Cholesterol Consensus Motif Research Articles

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.

Cortisol Interaction with Aquaporin-2 Modulates Its Water Permeability: Perspectives for Non-Genomic Effects of Corticosteroids.

Aquaporins (AQPs) are water channels widely distributed in living organisms and involved in many pathophysiologies as well as in cell volume regulations (CVR). In the present study, based on the structural homology existing between mineralocorticoid receptors (MRs), glucocorticoid receptors (GRs), cholesterol consensus motif (CCM) and the extra-cellular vestibules of AQPs, we investigated the binding of corticosteroids on the AQP family through in silico molecular dynamics simulations of AQP2 interactions with cortisol. We propose, for the first time, a putative AQPs corticosteroid binding site (ACBS) and discussed its conservation through structural alignment. Corticosteroids can mediate non-genomic effects; nonetheless, the transduction pathways involved are still misunderstood. Moreover, a growing body of evidence is pointing toward the existence of a novel membrane receptor mediating part of these rapid corticosteroids' effects. Our results suggest that the naturally produced glucocorticoid cortisol inhibits channel water permeability. Based on these results, we propose a detailed description of a putative underlying molecular mechanism. In this process, we also bring new insights on the regulatory function of AQPs extra-cellular loops and on the role of ions in tuning the water permeability. Altogether, this work brings new insights into the non-genomic effects of corticosteroids through the proposition of AQPs as the membrane receptor of this family of regulatory molecules. This original result is the starting point for future investigations to define more in-depth and in vivo the validity of this functional model.

Cholesterol Dependent Activity of the Adenosine A2A Receptor Is Modulated via the Cholesterol Consensus Motif.

Background: Membrane cholesterol dysregulation has been shown to alter the activity of the adenosine A2A receptor (A2AR), a G protein-coupled receptor, thereby implicating cholesterol levels in diseases such as Alzheimer’s and Parkinson’s. A limited number of A2AR crystal structures show the receptor interacting with cholesterol, as such molecular simulations are often used to predict cholesterol interaction sites. Methods: Here, we use experimental methods to determine whether a specific interaction between amino acid side chains in the cholesterol consensus motif (CCM) of full length, wild-type human A2AR, and cholesterol modulates activity of the receptor by testing the effects of mutational changes on functional consequences, including ligand binding, G protein coupling, and downstream activation of cyclic AMP. Results and conclusions: Our data, taken with previously published studies, support a model of receptor state-dependent binding between cholesterol and the CCM, whereby cholesterol facilitates both G protein coupling and downstream signaling of A2AR.

Structural dynamics of Smoothened (SMO) in the ciliary membrane and its interaction with membrane lipids

The Smoothened receptor (SMO, a 7 pass transmembrane domain, Class F GPCR family protein) plays a crucial role in the Hedgehog (HH) signaling pathway, which is involved in embryonic development and is implicated in various types of cancer throughout the animal kingdom. In the absence of HH signaling, SMO is inhibited by Patched 1 (PTC1; a 12 pass transmembrane domain protein), which is localized in the primary cilia. HH binding leads to the dislocation of PTC1 from the cilia, thus making way for SMO to localize in the primary cilia, as an essential prerequisite for its activation. We have carried out MARTINI coarse-grained molecular dynamics simulations of SMO in POPC and in ciliary membrane models, respectively, to study the interactions of SMO with cholesterol and other lipid molecules in the ciliary membrane, and to gain molecular-level insights into the role of the primary cilia in shaping the functional dynamics of SMO. We are able to identify the interaction of membrane cholesterols with definite sites and domains within SMO and relate them with known cholesterol-binding sequence and structure motifs. We show that cholesterol interactions with the transmembrane domain TMD, unlike those with the cysteine-rich domain (CRD) and the intracellular domain (ICD), are through residues belonging to known cholesterol-binding motifs. Notably, a few persistent interactions of cholesterol with lower TM cholesterol-binding domains are governed by the presence of multiple cholesterol-binding motifs. These analyses have also helped to identify and define a strict cholesterol consensus motif (CCM), which may well steer cholesterol into the hitherto identified binding sites within the TMD of SMO. We have also reported the interaction of phosphatidylinositol 4-phosphate with the intracellular region of transmembrane (TM) helices (TM1, TM3, TM4, and TM5), intracellular loop1, helix8, and Arg/Lys clusters of the ICD. Structural analysis of SMO domains shows significant changes in the CRD and ICD, during the course of the simulation. Further detailed analysis of the dynamics of the TMD reveals the movements of TM5, TM6, and TM7, linked with the helix8, which are possibly involved in shaping the conformational disposition of the ICD. The movement of these TM helices could possibly be a consequence of interactions involving the extracellular domain and extracellular loops. In addition, our analysis also shows that phosphatidylinositol-4-phosphate (PI4P), along with some ICD cholesterols, are implicated in anchoring SMO in the membrane.

