Putative Cholesterol Binding Sites Research Articles

Interaction with stomatin directs human proton channels into cholesterol-dependent membrane domains

Many membrane proteins are modulated by cholesterol. Here we report profound effects of cholesterol depletion and restoration on the human voltage-gated proton channel, hHV1, in excised patches but negligible effects in the whole-cell configuration. Despite the presence of a putative cholesterol-binding site, a CARC motif in hHV1, mutation of this motif did not affect cholesterol effects. The murine HV1 lacks a CARC sequence but displays similar cholesterol effects. These results argue against a direct effect of cholesterol on the HV1 protein. However, the data are fully explainable if HV1 preferentially associates with cholesterol-dependent lipid domains, or “rafts.” The rafts would be expected to concentrate in the membrane/glass interface and to be depleted from the electrically accessible patch membrane. This idea is supported by evidence that HV1 channels can diffuse between seal and patch membranes when suction is applied. Simultaneous truncation of the large intracellular N and C termini of hHV1 greatly attenuated the cholesterol effect, but C truncation alone did not; this suggests that the N terminus is the region of attachment to lipid domains. Searching for abundant raft-associated proteins led to stomatin. Co-immunoprecipitation experiment results were consistent with hHV1 binding to stomatin. The stomatin-mediated association of HV1 with cholesterol-dependent lipid domains provides a mechanism for cells to direct HV1 to subcellular locations where it is needed, such as the phagosome in leukocytes.

Biophysical Analysis to Assess the Interaction of CRAC and CARC Motif Peptides of Alpha Hemolysin of Escherichia coli with Membranes.

Alpha hemolysin of Escherichia coli (HlyA) is a pore-forming protein, which is a prototype of the "Repeat in Toxins" (RTX) family. It was demonstrated that HlyA-cholesterol interaction facilitates the insertion of the toxin into membranes. Putative cholesterol-binding sites, called cholesterol recognition/amino acid consensus (CRAC), and CARC (analogous to CRAC but with the opposite orientation) were identified in the HlyA sequence. In this context, two peptides were synthesized, one derived from a CARC site from the insertion domain of the toxin (residues 341-353) (PEP 1) and the other one from a CRAC site from the domain between the acylated lysines (residues 639-644) (PEP 2), to study their role in the interaction of HlyA with membranes. The interaction of peptides with membranes of different lipid compositions (pure POPC and POPC/Cho of 4:1 and 2:1 molar ratios) was analyzed by surface plasmon resonance and molecular dynamics simulations. Results demonstrate that both peptides interact preferentially with Cho-containing membranes, although PEP 2 presents a lower KD than PEP 1. Molecular dynamics simulation results indicate that the insertion and interaction of PEP 2 with Cho-containing membranes are more prominent than those caused by PEP 1. The hemolytic activity of HlyA in the presence of peptides indicates that PEP 2 was the only one that inhibits HlyA activity, interfering in the binding between the toxin and cholesterol.

Modeling the Transport Mechanism of MsbA and P‐glycoprotein

MsbA and P-glycoprotein (P-gp) are structurally, functionally, and mechanistically similar ABC transporter proteins. MsbA is found in gram-negative bacteria, while P-gp is found in intestinal and blood-brain membranes. The endogenous substrate for MsbA is membrane component lipopolysaccharide (LPS), whereas P-gp exports foreign xenobiotics, being responsible for drug efflux activity in acquired multidrug resistance to chemotherapeutics. Co-crystal structures have been determined for P-gp with bound ligand, but MsbA has yet to be solved in the presence of inhibitors or LPS. Elucidating the chemical characteristics of substrates and their protein binding pockets provides insight for designing inhibitors to combat drug resistance. We constructed three-dimensional models at key points along the LPS translocation pathway. A mechanism for transport is revealed involving clusters of basic arginine residues at the entry and exit portions of the transmembrane chamber. Lastly, sequence and structural analysis of P-gp revealed the location of putative cholesterol binding sites that are likely to influence drug transport.

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.

Cryo-EM structures of the human glutamine transporter SLC1A5 (ASCT2) in the outward-facing conformation.

