Cholesterol and the interaction of proteins with membrane domains

Cholesterol is not uniformly distributed in biological membranes. One of the factors influencing the formation of cholesterol-rich domains in membranes is the unequal lateral distribution of proteins in membranes. Certain proteins are found in cholesterol-rich domains. In some of these cases, it is as a consequence of the proteins interacting directly with cholesterol. There are several structural features of a protein that result in the protein preferentially associating with cholesterol-rich domains. One of the best documented of these is certain types of lipidations. In addition, however, there are segments of a protein that can preferentially sequester cholesterol. We discuss two examples of these cholesterol-recognition elements: the cholesterol recognition/interaction amino acid consensus (CRAC) domain and the sterol-sensing domain (SSD). The requirements for a CRAC motif are quite flexible and predict that a large number of sequences could recognize cholesterol. There are, however, certain proteins that are known to interact with cholesterol-rich domains of cell membranes that have CRAC motifs, and synthetic peptides corresponding to these segments also promote the formation of cholesterol-rich domains. Modeling studies have provided a rationale for certain requirements of the CRAC motif. The SSD is a larger protein segment comprising five transmembrane domains. The amino acid sequence YIYF is found in several SSD and in certain other proteins for which there is evidence that they interact with cholesterol-rich domains. The CRAC sequences as well as YIYF are generally found adjacent to a transmembrane helical segment. These regions appear to have a strong influence of the localization of certain proteins into domains in biological membranes. In addition to the SSD, there is also a domain found in soluble proteins, the START domain, that binds lipids. Certain proteins with START domains specifically bind cholesterol and are believed to function in intracellular cholesterol transport. One of these proteins is StAR-D1, that also has a mitochondrial targeting sequence and plays an important role in delivering cholesterol to the mitochondria of steroidogenic cells.     

Interactions of caveolin-1 scaffolding and intramembrane regions containing a CRAC motif with cholesterol in lipid bilayers

Caveolin-1 is a major structural protein of caveolae and specifically binds cholesterol (Chol). The caveolin scaffolding domain is thought to be involved in caveolin–Chol interaction through the sequence V94-T-K-Y-W-F-Y-R101, a motif that matches a cholesterol recognition amino-acid consensus (CRAC). In the present work, three CRAC-containing peptides, corresponding to caveolin-1 94–101, 82–101 and 93–126, were tested to study the role of the CRAC motif in the caveolin–Chol interaction in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers using differential scanning calorimetry (DSC), fluorescence and circular dichroism (CD). The Y97I substituents of the three peptides and one peptide segment corresponding to caveolin-1 101–126 that excludes the CRAC motif were also tested for comparison. Our results showed the potency of these CRAC-containing peptides in sequestering Chol into domains and the enhanced role of the intramembrane domain and scaffolding domain for the potency. Of the three CRAC-containing peptides, the peptide 93–126 was particularly effective in promoting Chol segregation, while the peptide 82–101 was less potent in promoting the formation of domains than the peptide 93–126, but was more potent than the peptide 94–101. The domain partition of DPPC/Chol bilayers was not observed in the presence of the peptide 101–126, in contrast to the case in the presence of the peptide 93–126 at the same concentrations of peptide and Chol. The potency of the CRAC motif in Chol segregation was lowered by the Y97I mutation. The difference in structure may be a factor that contributes to different effects of these peptides on the distribution of Chol in the lipid membrane.     

Juxtamembrane protein segments that contribute to recruitment of cholesterol into domains