Experimental studies of wild type A2A receptor and cholesterol-binding receptor variants support a model for cholesterol as an allosteric modulator of G protein coupling

G protein-coupled receptors (GPCRs) comprise the largest known family of human receptors and are the targets of approximately a third of presently approved pharmaceuticals. Cholesterol is present in the cell membrane of eukaryotic cells, and variations in cholesterol concentrations within a single cell type have been implicated in disease pathologies such as Alzheimer's disease. A2AR contains have specific cholesterol binding motifs, such as the cholesterol consensus motif (CCM), which are predicted sites where membrane cholesterol interacts with the receptor.

Allosteric modulation of cannabinoid receptors through transmembrane binding sites as a potential therapeutic intervention for pain and inflammation

Objective: Pain and inflammation co-exist in many pathological conditions and involve complex molecular and biochemical pathways. It is estimated that 20.4% of adults in the USA have chronic pain, and the economic burden associated with treating and managing pain is $635 billion annually. The Cannabinoid receptor 1 (CB1), a G protein-coupled receptor (GPCR), has been implicated in pain and inflammation management. However, many side effects associated with this receptor's orthosteric activation have led to failure in drug trials utilizing this receptor system. Allosteric modulation, which targets a different site in the receptor, offers a new avenue for generating a new class of drugs that can activate the receptor but with less or no incidence of off-target effects. A transmembrane allosteric site with a bound negative allosteric modulator was recently described for the CB1(6KQI). This extrahelical binding site spanning transmembrane helices 1, 2, and 4, is located in the membrane's inner leaflet and overlaps with a conserved cholesterol consensus motif described for other GPCRs. Our central hypothesis is that positive allosteric modulators that can bind to the described allosteric site, thereby stabilizing the receptor in its active state, can be identified. Using a combination of computational and experimental techniques, we have identified some positive allosteric modulators which bind stably to the allosteric site. Results obtained offer a structural insight into allosteric modulation of the CB1, key residues involved and the contribution of membrane lipids to ligand binding and stability in the allosteric site. Methods A structure-based pharmacophore was developed for the CB1 and was utilized for virtual screening of commercially available libraries using MOE software. Molecular docking of these screened compounds was then carried out using MOE. Employing a heterogeneous membrane setup (containing POPC, POPE, PSM, POPI24, POPS, and Cholesterol), these ligands' stability was evaluated using classical all-atom molecular dynamics simulations utilizing GROMACS software. Furthermore, funnel metadynamics was used to obtain the ligands' relative binding energies and rank order, using PLUMED software. Top scoring ligands were evaluated by multiple in vitro functional assays to assess positive allosteric modulation of CB1. Results : Multiple ligands with high affinity for the allosteric binding site were obtained. These compounds stabilized the key activation signatures of CB1 without affecting the agonist binding. Furthermore, the membrane was observed to play a critical role in ligand stability at the binding site as they formed stable bonds with the ligands. Future in vivo studies will be carried out to assess the utility of the ligands in treating pain and inflammation Conclusions: The obtained results provide useful insights into the structural basis and rational design of allosteric ligands with high affinity and the contribution of the membrane lipids to ligand stability and activity. These obtained ligands might translate into potential therapeutics in treating pain and providing alternatives to currently available medication.

