiscs (PDs)], and micelles. We find that direct ESI-MS analysis of CTB5 binding to GM1 is unreliable due to non-uniform response factors, incomplete extraction of bound GM1 in the gas phase, and nonspecific CTB5–GM1 interactions. Conversely, indirect proxy ligand ESI-MS measurements show that the intrinsic (per binding site) association constants of CTB5 for PDs, NDs, and SMALPs are similar and comparable to the affinity of soluble GM1 pentasaccharide (GM1os). The observed affinity decreases with increasing GM1 content due to molecular crowding stemming from GM1 clustering. Unlike the smaller model membranes, the observed affinity of CTB5 toward GM1 liposomes is ∼10-fold weaker than GM1os and relatively insensitive to the GM1 content. GM1 glycomicelles exhibit the lowest affinity, ∼35-fold weaker than GM1os. Together, the results highlight experimental design considerations for quantitative GBP–GSL binding studies involving multisubunit GBPs and factors to consider when comparing results obtained with different membrane systems. Notably, they suggest that bilayer islands with a low percentage of GSL, wherein clustering is minimized, are ideal for assessing intrinsic strength of GBP–GSL interactions in a membrane environment, while binding to liposomes, which is sub-optimal due to extensive clustering, may be more representative of authentic cellular environments.

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“…7A) (Crane and Tamm, 2004;De Meyer and Smit, 2009;Filippov et al, 2003;Hung et al, 2007). For instance, as much as 20% increase in thickness can be expected for a POPC bilayer when the cholesterol concentration is varied from 0 to 30% (Mouritsen and Bagatolli, 2016;Tharad et al, 2018). ) of Laurdan fluorescence is a consequence of solvent contact.…”

Section: Cholesterol Allostery In A2ar May Be a Results Of Indirect Membrane Effectsmentioning

confidence: 99%

Allosteric modulation of the adenosine A_2A receptor by cholesterol

Huang

Almurad

Pejana

et al. 2021

Preprint

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Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A2A receptor (A2AR) in reconstituted phospholipid bilayers. GTP hydrolysis assays show that cholesterol is a weak positive allosteric modulator of A2AR, as seen through enhanced basal signaling and a small decrease in agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy suggests that this enhancement arises from an increase in the receptor's active state populations and stronger G protein coupling. 19F NMR of fluorinated cholesterol analogs reveals transient and non-specific interactions with A2AR, indicating a lack of high-affinity binding sites or direct allosteric modulation. This is confirmed by computational analysis which suggests that cholesterol contacts confer a weak and possibly negative allosteric effect. The combined results suggest that the observed cholesterol allostery in A2AR is likely a result of indirect membrane effects through cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.

show abstract

Cholesterol Increases Lipid Binding Rate and Changes Binding Behavior of Bacillus thuringiensis Cytolytic Protein

Cited by 14 publications

References 37 publications

Allosteric modulation of the adenosine A2A receptor by cholesterol

Allosteric modulation of the adenosine A2A receptor by cholesterol

How Choice of Model Membrane Affects Protein–Glycosphingolipid Interactions: Insights from Native Mass Spectrometry

Allosteric modulation of the adenosine A_2A receptor by cholesterol

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