Alanine-serine-cysteine transporter 2 (ASCT2, SLC1A5) is the primary transporter of glutamine in cancer cells and regulates the mTORC1 signaling pathway. The SLC1A5 function involves finely tuned orchestration of two domain movements that include the substrate-binding transport domain and the scaffold domain. Here, we present cryo-EM structures of human SLC1A5 and its complex with the substrate, L-glutamine in an outward-facing conformation. These structures reveal insights into the conformation of the critical ECL2a loop which connects the two domains, thus allowing rigid body movement of the transport domain throughout the transport cycle. Furthermore, the structures provide new insights into substrate recognition, which involves conformational changes in the HP2 loop. A putative cholesterol binding site was observed near the domain interface in the outward-facing state. Comparison with the previously determined inward-facing structure of SCL1A5 provides a basis for a more integrated understanding of substrate recognition and transport mechanism in the SLC1 family.

Cholesterol Binding Sites in Inwardly Rectifying Potassium Channels.

Inwardly rectifying potassium (Kir) channels play a variety of critical cellular roles including modulating membrane excitability in neurons, cardiomyocytes and muscle cells, and setting the resting membrane potential, heart rate, vascular tone, insulin release, and salt flow across epithelia. These processes are regulated by a variegated list of modulators. In particular, in recent years, cholesterol has been shown to modulate a growing number of Kir channels. Subsequent to the discovery that members of the Kir2 subfamily were down-regulated by cholesterol, we have shown that members of several other Kir subfamilies were also modulated by cholesterol. However, not all cholesterol sensitive Kir channels were down-regulated by cholesterol. Our recent studies focused on three Kir channels: Kir2.1 (IRK1), Kir3.2^ (GIRK2^) and Kir3.4* (GIRK4*). Among these, Kir2.1 was down-regulated by cholesterol whereas Kir3.2^ and Kir3.4* were both up-regulated by cholesterol. Despite the opposite impact of cholesterol on these Kir3 channels compared to Kir2.1, putative cholesterol binding sites in all three channels were identified in equivalent transmembrane domains. Interestingly, however, there are intriguing differences in the specific residues that interact with the cholesterol molecule in these Kir channels. Here we compare and contrast the molecular characteristics of the putative cholesterol binding sites in the three channels, and discuss the potential implications of the differencesfor the impact of cholesterol on ion channels.

P.1.005 - Mutating the cholesterol binding site on amyloid precursor protein alters the processing and production of Aβ-amyloid peptides

Levels of cholesterol are elevated in the brains of Alzheimer’s disease (AD) patients [1,2]. While the precise pathophysiological consequences remain unclear, an increase in cholesterol at the plasma membrane promotes endocytosis of the amyloid precursor protein (APP) thereby leading to increased Aβ production [3]. An interaction between the Aβ fragment C99 (resulting from cleavage of APP by β-secretase), and cholesterol was recently proposed [4]. In the present work, we studied the nature of this putative cholesterol binding site (CBS) on APP by analyzing APP mutants within the CBS (located between 691-710 of APP770). We measured the influence of mutants on production of amyloid peptides, cleavage by secretases and the subcellular localization of APP mutants in HEK293T cells. A variety of mutants of the CBS of APP751-mCherry (APP isoform which contain 751 residues fused to the fluorescent protein mCherry) were constructed using site-directed mutagenesis: E693K, S697A, K699A, K699E, G700A, G704A, V710A, S697A/K699A and G700A/G704A (numbering according to APP770 sequence). The amounts of secreted Aβ40 and Aβ42 peptides were measured by ELISA (Meso Scale Discovery) and short peptides (28 to 42aa) by ELISA from IBL (Immuno-Biological Laboratories) in the culture medium of HEK293T cells transfected with various mutants. We studied the activity of secretases by analyzing the levels of C99 and AICD (APP Intracellular Domain) using western blot. In addition, we analyzed the subcellular localization of mutant APP-mCherry by confocal microscopy, using immunocytochemistry with anti-Early Endosome Antigen 1 antibodies. Levels of secreted Aβ40 and Aβ42 were significantly decreased in HEK293T cells transfected with all mutants (except for S697A) as compared to HEK293T cells transfected with APPwt (p In conclusion, these results suggest that the CBS is a key player in the processing of APP and generation of neurotoxic fragments like Aβ42. Further, a specific interaction between cholesterol and the APP CBS controls the size as well as amount of Aβ peptides. We hypothesize that CBS mutants are positioned more deeply in the membrane thus being processed differently leading to the formation of shorter Aβ peptides. These data demonstrate the functional importance of an interaction between cholesterol and APP, provide insights into the pathology of AD, and suggest potential routes to novel therapies.