Interplay of cholesterol, membrane bilayers and the AT1R: A cholesterol consensus motif on AT1R is revealed

Hypertension, mediated by the Angiotensin II receptor type 1 (AT1R), is still the major cause of premature death despite the discovery of novel therapeutics, highlighting the importance of an in depth understanding of the drug-AT1R recognition mechanisms coupled with the impact of the membrane environment on the interaction of drugs with AT1R. Herein, we examine the interplay of cholesterol-lipid-candesartan and the AT1R using Molecular Dynamics simulations of a model membrane consisting of 60:40 mol%. DPPC:cholesterol, candesartan and the AT1R, mimicking the physiological cholesterol concentration in sarcolemma membranes. The simulations of the model membrane of 60:40 mol%. DPPC:cholesterol were further validated using DOSY NMR experiments. Interestingly, our results suggest a significant role of cholesterol in the AT1R function imposed through a Cholesterol Consensus Motif (CCM) in the receptor, which could be crucial in the drug binding process. Candesartan diffusion towards AT1R through incorporation into lipid bilayers, appears to be retarded by the presence of cholesterol. However, its direct approach towards AT1R may be facilitated through the mobility induced on the N-terminus by the cholesterol binding on the CCM these novel insights could pave the way towards the development of more potent pharmaceutical agents to combat hypertension more effectively.

Tetraspanin CD82 interaction with cholesterol promotes extracellular vesicle–mediated release of ezrin to inhibit tumour cell movement

ABSTRACT Tumour metastasis suppressor KAI1/CD82 inhibits tumour cell movement. As a transmembrane protein, tetraspanin CD82 bridges the interactions between membrane microdomains of lipid rafts and tetraspanin-enriched microdomains (TEMs). In this study, we found that CD82 and other tetraspanins contain cholesterol recognition/interaction amino-acid consensus (CRAC) sequences in their transmembrane domains and revealed that cholesterol binding of CD82 determines its interaction with lipid rafts but not with TEMs. Functionally, CD82 needs cholesterol binding to inhibit solitary migration, collective migration, invasion and infiltrative outgrowth of tumour cells. Importantly, CD82–cholesterol/–lipid raft interaction not only promotes extracellular release of lipid raft components such as cholesterol and gangliosides but also facilitates extracellular vesicle (EV)–mediated release of ezrin–radixin–moesin (ERM) protein Ezrin. Since ERM proteins link actin cytoskeleton to the plasma membrane, we show for the first time that cell movement can be regulated by EV-mediated releases, which disengage the plasma membrane from cytoskeleton and then impair cell movement. Our findings also conceptualize that interactions between membrane domains, in this case converge of lipid rafts and TEMs by CD82, can change cell movement. Moreover, CD82 coalescences with both lipid rafts and TEMs are essential for its inhibition of tumour cell movement and for its enhancement of EV release. Finally, our study underpins that tetraspanins as a superfamily of functionally versatile molecules are cholesterol-binding proteins. Abbreviations: Ab: antibody; CBM: cholesterol-binding motif; CCM: cholesterol consensus motif; CRAC/CARC: cholesterol recognition or interaction amino-acid consensus; CTxB: cholera toxin B subunit; ECM: extracellular matrix; ERM: ezrin, radixin and moesin; EV: extracellular vesicles; FBS: foetal bovine serum; mAb: monoclonal antibody; MST: microscale thermophoresis; pAb: polyclonal antibody; and TEM: tetraspanin-enriched microdomain

Membrane cholesterol depletion reduces downstream signaling activity of the adenosine A2A receptor

Cholesterol has been shown to modulate the activity of multiple G Protein-coupled receptors (GPCRs), yet whether cholesterol acts through specific interactions, indirectly via modifications to the membrane, or via both mechanisms is not well understood. High-resolution crystal structures of GPCRs have identified bound cholesterols; based on a β2-adrenergic receptor (β2AR) structure bound to cholesterol and the presence of conserved amino acids in class A receptors, the cholesterol consensus motif (CCM) was identified. Here in mammalian cells expressing the adenosine A2A receptor (A2AR), ligand dependent production of cAMP is reduced following membrane cholesterol depletion with methyl-beta-cyclodextrin (MβCD), indicating that A2AR signaling is dependent on cholesterol. In contrast, ligand binding is not dependent on cholesterol depletion. All-atom molecular simulations suggest that cholesterol interacts specifically with the CCM when the receptor is in an active state, but not when in an inactive state. Taken together, the data support a model of receptor state-dependent binding between cholesterol and the CCM, which could facilitate both G-protein coupling and downstream signaling of A2AR.

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.