We investigated the properties of several peptides with sequences related to LWYIK, a segment found in the gp41 protein of HIV and believed to play a role in sequestering this protein to a cholesterol-rich domain in the membrane. This segment fulfills the requirements to be classified as a CRAC motif that has been suggested to predict those proteins that will partition into cholesterol-rich regions of the membrane. All of the peptides were studied with the terminal amino and carboxyl groups blocked, i.e., as N-acetyl-peptide-amides. Effects of cholesterol on the intensity of W emission generally parallel DSC evidence of sequestration of cholesterol. Modeling studies indicate that all of these peptides tend to partition with their mass center at the membrane interface at the level of the hydroxyl of cholesterol. Interaction with cholesterol is dual: van der Waals interactions between mainly hydrophobic surfaces and electrostatic stabilization of the cholesterol OH group. Thus, both experiments and modeling studies indicate that the preference of CRAC motifs for cholesterol-rich domains might be related to a membrane interfacial preference of the motif, to a capacity to wrap and block the cholesterol polar OH group by H-bond interactions, and to a capacity for peptide aromatic side chains to stack with cholesterol. These results were supported by studies of single mutations in the gp41 protein of HIV-1, in which L(679) is replaced with I. Despite the similarity of the properties of these amino acid residues, this single substitution resulted in a marked attenuation of the ability of JC53-BL HeLa-based HIV-1 indicator cells to form syncytia.     

Proteins and cholesterol-rich domains

Biological membranes are composed of many molecular species of lipids and proteins. These molecules do not mix ideally. In the plane of the membrane components are segregated into domains that are enriched in certain lipids and proteins. Cholesterol is a membrane lipid that is not uniformly distributed in the membrane. Proteins play an important role in determining cholesterol distribution. Certain types of protein lipidation are known to cause the lipoprotein to sequester with cholesterol and to stabilize cholesterol-rich domains. However, proteins that are excluded from such domains also contribute to the redistribution of cholesterol. One of the motifs that favor interaction with cholesterol is the CRAC motif. The role of the CRAC motif of the gp41 fusogenic protein of HIV is discussed. The distribution of the multianionic lipid, phosphatidylinositol(4,5)bis-phosphate (PtnIns(4,5)P2), is also not uniform in cell membranes. This lipid has several functions in the cell, including a morphological role in determining the sites of attachment of the actin cytoskeleton to the plasma membrane. PtnIns(4,5)P2 is sequestered by proteins having clusters of cationic residues in their sequence. Certain proteins containing cationic clusters also contain moieties such as myristoylation or a CRAC segment that would also endow them with the ability to sequester to a cholesterol-rich domain. These proteins interact with PtnIns(4,5)P2 in a cholesterol-dependent manner forming domains that are enriched in both cholesterol and in PtnIns(4,5)P2 but can also be distinct from liquid-ordered raft-like domains.     

A Cholesterol Recognition Motif in Human Phospholipid Scramblase 1

Human phospholipid scramblase 1 (SCR) catalyzes phospholipid transmembrane (flip-flop) motion. This protein is assumed to bind the membrane hydrophobic core through a transmembrane domain (TMD) as well as via covalently bound palmitoyl residues. Here, we explore the possible interaction of the SCR TMD with cholesterol by using a variety of experimental and computational biophysical approaches. Our findings indicate that SCR contains an amino acid segment at the C-terminal region that shows a remarkable affinity for cholesterol, although it lacks the CRAC sequence. Other 3-OH sterols, but not steroids lacking the 3-OH group, also bind this region of the protein. The newly identified cholesterol-binding region is located partly at the C-terminal portion of the TMD and partly in the first amino acid residues in the SCR C-terminal extracellular coil. This finding could be related to the previously described affinity of SCR for cholesterol-rich domains in membranes.     

A Cholesterol Recognition Motif in Human Phospholipid Scramblase 1

Human phospholipid scramblase 1 (SCR) catalyzes phospholipid transmembrane (flip-flop) motion. This protein is assumed to bind the membrane hydrophobic core through a transmembrane domain (TMD) as well as via covalently bound palmitoyl residues. Here, we explore the possible interaction of the SCR TMD with cholesterol by using a variety of experimental and computational biophysical approaches. Our findings indicate that SCR contains an amino acid segment at the C-terminal region that shows a remarkable affinity for cholesterol, although it lacks the CRAC sequence. Other 3-OH sterols, but not steroids lacking the 3-OH group, also bind this region of the protein. The newly identified cholesterol-binding region is located partly at the C-terminal portion of the TMD and partly in the first amino acid residues in the SCR C-terminal extracellular coil. This finding could be related to the previously described affinity of SCR for cholesterol-rich domains in membranes.     