Cholesterol-Binding Sites in GIRK Channels: The Devil is in the Details.

In recent years, it has become evident that cholesterol plays a direct role in the modulation of a variety of ion channels. In most cases, cholesterol downregulates channel activity. In contrast, our earlier studies have demonstrated that atrial G protein inwardly rectifying potassium (GIRK) channels are upregulated by cholesterol. Recently, we have shown that hippocampal GIRK currents are also upregulated by cholesterol. A combined computational-experimental approach pointed to putative cholesterol-binding sites in the transmembrane domain of the GIRK2 channel, the primary subunit in hippocampal GIRK channels. In particular, the principal cholesterol-binding site was located in the center of the transmembrane domain in between the inner and outer α-helices of 2 adjacent subunits. Further studies pointed to a similar cholesterol-binding site in GIRK4, a major subunit in atrial GIRK channels. However, a close look at a sequence alignment of the transmembrane helices of the 2 channels reveals surprising differences among the residues that interact with the cholesterol molecule in these 2 channels. Here, we compare the residues that form putative cholesterol-binding sites in GIRK2 and GIRK4 and discuss the similarities and differences among them.

Cholesterol up-regulates neuronal G protein-gated inwardly rectifying potassium (GIRK) channel activity in the hippocampus

Hypercholesterolemia is a well known risk factor for the development of neurodegenerative disease. However, the underlying mechanisms are mostly unknown. In recent years, it has become increasingly evident that cholesterol-driven effects on physiology and pathophysiology derive from its ability to alter the function of a variety of membrane proteins including ion channels. Yet, the effect of cholesterol on G protein-gated inwardly rectifying potassium (GIRK) channels expressed in the brain is unknown. GIRK channels mediate the actions of inhibitory brain neurotransmitters. As a result, loss of GIRK function can enhance neuron excitability, whereas gain of GIRK function can reduce neuronal activity. Here we show that in rats on a high-cholesterol diet, cholesterol levels in hippocampal neurons are increased. We also demonstrate that cholesterol plays a critical role in modulating neuronal GIRK currents. Specifically, cholesterol enrichment of rat hippocampal neurons resulted in enhanced channel activity. In accordance, elevated currents upon cholesterol enrichment were also observed in Xenopus oocytes expressing GIRK2 channels, the primary GIRK subunit expressed in the brain. Furthermore, using planar lipid bilayers, we show that although cholesterol did not affect the unitary conductance of GIRK2, it significantly enhanced the frequency of channel openings. Last, combining computational and functional approaches, we identified two putative cholesterol-binding sites in the transmembrane domain of GIRK2. These findings establish that cholesterol plays a critical role in modulating GIRK activity in the brain. Because up-regulation of GIRK function can reduce neuronal activity, our findings may lead to novel approaches for prevention and therapy of cholesterol-driven neurodegenerative disease.