Automated Identification of Cholesterol Interaction Sites on G-Protein Coupled Receptors by Coarse-Grained Simulation

Extensive experimental evidence suggests that cholesterol influences the function of G-protein coupled receptors (GPCRs), perhaps by direct and specific interactions with the protein surface. Interaction motifs (cholesterol recognition amino acid consensus motif and cholesterol consensus motif) have been identified by bioinformatic analysis and inspection of crystal structures. Here we report an approach based on analysis of molecular simulation data to identify other putative interaction sites. In unbiased simulations of GPCRs in cholesterol containing membranes, cholesterol samples the protein surface. This data is then analyzed to identify localized, specific, and reproducible interaction sites. The result of the calculation is a ranking of membrane-facing residues in terms of a cholesterol interaction “score,” which may then be used to guide experimental tests based on mutagenesis. Results based on coarse-grained “Martini” simulations of the A2A adenosine receptor are presented, which predict that cholesterol is most likely to bind in three distinct areas on the surface of the protein.

Membrane Cholesterol and the Adenosine A2a Receptor

G-protein coupled receptors (GPCRs) represent the largest family of receptor proteins in the living world, having approximately 800 human genes predicted; however the high-resolution crystal structures of only 26 GPCRs have been reported (Ghosh et al., 2015). The first human GPCR to be crystallized was the β2-adrenergic receptor (β2AR) in 2007 (Cherezov et al., 2007; Rasmussen et al., 2007; Rosenbaum et al., 2007). Shortly thereafter an alternate crystal form of the β2AR revealed a specific cholesterol binding site between helices I, II, III and IV. From this work a cholesterol consensus motif (CCM) was established, which defined specific interactions between cholesterol and the receptor. Utilization of this CCM predicted that as many as 25% of all class A GPCRs could have a specific interaction with cholesterol (Hanson et al., 2008).Our focus is the effects of cholesterol on the activity of the Adenosine A2a receptor (A2aR), a class A GPCR. Membrane cholesterol concentrations were varied in Human embryonic kidney (HEK-293) cells by the use of methyl- β-cyclodextrin (MβCD), which is capable of capturing cholesterol in its inner cavity (Pucadyil et al., 2006). We tested the role that bulk cholesterol depletion played in expression, ligand binding and downstream synthesis of cyclic AMP (cAMP) in HEK-293 cells.Continuing work from our lab will study the specific interaction between cholesterol and A2aR by making point mutations in the residues of the CCM. In A2aR those residues include Tyr43(2.41), Ser47(2.45), Lys122(4.43), Ile125(4.46) and Trp129(4.50) (Lee et al., 2013). Our lab will investigate whether these point mutations effect the interaction between cholesterol and purified receptor, as well as the receptors activity in both yeast and mammalian cell model systems.

A novel fuzzy C-means approach for uncovering cholesterol consensus motif from human G-protein coupled receptors (GPCR)

Membrane cholesterol plays an important role in modulating the function of several membrane proteins. From these proteins, a special cholesterol binding motif is reported to which the membrane cholesterol binds and modulates their activity. This consensus motif is either seen as a forward pattern known as CRAC (L/V-X(1-5)-Y-X(1-5)-R/K) and/or as a backward pattern CARC (R/K-X(1-5)-Y-X(1-5)-L/V). This as such is a low consensus motif as substituting amino acid in the unconserved positions of the motif (‘X’) yields many combinations. In order to obtain a better consensus motif for cholesterol binding, it is worthwhile to look for the same within a membrane proteins superfamily (ABC transporters, GPCRs, etc.) and assign them as a signature motif. Therefore, in the current work an attempt was made to identify the distribution of this motif in all seven helices of GPCR family and assign a consensus signature motif for an individual helix using a novel Fuzzy C-Means (FCM) approach. The workflow proceeds in four phases; first, GPCR protein sequences were extracted from UniProt database that contains seven transmembrane (TM) helices and a cholesterol dictionary has been designed for different window sizes. In second phase; those sequences are filtered which starts with R/K or L/V using both CRAC and CARC cholesterol recognition methods leading to discovery of filtered cholesterol motifs. Third phase leads to identification of significant cholesterol motifs using FCM algorithm by computing the membership of sequences to different motifs and pattern matching with different helices. Finally those uncovered cholesterol motifs that matched with TM helices were analyzed. From the results we report an algorithm that can efficiently identify and assign cholesterol signature motifs in GPCR protein sequences that can be further extended to other membrane proteins.

Endogenous vs Exogenous Allosteric Modulators in GPCRs: A dispute for shuttling CB1 among different membrane microenvironments.

