Cholesterol Recognition Amino Acid Consensus Research Articles

TRPV4-dependent Ca2+ influx determines cholesterol dynamics at the plasma membrane

The activities of the transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable nonselective cation channel, are controlled by its surrounding membrane lipids (e.g., cholesterol, phosphoinositides). The transmembrane region of TRPV4 contains a cholesterol recognition amino acid consensus (CRAC) motif and its inverted (CARC) motif located in the plasmalemmal cytosolic leaflet. TRPV4 localizes in caveolae, a bulb-shaped cholesterol-rich domain at the plasma membrane. Here, we visualized the spatiotemporal interactions between TRPV4 and cholesterol at the plasma membrane in living cells by dual-color single-molecule imaging using total internal reflection fluorescence microscopy. To this aim, we labeled cholesterol at the cytosolic leaflets of the plasma membrane using a cholesterol biosensor, D4H. Our single-molecule tracking analysis showed that the TRPV4 molecules colocalize with D4H-accessible cholesterol molecules mainly in the low fluidity membrane domains in which both molecules are highly clustered. Colocalization of TRPV4 and D4H-accessible cholesterol was observed both inside and outside of caveolae. Agonist-evoked TRPV4 activation remarkably decreased colocalization probability and association rate between TRPV4 and D4H-accessible cholesterol molecules. Interestingly, upon TRPV4 activation, the particle density of D4H-accessible cholesterol molecules was decreased and the D4H-accessible cholesterol molecules in the fast-diffusing state were increased at the plasma membrane. The introduction of skeletal dysplasia-associated R616Q mutation into the CRAC/CARC motif of TRPV4, which reduced the interaction with cholesterol clusters, could not alter the D4H-accessible cholesterol dynamics. Mechanistically, TRPV4-mediated Ca2+ influx and the C-terminal calmodulin-binding site of TRPV4 are essential for modulating the plasmalemmal D4H-accessible cholesterol dynamics. We propose that TRPV4 remodels its surrounding plasmalemmal environment by manipulating cholesterol dynamics through Ca2+ influx.

Identification of structural motifs critical for human G6PC2 function informed by sequence analysis and an AlphaFold2-predicted model.

G6PC2 encodes a glucose-6-phosphatase (G6Pase) catalytic subunit, primarily expressed in pancreatic islet β cells, which modulates the sensitivity of insulin secretion to glucose and thereby regulates fasting blood glucose (FBG). Mutational analyses were conducted to validate an AlphaFold2 (AF2)-predicted structure of human G6PC2 in conjunction with a novel method to solubilize and purify human G6PC2 from a heterologous expression system. These analyses show that residues forming a predicted intramolecular disulfide bond are essential for G6PC2 expression and that residues forming part of a type 2 phosphatidic acid phosphatase (PAP2) motif are critical for enzyme activity. Additional mutagenesis shows that residues forming a predicted substrate cavity modulate enzyme activity and substrate specificity and residues forming a putative cholesterol recognition amino acid consensus (CRAC) motif influence protein expression or enzyme activity. This CRAC motif begins at residue 219, the site of a common G6PC2 non-synonymous single-nucleotide polymorphism (SNP), rs492594 (Val219Leu), though the functional impact of this SNP is disputed. In microsomal membrane preparations, the L219 variant has greater activity than the V219 variant, but this difference disappears when G6PC2 is purified in detergent micelles. We hypothesize that this was due to a differential association of the two variants with cholesterol. This concept was supported by the observation that the addition of cholesteryl hemi-succinate to the purified enzymes decreased the Vmax of the V219 and L219 variants ∼8-fold and ∼3 fold, respectively. We anticipate that these observations should support the rational development of G6PC2 inhibitors designed to lower FBG.

Molecular Biophysics of Class A G Protein Coupled Receptors-Lipids Interactome at a Glance-Highlights from the A2A Adenosine Receptor.