Up‐regulation of atrial and neuronal Kir3 activity by cholesterol

Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells, and its excess is associated with multiple pathological conditions including cardiovascular and neurodegenerative disease. In recent years, cholesterol has been emerging as a major regulator of ion channel function. Here we focus on G protein‐gated inwardly rectifying K+ (GIRK or Kir3) channels that play a key role in the regulation of membrane excitability in neuronal and cardiac cells.The most common effect of cholesterol on ion channels is a decrease in channel activity. In contrast, we have recently shown that unexpectedly cholesterol enrichment up‐regulates Kir3 activity in atrial myocytes. Notably, we show here that also in freshly isolated CA1 pyramidal neurons from hippocampus, in‐vitro cholesterol enrichment results in a strong increase in physiological Kir3 currents.To obtain mechanistic insights into the up‐regulation of cardiac and neuronal Kir3 channels by cholesterol, we employed planar lipid bilayers, mutagenesis studies using the Xenopus oocytes heterologous expression system, and molecular modeling. We first investigated the biophysical properties of the channel. Our data showed that the increase in channel activity following an increase in cholesterol levels is a result of an increase in the open probability of the channel and not in the unitary current amplitude and channel conductance. Next, we investigated why and how cholesterol up‐regulates Kir3 channels. Our studies traced the unique impact of cholesterol on cardiac Kir3 channels (up‐regulation and not down‐regulation) to a transmembrane region that includes residues in both the inner and outer alpha helices. Moreover, we identified putative cholesterol binding sites that overlap with the gating hinge region in the transmembrane domains of both cardiac and neuronal channels. Together, our studies provide novel insights into the molecular mechanism that underlies cholesterol‐driven up‐regulation of Kir3 currents in the heart and in the brain.Support or Funding InformationScientist Development Grant 11SDG5190025 from the American Heart Association (to ARD)

Depletion of Membrane Cholesterol Suppresses Drosophila Transient Receptor Potential-Like (TRPL) Channel Activity.

Cholesterol is an essential compound of higher eukaryotic cell membranes and a known modulator of ion channel activity. Changes in phospholipids and cholesterol composition of cell membranes are known to alter the activity of ion channels. However, there is little knowledge on the effects of cholesterol on transient receptor potential (TRP) channels. In this study, we explore the effects of cholesterol depletion on the Drosophila photoreceptor channel TRP-like (TRPL), when expressed in tissue culture cells. Depletion of membrane cholesterol with methyl-β-cyclodextrin (MβCD) induced fast (<100s) suppression of spontaneous TRPL channel activity, a typical state of expressed TRPL channels in Drosophila S2 cells. An equally fast suppression of receptor-induced TRPL channel activity in HEK293 cells, downstream of phospholipase C (PLC) activation, was also induced by MβCD. Biochemical experiments showed binding of TRPL to immobilized cholesterol, suggesting direct binding of cholesterol to TRPL. Exploring the effects of several mutations in a putative cholesterol-binding site of TRPL was inconclusive as some did not render the channel insensitive to cholesterol depletion while others rendered the channel inactive. We conclude that (i) cholesterol is essential for TRPL channel activity, (ii) TRPL channels interact with cholesterol, and (iii) the binding site of cholesterol in TRPL differs from the putative binding site of TRPV1. Thus, the fast and strong effects of cholesterol depletion on the TRPL channel activity suggest that cholesterol is an important component of fly photoreceptor signaling membrane.

Cholesterol-mediated Degradation of 7-Dehydrocholesterol Reductase Switches the Balance from Cholesterol to Vitamin D Synthesis

Cholesterol is detrimental to human health in excess but is also essential for normal embryogenesis. Hence, enzymes involved in its synthesis possess many layers of regulation to achieve balanced cholesterol levels. 7-Dehydrocholesterol reductase (DHCR7) is the terminal enzyme of cholesterol synthesis in the Kandutsch-Russell pathway, converting 7-dehydrocholesterol (7DHC) to cholesterol. In the absence of functional DHCR7, accumulation of 7DHC and a lack of cholesterol production leads to the devastating developmental disorder, Smith-Lemli-Opitz syndrome. This study identifies that statin treatment can ameliorate the low DHCR7 expression seen with common Smith-Lemli-Opitz syndrome mutations. Furthermore, we show that wild-type DHCR7 is also relatively labile. In an example of end-product inhibition, cholesterol accelerates the proteasomal degradation of DHCR7, resulting in decreased protein levels and activity. The loss of enzymatic activity results in the accumulation of the substrate 7DHC, which leads to an increased production of vitamin D. Thus, these findings highlight DHCR7 as an important regulatory switch between cholesterol and vitamin D synthesis.

Cholesterol binding to ion channels.