Structural characterization of the caveolin scaffolding domain in association with cholesterol-rich membranes

Members of the caveolin protein family are implicated in the formation of caveolae and play important roles in a number of signaling pathways and in the regulation of various proteins. We employ complementary spectroscopic methods to study the structure of the caveolin scaffolding domain (CSD) in caveolin-1 fragments, while bound to cholesterol-rich membranes. This key domain is thought to be involved in multiple critical functions that include protein recognition, oligomerization, and cholesterol binding. In our membrane-bound peptides, residues within the flanking intramembrane domain (IMD) are found to adopt an α-helical structure, consistent with its commonly believed helical hairpin conformation. Intriguingly, in these same peptides, we observe a β-stranded conformation for residues in the CSD, contrasting with earlier reports, which commonly do not reflect β-structure. Our experimental data based on solid-state NMR, CD, and FTIR are found to be consistent with computational analyses of the secondary structure preference of the primary sequence. We discuss how our structural data of membrane binding Cav fragments may match certain general features of cholesterol-binding domains and could be consistent with the role for CSD in protein recognition and homo-oligomerization.     

Do proteins facilitate the formation of cholesterol-rich domains?

Both biological and model membranes can exhibit the formation of domains. A brief review of some of the diverse methodologies used to identify the presence of domains in membranes is given. Some of these domains are enriched in cholesterol. The segregation of lipids into cholesterol-rich domains can occur in both pure lipid systems as well as membranes containing peptides and proteins. Peptides and proteins can promote the formation of cholesterol-rich domains not only by preferentially interacting with cholesterol and being sequestered into these regions of the membrane, but also indirectly as a consequence of being excluded from cholesterol-rich domains. The redistribution of components is dictated by the thermodynamics of the system. The formation of domains in a biological membrane is a consequence of all of the intermolecular interactions including those among lipid molecules as well as between lipids and proteins.     

Structural studies of the transmembrane C-terminal domain of the amyloid precursor protein (APP): does APP function as a cholesterol sensor?

The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-beta (Abeta) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by beta-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein. C99 was purified into model membranes where it was observed to homodimerize. NMR data show that the transmembrane domain of C99 is an alpha-helix that is flanked on both sides by mostly disordered extramembrane domains, with two exceptions. First, there is a short extracellular surface-associated helix located just after the site of alpha-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Abeta production. Second, there is a surface-associated helix located at the cytosolic C-terminus, adjacent to the YENPTY motif that plays critical roles in APP trafficking and protein-protein interactions. Cholesterol was seen to participate in saturable interactions with C99 that are centered at the critical loop connecting the extracellular helix to the transmembrane domain. Binding of cholesterol to C99 and, most likely, to APP may be critical for the trafficking of these proteins to cholesterol-rich membrane domains, which leads to cleavage by beta- and gamma-secretase and resulting amyloid-beta production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake.     

Membrane topology of gp41 and amyloid precursor protein: Interfering transmembrane interactions as potential targets for HIV and Alzheimer treatment

The amyloid precursor protein (APP), that plays a critical role in the development of senile plaques in Alzheimer disease (AD), and the gp41 envelope protein of the human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome (AIDS), are single-spanning type-1 transmembrane (TM) glycoproteins with the ability to form homo-oligomers. In this review we describe similarities, both in structural terms and sequence determinants of their TM and juxtamembrane regions. The TM domains are essential not only for anchoring the proteins in membranes but also have functional roles. Both TM segments contain GxxxG motifs that drive TM associations within the lipid bilayer. They also each possess similar sequence motifs, positioned at the membrane interface preceding their TM domains. These domains are known as cholesterol recognition/interaction amino acid consensus (CRAC) motif in gp41 and CRAC-like motif in APP. Moreover, in the cytoplasmic domain of both proteins other α-helical membranotropic regions with functional implications have been identified. Recent drug developments targeting both diseases are reviewed and the potential use of TM interaction modulators as therapeutic targets is discussed.     