G protein-coupled receptors (GPCRs) are embedded in phospholipid membrane bilayers with cholesterol representing 34% of the total lipid content in mammalian plasma membranes. Membrane lipids interact with GPCRs structures and modulate their function and drug-stimulated signaling through conformational selection. It has been shown that anionic phospholipids form strong interactions between positively charged residues in the G protein and the TM5-TM6-TM 7 cytoplasmic interface of class A GPCRs stabilizing the signaling GPCR-G complex. Cholesterol with a high content in plasma membranes can be identified in more specific sites in the transmembrane region of GPCRs, such as the Cholesterol Consensus Motif (CCM) and Cholesterol Recognition Amino Acid Consensus (CRAC) motifs and other receptor dependent and receptor state dependent sites. Experimental biophysical methods, atomistic (AA) MD simulations and coarse-grained (CG) molecular dynamics simulations have been applied to investigate these interactions. We emphasized here the impact of phosphatidyl inositol-4,5-bisphosphate (PtdIns(4,5)P2 or PIP2), a minor phospholipid component and of cholesterol on the function-related conformational equilibria of the human A2A adenosine receptor (A2AR), a representative receptor in class A GPCR. Several GPCRs of class A interacted with PIP2 and cholesterol and in many cases the mechanism of the modulation of their function remains unknown. This review provides a helpful comprehensive overview for biophysics that enter the field of GPCRs-lipid systems.

Cholesterol affects voltage-gated proton channels indirectly.

A number of membrane proteins, including ion channels, interact directly with cholesterol within the plasma membrane. This interaction may affect physiologically relevant properties of the protein. Often, protein-sterol interaction is facilitated by a cholesterol recognition amino acid consensus sequence (CRAC) domain with (L/V)-X1-5-(Y)-X1-5-(K/R) or the inverse sequence CARC (K/R)-X1-5-(Y/F)-X1-5-(L/V) (Fantini & Barrantes, 2013, Front. Physiol. 4:31). The human proton channel hHV1 contains a putative CARC sequence KNNYAAMV (amino-acid residues 131-138) within the extrafacial part of its S2 transmembrane domain. We explored effects of cholesterol depletion from or addition to the membrane. Modulation of cholesterol levels using methyl-β-cyclodextrin (MBCD) dramatically altered current amplitude in inside-out membrane patches. Mutations to the CARC domain did not alter the effects of cholesterol, indicating that cholesterol binding to the CARC domain is not responsible. We propose that hHV1 molecules preferentially associate with cholesterol-dependent domains within the membrane. Cholesterol might affect proton currents if varying cholesterol changes the distribution of hHV1 molecules in the membrane between the cholesterol-dependent lipid domains and the rest of the membrane. A previous study reported that suction increased proton currents in excised patches and this was interpreted as evidence for mechanosensitivity of the HV1 protein (Pathak et al, 2016, J. Gen. Physiol. 148:405). We have confirmed that suction applied to the pipette increases proton currents. This observation is consistent with our hypothesis since membrane tension will undoubtedly affect the association of individual Hv1 molecules with lipid domains without the need for bona fide channel mechanosensitivity. Increase in membrane tension would result in the increased proton currents due to the dispersal of condensed lipid domains in a similar way to the cholesterol depletion. This study provides the first indication that HV1 associates with membrane lipid domains, sometimes called lipid rafts.

Cholesterol binding to BK channel-forming proteins involves the central tyrosine of a CRAC domain located at the channel core-cytosolic tail interface.

Cholesterol (CLR) decreases the open probability of voltage/Ca2+-gated, large conductance K+ (BK, cbv1) channels. Previous studies strongly favored the possibility of cholesterol-cbv1 direct interactions, yet CLR binding to cbv1 has not been tested. The Tyr450Phe substitution in the CLR recognition amino acid consensus motif 4 (CRAC4) blunts CLR inhibition of channel activity. CRAC4 is located at the interface of two structural domains of cbv1: the core and the cytosolic tail domain (CTD). Here, we set out to determine whether CLR binds to cbv1, and the importance of CRAC4 for this binding. We performed equilibrium dialysis binding experiments with recombinant cbv1 protein from Chinese hamster ovary (CHO) cell cultures at 5.8, 3, 1.5, 0.65, 0.3, 0.03 and zero mM CLR. CLR was delivered in a 1:8 molar complex with methyl-β-cyclodextrin. The protein constructs of study included full-length wild-type cbv1, cbv1 core following truncation at Ile327, cbv1 CTD alone, and full-length cbv1Tyr450Phe. Cbv1 denatured by heating to 75oC for 5 minutes prior to loading the dialysis tubes (denatured cbv1) was used as a negative control. Proper folding of cbv1Tyr450Phe was confirmed by nanoscale differential scanning fluorimetry. CLR concentrations present in the protein sample after 20-24 hours were measured by fluorimetry. Results showed that CLR affinity was greatly diminished in all experimental constructs. This suggests that Tyr450 located at the core-CTD interface either participates directly in CLR-cbv1 interaction or transduces allosteric communication between the core and CTD to enable CLR binding elsewhere. In summary, we detected CLR binding to cbv1 and established a critical role for Tyr450 within CRAC4 in this binding. Support: NIH grant HL148941 (ANB and AMD) and the UTHSC Frank W. Dugan Fellowship (EHS).