A Cannabinoid Receptor 1 (CB1) binding site for the selective allosteric modulator ORG27569 is here identified through an integrate approach of consensus pocket prediction, mutagenesis studies and Mass Spectrometry. This unprecedented ORG27569 pocket presents the structural features of a Cholesterol Consensus Motif, a cholesterol interacting region already found in other GPCRs. ORG27569 and cholesterol affects oppositely CB1 affinity for orthosteric ligands. Moreover, the rise in cholesterol intracellular level results in CB1 trafficking to the axonal region of neuronal cells, while, on the contrary, ORG27568 binding induces CB1 enrichment at the soma. This control of receptor migration among functionally different membrane regions of the cell further contributes to downstream signalling and adds a previously unknown mechanism underpinning CB1 modulation by ORG27569 , that goes beyond a mere control of receptor affinity for orthosteric ligands.

Amphipathic alpha-helices and putative cholesterol binding domains of the influenza virus matrix M1 protein are crucial for virion structure organisation

The influenza virus matrix M1 protein is an amphitropic membrane-associated protein, forming the matrix layer immediately beneath the virus raft membrane, thereby ensuring the proper structure of the influenza virion. The objective of this study was to elucidate M1 fine structural characteristics, which determine amphitropic properties and raft membrane activities of the protein, via 3D in silico modelling with subsequent mutational analysis. Computer simulations suggest the amphipathic nature of the M1 α-helices and the existence of putative cholesterol binding (CRAC) motifs on six amphipathic α-helices. Our finding explains for the first time many features of this protein, particularly the amphitropic properties and raft/cholesterol binding potential. To verify these results, we generated mutants of the A/WSN/33 strain via reverse genetics. The M1 mutations included F32Y in the CRAC of α-helix 2, W45Y and W45F in the CRAC of α-helix 3, Y100S in the CRAC of α-helix 6, M128A and M128S in the CRAC of α-helix 8 and a double L103I/L130I mutation in both a putative cholesterol consensus motif and the nuclear localisation signal. All mutations resulted in viruses with unusual filamentous morphology. Previous experimental data regarding the morphology of M1-gene mutant influenza viruses can now be explained in structural terms and are consistent with the pivotal role of the CRAC-domains and amphipathic α-helices in M1-lipid interactions.

A cholesterol consensus motif is required for efficient intracellular transport and raft association of a group 2 HA from influenza virus.

The HA (haemagglutinin) of influenza viruses must be recruited to membrane rafts to perform its function in membrane fusion and virus budding. We previously showed using FRET that deletion of the two raft-targeting features of HA, S-acylation at the cytoplasmic tail and the hydrophobic amino acids VIL (Val-Ile-Leu) in the outer part of the TMR (transmembrane region), lead to reduced raft association. In addition, exchange of VIL, but not of the S-acylation sites severely retards transport of HA through the Golgi. In the present study, we have further characterized the ill-defined signal in the TMR.A sequence comparison suggests that the leucine residue of VIL might be part of a CCM (cholesterol consensus motif) that is known to bind cholesterol to seven-transmembrane receptors. The signal also comprises a lysine residue and a tryptophan residue on one and a tyrosine residue on another TMR helix and is conserved in group 2 HAs. Mutations in the CCM retard Golgi-localized processing of HA, such as acquisition of Endo H (endoglycosidase H)-resistant carbohydrates in the medial Golgi and proteolytic cleavage in the TGN (trans-Golgi network). The delay in transport of HA to and from the medial Golgi varied with the mutation, suggesting that different transport steps are affected. All mutants analysed by FRET also showed reduced association with rafts at the plasma membrane. Thus the raft-targeting signal of HA encompasses not only hydrophobic, but also aromatic and positively charged, residues. We speculate that binding to cholesterol might facilitate intracellular transport of HA and association with rafts.

Abstract 412: In Silico Structural and Metabolic Profiling of Non-Synonymous Single Nucleotide Polymorphisms in APOA-I Gene