Characterization of the cholesterol recognition amino acid consensus sequence of the peripheral-type benzodiazepine receptor

We previously defined a cholesterol recognition/interaction amino acid consensus sequence [CRAC: L/V-X (1-5)-Y-X (1-5)-R/K] in the carboxyl terminus of the peripheral-type benzodiazepine receptor (PBR), a high-affinity drug and cholesterol-binding protein present in the outer mitochondrial membrane protein. This protein is involved in the regulation of cholesterol transport into the mitochondria, the rate-determining step in steroid biosynthesis. Reconstituted wild-type recombinant PBR into proteoliposomes demonstrated high-affinity 2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide and cholesterol binding. In the present work, we functionally and structurally characterized this CRAC motif using reconstituted recombinant PBR and nuclear magnetic resonance. Deletion of the C-terminal domain of PBR and mutation of the highly conserved among all PBR amino acid sequences Y152 of the CRAC domain resulted in loss of the ability of mutant recPBR to bind cholesterol. Nuclear magnetic resonance analysis of a PBR C-terminal peptide (144-169) containing the CRAC domain indicated a helical conformation for the L144-S159 fragment. As a result of the side-chain distribution, a groove that could fit a cholesterol molecule is delineated, on one hand, by Y152, T148, and L144, and, on the other hand, by Y153, M149, and A145. The aromatic rings of Y152 and Y153 assigned as essential residues for cholesterol binding constitute the gate of the groove. Furthermore, the side chain of R156 may cap the groove by interacting with the sterol hydroxyl group. These results provide structural and functional evidence supporting the finding that the CRAC domain in the cytosolic carboxyl-terminal domain of PBR might be responsible for the uptake and translocation of cholesterol into the mitochondria.     

The tryptophan-rich region of HIV gp41 and the promotion of cholesterol-rich domains

The peptide N-acetyl-KWASLWNWFNITNWLWYIK-amide has a sequence that corresponds to the juxtamembrane region of the HIV-1 gp41 fusion protein. We have studied how cholesterol modulates the interaction of this peptide with membranes containing cholesterol using differential scanning calorimetry, circular dichroism, fluorescence spectroscopy, and nuclear magnetic resonance. We find that this peptide is less able to sequester cholesterol into domains than is N-acetyl-LWYIK-amide. On the other hand, the peptide N-acetyl-LASWIK-amide, which corresponds to a segment of HIV-2 and SIV gp41 fusion proteins, has intermediate potency between N-acetyl-KWASLWNWFNITNWLWYIK-amide and N-acetyl-LWYIK-amide in forming areas enriched in cholesterol, even though it does not have a cholesterol recognition/interaction amino acid consensus sequence (CRAC). We suggest that the difference between HIV-1 and HIV-2 in their requirements for glycosphingolipids in determining their tropism is related to their difference in partitioning to cholesterol-rich domains in biological membranes.     

CUP-1 is a novel protein involved in dietary cholesterol uptake in Caenorhabditis elegans

Sterols transport and distribution are essential processes in all multicellular organisms. Survival of the nematode Caenorhabditis elegans depends on dietary absorption of sterols present in the environment. However the general mechanisms associated to sterol uptake in nematodes are poorly understood. In the present work we provide evidence showing that a previously uncharacterized transmembrane protein, designated Cholesterol Uptake Protein-1 (CUP-1), is involved in dietary cholesterol uptake in C. elegans. Animals lacking CUP-1 showed hypersensitivity to cholesterol limitation and were unable to uptake cholesterol. A CUP-1-GFP fusion protein colocalized with cholesterol-rich vesicles, endosomes and lysosomes as well as the plasma membrane. Additionally, by FRET imaging, a direct interaction was found between the cholesterol analog DHE and the transmembrane "cholesterol recognition/interaction amino acid consensus" (CRAC) motif present in C. elegans CUP-1. In-silico analysis identified two mammalian homologues of CUP-1. Most interestingly, CRAC motifs are conserved in mammalian CUP-1 homologous. Our results suggest a role of CUP-1 in cholesterol uptake in C. elegans and open up the possibility for the existence of a new class of proteins involved in sterol absorption in mammals.     