Regulation of PD-L1 through direct binding of cholesterol to CRAC motifs.

Cholesterol, an essential molecule for cell structure, function, and viability, plays crucial roles in the development, progression, and survival of cancer cells. Earlier studies have shown that cholesterol-lowering drugs can inhibit the high expression of programmed-death ligand 1 (PD-L1) that contributes to immunoevasion in cancer cells. However, the regulatory mechanism of cell surface PD-L1 abundance by cholesterol is still controversial. Here, using nuclear magnetic resonance and biochemical techniques, we demonstrated that cholesterol can directly bind to the transmembrane domain of PD-L1 through two cholesterol-recognition amino acid consensus (CRAC) motifs, forming a sandwich-like architecture and stabilizing PD-L1 to prevent downstream degradation. Mutations at key binding residues prohibit PD-L1–cholesterol interactions, decreasing the cellular abundance of PD-L1. Our results reveal a unique regulatory mechanism that controls the stability of PD-L1 in cancer cells, providing an alternative method to overcome PD-L1–mediated immunoevasion in cancers.

The TSPO ligand 3,17,19‐androsten‐5‐triol attenuates NASH by increasing FXR expression and autophagy

Translocator protein (TSPO; 18 kDa) is a ubiquitous high‐affinity cholesterol‐ and drug‐binding protein located in the outer mitochondrial membrane. 3,17,19‐androsten‐5‐triol (19‐Atriol) is a ligand binding at the cholesterol recognition amino acid consensus domain of TSPO. Our previous study suggested that TSPO deficiency ameliorated methionine‐choline‐deficient induced non‐alcoholic steatohepatitis (NASH) through downregulation of bile acid synthesis (Li et al., iScience 24: 102457, 2021). We examined the role of cholesterol binding to TSPO in NASH using the 19‐Atriol on high‐fat‐fructose‐cholesterol diet (HFFC)‐induced NASH in rats. After HFFC‐feeding for 13weeks, the rats were subcutaneously administered daily 19‐Atriol (20mg/kg) for 2 weeks (weeks 13‐15). The ratio of liver/body weight and serum triacylglycerol content were significantly reduced in 19‐Atriol‐ compared to vehicle‐treated rats. Immunoblot analyses indicated that liver inflammation (TNFα) and fibrosis markers (COL1A1, ACTA2) were reduced following 19‐Atriol treatment. Farnesoid X receptor (FXR), a bile acid receptor, was upregulated in response to 19‐Atriol treatment in HFFC‐fed rats. In search of the mechanism mediating the effects of 19‐Atriol we observed an increase in autophagy flux and the levels of phosphorylated transcription factor EB (TFEB), a major regulator of autophagy and lysosomal biogenesis. Taken together these results suggest that blocking cholesterol binding to TSPO by 19‐Atriol leads to the attenuation of NASH by increasing FXR expression levels and activating of autophagic degradation as was the case in TSPO‐deficient rats.

Prediction of Plasma Membrane Cholesterol from 7-Transmembrane Receptor Using Hybrid Machine Learning Algorithm

The researches have been made on G-protein coupled receptors (GPCRs) over the long-ago decades. GPCR is also named as 7-transmembrane (7TM) receptor. According to biological prospective GPCRs consist of large protein family with respective subfamilies and are mediated by different physiological phenomena like taste, smell, vision etc. The main functionality of these 7TM receptors is signal transduction among various cells. In human genome, cell membrane plays significant role. All cells are made up of trillion of cells and have dissimilar functionality. Cell membrane composed of different components. GPCRs are reported to be modulated by membrane cholesterol by interacting with cholesterol recognition amino acid consensus (L/V-X (1-5)-Y-X (1-5)-R/K) (CRAC) or reverse orientation of CRAC (R/K-X (1-5)-Y-X (1-5)-L/V) (CARC) motifs present in the TM helices. Among all, cholesterol is one who is regulated by membrane proteins. Here we took GPCR as membrane proteins and this protein modulates membrane cholesterol. According to cell biology, GPCR regulates a wide diversity of vital cellular processes and are targeted by a huge fraction of approved drugs. In this paper we have concentrated our investigation on membrane protein with membrane cholesterol. A hybrid algorithm consisting of spectral clustering and support vector machine is proposed for prediction of membrane cholesterol with GPCR. Spectral clustering uses graph nodes for calculating the cluster points and also it considers other concept such as similarity matrix, low-dimensional space for projecting the data points and upon this parameter at last construct the cluster centre. Supervised learning method is used for solving regression and classification problems. From the analysis we found that our result shows better prediction accuracy in terms of time complexity when compared with two existing models such as fuzzy c-means (FCM) and rough set with FCM model