Mature apoA-I contains 243 residues organized in two-domains as N-terminal four-helix bundle and C-terminal two helices. Cholesterol interaction with both lipid-free and lipid-bound apoA-I may be one of the key events in amyloidosis and reverse cholesterol transport (RCT). By the SIFT score, 20 non-synonymous missense mutations in APOA-I gene, located almost exclusively in N-domain, may impair apoA-I function. A majority (4 from 5) of putative cholesterol recognition amino acid consensus (CRAC: L/V-X(1-5)-Y-X(1-5)-R/K) and a single cholesterol consensus motif (CCM: R/K-X(2-6)-I/L/V-X(3)-W/Y) aligned within/between helices in the bundle. Importantly, CRAC2 in apoA-I co-aligned with CRAC2 in apoE and CRAC4 in apoA-IV. A short four amino acid sequence L44-L47 may present disorder-to-order transitioning binding region in apoA-I as predicted by molecular recognition feature (MoRF). Indeed, CCM corresponded to unstructured region with low stability in lipid-free apoA-I. Stabilizing effect due to the direct cholesterol-peptide interaction with the involvement of CRAC1 (L14-K23) and/or CCM (K40-W50) in amyloidogenic peptide 1-93 is suggested to underlie the known effect of cholesterol on the induction of helix formation in this peptide. Cholesterol may be speculated to act in helix formation as a chemical chaperone thus triggering disorder-to-order transition within MoRF; without cholesterol, this MoRF may be involved into protein-protein interaction. Indeed, the association of W50R substitution with the accelerated mutant aggregation rate and the involvement of L44-S55 segment into cross-beta-sheet formation were demonstrated by others. Also, D102H substitution in CRAC2 (V97-K106) may be harmful in RCT. With PDBe Motifs and Sites algorithm, cholesterol is a ligand for L101, F104 and W108 residues in CRAC2 motif of apoA-I in nascent HDL. Besides, the resistance of two-domain apoA-I structure to denaturation by GuHCl is sensitive to cholesterol level in discoidal HDL. Overall, different CRACs may contribute into amyloidosis-connected apoA-I misfolding and cholesterol efflux for lipid-free apoA-I. Cholesterol binding to lipid-bound apolipoprotein may change the structural plasticity of two domains and/or their relative dimensions.

Ligand-dependent cholesterol interactions with the human A2A adenosine receptor

We present nearly 10μs of all-atom simulation data of a G-protein coupled receptor, the human A2A adenosine receptor, bound to four different ligands. Our focus is on binding of cholesterol to the “cholesterol consensus motif,” a cluster of five amino acids on the second and fourth transmembrane helices, which interact with two cholesterols in the intracellular leaflet of the bilayer. We find evidence for a ligand-specific interaction between the CCM and cholesterol, mediated by the rotameric dynamics and configuration of Trp129. Binding of the synthetic agonist UK432097 disrupts hydrogen bonding between Trp129 and Ser47, which activates the rotameric dynamics of Trp129 and disrupts the interaction with one of the two cholesterols. We also investigate the effect of four thermostabilizing mutations, three of which are located on helix two. The conformational stability of helix two has been proposed to be sensitive to interaction with cholesterol in the CCM, suggesting a mechanism for the thermostabilization. However, our data are instead suggestive of a force-field dependent “straightening” of helix two, and therefore offer no basis for rationalizing the effect of the quadruple mutant.

Lipid-binding surfaces of membrane proteins: Evidence from evolutionary and structural analysis

Membrane proteins function in the diverse environment of the lipid bilayer. Experimental evidence suggests that some lipid molecules bind tightly to specific sites on the membrane protein surface. These lipid molecules often act as co-factors and play important functional roles. In this study, we have assessed the evolutionary selection pressure experienced at lipid-binding sites in a set of α-helical and β-barrel membrane proteins using posterior probability analysis of the ratio of synonymous vs. nonsynonymous substitutions (ω-ratio). We have also carried out a geometric analysis of the membrane protein structures to identify residues in close contact with co-crystallized lipids. We found that residues forming cholesterol-binding sites in both β2-adrenergic receptor and Na+–K+-ATPase exhibit strong conservation, which can be characterized by an expanded cholesterol consensus motif for GPCRs. Our results suggest the functional importance of aromatic stacking interactions and interhelical hydrogen bonds in facilitating protein–cholesterol interactions, which is now reflected in the expanded motif. We also find that residues forming the cardiolipin-binding site in formate dehydrogenase-N γ-subunit and the phosphatidylglycerol binding site in KcsA are under strong purifying selection pressure. Although the lipopolysaccharide (LPS)-binding site in ferric hydroxamate uptake receptor (FhuA) is only weakly conserved, we show using a statistical mechanical model that LPS binds to the least stable FhuA β-strand and protects it from the bulk lipid. Our results suggest that specific lipid binding may be a general mechanism employed by β-barrel membrane proteins to stabilize weakly stable regions. Overall, we find that the residues forming specific lipid binding sites on the surfaces of membrane proteins often experience strong purifying selection pressure.