Numerous studies demonstrated that membrane cholesterol is a major regulator of ion channel function. The goal of this review is to discuss significant advances that have been recently achieved in elucidating the mechanisms responsible for cholesterol regulation of ion channels. The first major insight that comes from growing number of studies that based on the sterol specificity of cholesterol effects, show that several types of ion channels (nAChR, Kir, BK, TRPV) are regulated by specific sterol-protein interactions. This conclusion is supported by demonstrating direct saturable binding of cholesterol to a bacterial Kir channel. The second major advance in the field is the identification of putative cholesterol binding sites in several types of ion channels. These include sites at locations associated with the well-known cholesterol binding motif CRAC and its reversed form CARC in nAChR, BK, and TRPV, as well as novel cholesterol binding regions in Kir channels. Notably, in the majority of these channels, cholesterol is suggested to interact mainly with hydrophobic residues in non-annular regions of the channels being embedded in between transmembrane protein helices. We also discuss how identification of putative cholesterol binding sites is an essential step to understand the mechanistic basis of cholesterol-induced channel regulation. Clearly, however, these are only the first few steps in obtaining a general understanding of cholesterol-ion channels interactions and their roles in cellular and organ functions.

Putative cholesterol‐binding sites in human immunodeficiency virus (HIV) coreceptors CXCR4 and CCR5

Using molecular docking, we identified a cholesterol-binding site in the groove between transmembrane helices 1 and 7 near the inner membrane-water interface of the G protein-coupled receptor CXCR4, a coreceptor for HIV entry into cells. In this docking pose, the amino group of lysine K67 establishes a hydrogen bond with the hydroxyl group of cholesterol, whereas tyrosine Y302 stacks with cholesterol by its aromatic side chain, and a number of residues form hydrophobic contacts with cholesterol. Sequence alignment showed that a similar putative cholesterol-binding site is also present in CCR5, another HIV coreceptor. We suggest that the interaction of cholesterol with these putative cholesterol-binding sites in CXCR4 and CCR5 is responsible for the presence of these receptors in lipid rafts, for the effect of cholesterol on their conformational stability and function, and for the role that cell cholesterol plays in the cell entry of HIV strains that use these membrane proteins as coreceptors. We propose that mutations of residues that are involved in cholesterol binding will make CXCR4 and CCR5 insensitive to membrane cholesterol content. Cholesterol-binding sites in HIV coreceptors are potential targets for steroid drugs that bind to CXCR4 and CCR5 with higher binding affinity than cholesterol, but do not stabilize the native conformation of these proteins.

Holo-sterol Carrier Protein-2: 13C NMR INVESTIGATION OF CHOLESTEROL AND FATTY ACID BINDING SITES

Although sterol carrier protein-2 (SCP-2) stimulates sterol transfer in vitro, almost nothing is known regarding the identity of the putative cholesterol binding site. Furthermore, the interrelationship(s) between this SCP-2 ligand binding site and the recently reported SCP-2 long chain fatty acid (LCFA) and long chain fatty acyl-CoA (LCFA-CoA) binding site(s) remains to be established. In the present work, two SCP-2 ligand binding sites were identified. First, both [4-(13)C]cholesterol and 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol) binding assays were consistent with a single cholesterol binding site in SCP-2. This ligand binding site had high affinity for NBD-cholesterol, K(d) = 4.15 +/- 0.71 nM. (13)C NMR-labeled ligand competition studies demonstrated that the SCP-2 high affinity cholesterol binding site also bound LCFA or LCFA-CoA. However, only the LCFA-CoA was able to effectively displace the SCP-2-bound [4-(13)C]cholesterol. Thus, the ligand affinities at this SCP-2 binding site were in the relative order cholesterol = LCFA-CoA > LCFA. Second, (13)C NMR studies demonstrated the presence of another ligand binding site on SCP-2 that bound either LCFA or LCFA-CoA but not cholesterol. Photon correlation spectroscopy was consistent with SCP-2 being monomeric in both liganded and unliganded states. In summary, both (13)C NMR and fluorescence techniques demonstrated for the first time that SCP-2 had a single high affinity binding site that bound cholesterol, LCFA, or LCFA-CoA. Furthermore, results with (13)C NMR supported the presence of a second SCP-2 ligand binding site that bound either LCFA or LCFA-CoA but not cholesterol. These data contribute to our understanding of a role for SCP-2 in both cellular cholesterol and LCFA metabolism.