Two Cholesterol Recognition Amino Acid Consensus Motifs of GP64 with Uncleaved Signal Peptide Are Required for Bombyx mori Nucleopolyhedrovirus Infection.

ABSTRACTThe signal peptide (SP) of integrated membrane proteins is removed cotranslationally or posttranslationally in the endoplasmic reticulum, while GP64, a membrane fusion protein of Bombyx mori nucleopolyhedrovirus (BmNPV), retains its SP in the mature protein and virion. In this study, we revealed that uncleaved SP is a key determinant with additional functions in infection. First, uncleaved SP endows BmNPV with strong virulence; second, SP retention-induced BmNPV infection depends on cholesterol recognition amino acid consensus domain 1 (CRAC1) and CRAC2. In contrast, the recombinant virus with SP-cleaved GP64 has reduced infectivity, and only CRAC2 is required for BmNPV infection. Furthermore, we showed that cholesterol in the plasma membrane is an important fusion receptor that interacts with CRAC2 of GP64. Our study suggested that BmNPV GP64 is a key cholesterol-binding protein and uncleaved SP determines GP64's unique dependence on the CRAC domains.IMPORTANCE BmNPV is a severe pathogen that mainly infects silkworms. GP64 is the key membrane fusion protein that mediates BmNPV infection, and some studies have indicated that cholesterol and lipids are involved in BmNPV infection. A remarkable difference from other membrane fusion proteins is that BmNPV GP64 retains its SP in the mature protein, but the cause is still unclear. In this study, we investigated the reason why BmNPV retains this SP, and its effects on protein targeting, virulence, and CRAC dependence were revealed by comparison of recombinant viruses harboring SP-cleaved or uncleaved GP64. Our study provides a basis for understanding the dependence of BmNPV infection on cholesterol/lipids and host specificity.

HRM and CRAC in MxIRT1 act as iron sensors to determine MxIRT1 vesicle-PM fusion and metal transport

ABSTRACT The IRON-REGULATED TRANSPORTER1 (IRT1) is critical for iron uptake in roots, and its exocytosis to the plasma membrane (PM) is regulated by detergent-resistant membranes. However, studies on IRT1 exocytosis and function in response to iron status are limited. Presently, we found that the histidine-rich motif (HRM) of MxIRT1 could bind to iron directly and HRM determined the delivery of MxIRT1 to the PM, after which the cholesterol recognition amino acid consensus (CRAC) motif-regulated MxIRT1 mediated metal transport. IMAC assay revealed that H192 was the vital site for HRM binding to Fe2+, and metal-binding activity was stopped after the deletion of HRM (MxIRT1∆HM) or in H192 site-directed mutants (H192A). MxIRT1∆HM or H192A in transgenic yeast and Arabidopsis failed to localize in the PM and displayed impaired iron absorption. In the PM, Y266 in CRAC was required for metal transport; Y266A transgenic Arabidopsis displayed the same root length, Cd2+ flux, and Fe concentration as Arabidopsis mutant irt1 under iron-deficient conditions. Therefore, H192 in HRM may be an iron sensor to regulate delivery of MxIRT1 vesicles to the PM after binding with iron; Y266 in CRAC acts as an iron sensor for active metal transport under iron-deficient conditions.

Cholesterol-recognition motifs in the transmembrane domain of the tyrosine kinase receptor family: The case of TRKB.

Cholesterol is an essential constituent of cell membranes. The discovery of cholesterol-recognition amino acid consensus (CRAC) motif in proteins indicated a putative direct, non-covalent interaction between cholesterol and proteins. In the present study, we evaluated the presence of a CRAC motif and its inverted version (CARC) in the transmembrane region (TMR) of the tyrosine kinase receptor family (RTK) in several species using in silico methods. CRAC motifs were found across all species analyzed, while CARC was found only in vertebrates. The tropomyosin-related kinase B (TRKB), a member of the RTK family, through interaction with its endogenous ligand brain-derived neurotrophic factor (BDNF) is a core participant in the neuronal plasticity process and exhibits a CARC motif in its TMR. Upon identifying the conserved CARC motif in the TRKB, we performed molecular dynamics simulations of the mouse TRKB.TMR. The simulations indicated that cholesterol interaction with the TRKB CARC motif occurs mainly at the central Y433 residue. Our binding assay suggested a bell-shaped effect of cholesterol on BDNF interaction with TRKB receptors, and our results suggest that CARC/CRAC motifs may play a role in the function of the RTK family TMR.

Evidence that specific interactions play a role in the cholesterol sensitivity of G protein-coupled receptors

G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol's general effects on the membrane is not well-understood. We performed coarse-grained molecular dynamics (CGMD) simulations coupled with structural bioinformatics approaches on the β2-adrenergic receptor (β2AR) and the cholecystokinin (CCK) receptor subfamily. The β2AR has been shown to be sensitive to membrane cholesterol and cholesterol molecules have been clearly resolved in numerous β2AR crystal structures. The two CCK receptors are highly homologous and preserve similar cholesterol recognition motifs but despite their homology, CCK1R shows functional sensitivity to membrane cholesterol while CCK2R does not. Our results offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with β2AR that agree with previously published data; additionally, we observe differential and specific cholesterol binding in the CCK receptor subfamily while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. A thermal denaturation assay (LCP-Tm) shows that mutation of a conserved CRAC sequence on TM7 of the β2AR affects cholesterol stabilization of the receptor in a lipid bilayer. The results of this study provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases.

Structure, membrane topology and influence of cholesterol of the membrane proximal region: transmembrane helical anchor sequence of gp41 from HIV

During the first steps of HIV infection the Env subunit gp41 is thought to establish contact between the membranes and to be the main driver of fusion. Here we investigated in liquid crystalline membranes the structure and cholesterol recognition of constructs made of a gp41 external region carrying a cholesterol recognition amino acid consensus (CRAC) motif and a hydrophobic membrane anchoring sequence. CD- und ATR-FTIR spectroscopies indicate that the constructs adopt a high degree of helical secondary structure in membrane environments. Furthermore, 15N and 2H solid-state NMR spectra of gp41 polypeptides reconstituted into uniaxially oriented bilayers agree with the CRAC domain being an extension of the transmembrane helix. Upon addition of cholesterol the CRAC NMR spectra remain largely unaffected when being associated with the native gp41 transmembrane sequence but its topology changes when anchored in the membrane by a hydrophobic model sequence. The 2H solid-state NMR spectra of deuterated cholesterol are indicative of a stronger influence of the model sequence on this lipid when compared to the native gp41 sequence. These observations are suggestive of a strong coupling between the transmembrane and the membrane proximal region of gp41 possibly enforced by oligomerization of the transmembrane helical region.

Cholesterol Interaction with the Trimeric HIV Fusion Protein gp41 in Lipid Bilayers Investigated by Solid-State NMR Spectroscopy and Molecular Dynamics Simulations

HIV-1 entry into cells is mediated by the fusion protein gp41. Cholesterol plays an important role in this virus–cell fusion, but molecular structural information about cholesterol–gp41 interaction is so far absent. Here, we present experimental and computational data about cholesterol complexation with gp41 in lipid bilayers. We focus on the C-terminal region of the protein, which comprises a membrane-proximal external region (MPER) and the transmembrane domain (TMD). We measured peptide–cholesterol contacts in virus-mimetic lipid bilayers using solid-state NMR spectroscopy, and augmented these experimental data with all-atom molecular dynamics simulations. 2D 19F NMR spectra show correlation peaks between MPER residues and the cholesterol isooctyl tail, indicating that cholesterol is in molecular contact with the MPER–TMD trimer. 19F–13C distance measurements between the peptide and 13C-labeled cholesterol show that C17 on the D ring and C9 at the intersection of B and C rings are ~7.0 Å from the F673 side-chain 4-19F. At high peptide concentrations in the membrane, the 19F–13C distance data indicate three cholesterol molecules bound near F673 in each trimer. Mutation of a cholesterol recognition amino acid consensus motif did not change these distances, indicating that cholesterol binding does not require this sequence motif. Molecular dynamics simulations further identify two hotspots for cholesterol interactions. Taken together, these experimental data and simulations indicate that the helix-turn-helix conformation of the MPER–TMD is responsible for sequestering cholesterol. We propose that this gp41–cholesterol interaction mediates virus–cell fusion by recruiting gp41 to the boundary of the liquid-disordered and liquid-ordered phases to incur membrane curvature.

Lipid rafts are required for effective renal D1 dopamine receptor function.

Effective receptor signaling is anchored on the preferential localization of the receptor in lipid rafts, which are plasma membrane platforms replete with cholesterol and sphingolipids. We hypothesized that the dopamine D1 receptor (D1 R) contains structural features that allow it to reside in lipid rafts for its activity. Mutation of C347 palmitoylation site and Y218 of a newly identified Cholesterol Recognition Amino Acid Consensus motif resulted in the exclusion of D1 R from lipid rafts, blunted cAMP response, impaired sodium transport, and increased oxidative stress in renal proximal tubule cells (RPTCs). Kidney-restricted silencing of Drd1 in C57BL/6J mice increased blood pressure (BP) that was normalized by renal tubule-restricted rescue with D1 R-wild-type but not the mutant D1 R 347A that lacks a palmitoylation site. Kidney-restricted disruption of lipid rafts by β-MCD jettisoned the D1 R from the brush border, decreased sodium excretion, and increased oxidative stress and BP in C57BL/6J mice. Deletion of the PX domain of the novel D1 R-binding partner sorting nexin 19 (SNX19) resulted in D1 R partitioning solely to non-raft domains, while silencing of SNX19 impaired D1 R function in RPTCs. Kidney-restricted silencing of Snx19 resulted in hypertension in C57BL/6J mice. Our results highlight the essential role of lipid rafts for effective D1 R signaling.

PIRT the TRP Channel Regulating Protein Binds Calmodulin and Cholesterol-Like Ligands.

Transient receptor potential (TRP) ion channels are polymodal receptors that have been implicated in a variety of pathophysiologies, including pain, obesity, and cancer. The capsaicin and heat sensor TRPV1, and the menthol and cold sensor TRPM8, have been shown to be modulated by the membrane protein PIRT (Phosphoinositide-interacting regulator of TRP). The emerging mechanism of PIRT-dependent TRPM8 regulation involves a competitive interaction between PIRT and TRPM8 for the activating phosphatidylinositol 4,5-bisphosphate (PIP2) lipid. As many PIP2 modulated ion channels also interact with calmodulin, we investigated the possible interaction between PIRT and calmodulin. Using microscale thermophoresis (MST), we show that calmodulin binds to the PIRT C-terminal α-helix, which we corroborate with a pull-down experiment, nuclear magnetic resonance-detected binding study, and Rosetta-based computational studies. Furthermore, we identify a cholesterol-recognition amino acid consensus (CRAC) domain in the outer leaflet of the first transmembrane helix of PIRT, and with MST, show that PIRT specifically binds to a number of cholesterol-derivatives. Additional studies identified that PIRT binds to cholecalciferol and oxytocin, which has mechanistic implications for the role of PIRT regulation of additional ion channels. This is the first study to show that PIRT specifically binds to a variety of ligands beyond TRP channels and PIP2.

Probing cholesterol binding and translocation in P-glycoprotein

P-glycoprotein (Pgp) is a biomedically important member of the ABC transporter superfamily that mediates multidrug resistance in various cancer types. Substrate binding and transport in Pgp are modulated by the presence of cholesterol in the membrane. Structural information on cholesterol binding sites and mechanistic details of its redistribution are, however, largely unknown. In this study, a set of 40 independent molecular dynamics (MD) simulations of Pgp embedded in cholesterol-rich lipid bilayers are reported, totaling 8 μs, enabling extensive sampling of cholesterol-protein interactions in Pgp. Clustering analyses of the ensemble of cholesterol molecules (∼5740) sampled around Pgp in these simulations reveal specific and asymmetric cholesterol-binding regions formed by the transmembrane (TM) helices TM1–6 and TM8. Notably, not all the putative cholesterol binding sites identified by MD can be predicted by the primary sequence based cholesterol-recognition amino acid consensus (CRAC) or inverted CRAC (CARC) motifs, an observation that we attribute to inadequacy of these motifs to account for binding sites formed by remote amino acids in the sequence that can still be spatially adjacent to each other. Binding of cholesterol to Pgp occurs more frequently through its rough β-face formed by the two protruding methyl groups, whereas the opposite smooth α-face prefers packing alongside the membrane lipids. One full and two partial cholesterol flipping events between the two leaflets of the bilayer mediated by the surface of Pgp are also captured in these simulations. All flipping events are observed in a region formed by helices TM1, TM2, and TM11, featuring two full and two partial CRAC/CARC motifs, with Tyr49 and Tyr126 identified as key residues interacting with cholesterol during this event. Our study is the first to report direct observation of unconventional cholesterol translocation on the surface of Pgp, providing a secondary transport model for the known flippase activity of ABC exporters of cholesterol.This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.

A novel rough set based affinity propagation method for cholesterol prediction from ABC transporter

In biological science, all functions of membrane protein are being modulated by membrane cholesterol. Cholesterol plays a prime role in plasma membrane. The present research emphasized on design of a hybrid rough set based affinity propagation model for prediction of cholesterol sequence from Adenosine Triphosphate Binding Cassette transporters (ABC transporters). Each and every time trans-membrane helix sequences of ABC are evaluated and targeted with cholesterol to find out new valid motif signature. Cholesterol sequences are generated using the formula like Cholesterol Recognition Aminoacid Consensus (CRAC) for forward pattern and reverse of CRAC is CARC for backward pattern. The hypothesis of cholesterol binding motif modulates the signalling pathway of helical membrane protein. Many types of membrane proteins are reported till now, among them G-Protein Coupled Receptors (GPCR) and ABC are important as they are needed for drug discovery. ABC transporter super family is aimed in this work. In experimental study, we explored our hybrid method to find out motif consensus. Finally, from the results we report that our novel technique work efficiently identifying valid motif consensus from ABC protein sequences and is helpful for clinical drug target for human diseases.

Molecular Signatures of Cholesterol Interaction with Serotonin Receptors.

The interaction of G protein-coupled receptors (GPCRs) with cholesterol is a hallmark of their function, organization, and structural dynamics. Several cholesterol interaction sites, such as the cholesterol recognition amino acid consensus (CRAC) and cholesterol consensus motif (CCM), have been mapped from crystallography, bioinformatics, and simulation studies. In this article, we characterize common descriptors for cholesterol interaction sites in the serotonin1A receptor from a series of coarse-grain simulations. We have identified a novel interaction mode for cholesterol in which the cholesterol polar headgroup interacts with aromatic amino acid residues, such as tryptophan and tyrosine. The cholesterol rings interact with both aromatic residues and nonpolar residues, thereby constituting a signature aromatic interaction site. In addition, we report a similar binding mode in the crystal structures of the serotonin2B receptor, suggesting that this binding mode could be a general feature of the serotonin receptor family. Interestingly, this signature aromatic interaction site is present along with one of the CRAC motifs in the serotonin1A receptor. Our results represent an important step toward mapping out the diversity of cholesterol-GPCR interaction sites.

The conserved tyrosine residue 940 plays a key structural role in membrane interaction of Bordetella adenylate cyclase toxin

The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) translocates its adenylate cyclase (AC) enzyme domain into target cells in a step that depends on membrane cholesterol content. We thus examined what role in toxin activities is played by the five putative cholesterol recognition amino acid consensus (CRAC) motifs predicted in CyaA hemolysin moiety. CRAC-disrupting phenylalanine substitutions had no impact on toxin activities and these were not inhibited by free cholesterol, showing that the putative CRAC motifs are not involved in cholesterol binding. However, helix-breaking proline substitutions in these segments uncovered a structural role of the Y632, Y658, Y725 and Y738 residues in AC domain delivery and pore formation by CyaA. Substitutions of Y940 of the fifth motif, conserved in the acylated domains of related RTX toxins, did not impact on fatty-acylation of CyaA by CyaC and the CyaA-Y940F mutant was intact for toxin activities on erythrocytes and myeloid cells. However, the Y940A or Y940P substitutions disrupted the capacity of CyaA to insert into artificial lipid bilayers or target cell membranes. The aromatic ring of tyrosine 940 side chain thus appears to play a key structural role in molecular interactions that initiate CyaA penetration into target membranes.