Specificity of Membrane Binding of the Neuronal Protein NAP-22

NAP-22, a major protein of neuronal rafts is known to preferentially bind to membranes containing cholesterol. In this work we establish the requirements for membrane binding of NAP-22. We find that other sterols can replace cholesterol to promote binding. In addition, bilayers containing phosphatidylethanolamine bind NAP-22 in the absence of cholesterol. Thus, there is not a specific interaction of NAP-22 with cholesterol that determines its binding to membranes. Addition of a mol fraction of phosphatidylserine of 0.05 to membranes of phosphatidylcholine and cholesterol enhances the membrane binding of NAP-22. The dependence of binding on the mol fraction of phosphatidylserine indicates that NAP-22 binds to membranes with its amino-terminal segment closer to the membrane than the remainder of the protein. We have also determined which segments of NAP-22 are required for membrane binding. A non-myristoylated form binds only weakly to membranes. Truncating the protein from 226 amino acids to the myristoylated amino-terminal 60 amino acids does not prevent binding to membranes in a cholesterol-dependent manner, but this binding is of weaker affinity. However, myristoylation is not sufficient to promote binding to cholesterol-rich domains. An N-terminal 19-amino-acid, myristoylated peptide binds to membranes but without requiring specific lipids. Thus, the remainder of the protein contributes to the lipid specificity of the membrane binding of NAP-22.     

Novel androstenetriol interacts with the mitochondrial translocator protein and controls steroidogenesis

Steroid hormones are metabolically derived from multiple enzymatic transformations of cholesterol. The controlling step in steroid hormone biogenesis is the delivery of cholesterol from intracellular stores to the cytochrome P450 enzyme CYP11A1 in the mitochondrial matrix. The 18-kDa translocator protein (TSPO) plays an integral part in this mitochondrial cholesterol transport. Consistent with its role in intracellular cholesterol movement, TSPO possesses a cholesterol recognition/interaction amino acid consensus (CRAC) motif that has been demonstrated to bind cholesterol. To further investigate the TSPO CRAC motif, we performed molecular modeling studies and identified a novel ligand, 3,17,19-androsten-5-triol (19-Atriol) that inhibits cholesterol binding at the CRAC motif. 19-Atriol could bind a synthetic CRAC peptide and rapidly inhibited hormonally induced steroidogenesis in MA-10 mouse Leydig tumor cells and constitutive steroidogenesis in R2C rat Leydig tumor cells at low micromolar concentrations. Inhibition at these concentrations was not due to toxicity or inhibition of the CYP11A1 enzyme and was reversed upon removal of the compound. In addition, 19-Atriol was an even more potent inhibitor of PK 11195-stimulated steroidogenesis, with activity in the high nanomolar range. This was accomplished without affecting PK 11195 binding or basal steroidogenesis. Finally, 19-Atriol inhibited mitochondrial import and processing of the steroidogenic acute regulatory protein without any effect on TSPO protein levels. In conclusion, we have identified a novel androstenetriol that can interact with the CRAC domain of TSPO, can control hormonal and constitutive steroidogenesis, and may prove to be a useful tool in the therapeutic control of diseases of excessive steroid formation.     

Identification of cholesterol recognition amino acid consensus (CRAC) motif in G-protein coupled receptors

G-protein coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes, and represent major targets in the development of novel drug candidates in all clinical areas. Membrane cholesterol has been reported to have an important role in the function of a number of GPCRs. Several structural features of proteins, believed to result in preferential association with cholesterol, have been recognized. Cholesterol recognition/interaction amino acid consensus (CRAC) sequence represents such a motif. Many proteins that interact with cholesterol have been shown to contain the CRAC motif in their sequence. We report here the presence of CRAC motifs in three representative GPCRs, namely, rhodopsin, the β(2)-adrenergic receptor, and the serotonin(1A) receptor. Interestingly, the function of these GPCRs has been previously shown to be dependent on membrane cholesterol. The presence of CRAC motifs in GPCRs indicates that interaction of cholesterol with GPCRs could be specific in nature. Further analysis shows that CRAC motifs are inherent characteristic features of the serotonin(1A) receptor and are conserved over natural evolution. These results constitute the first report of the presence of CRAC motifs in GPCRs and provide novel insight in the molecular nature of GPCR-cholesterol interaction.     

Intrinsic membrane association of the cytoplasmic tail of influenza virus M2 protein and lateral membrane sorting regulated by cholesterol binding and palmitoylation

The influenza virus transmembrane protein M2 is a proton channel, but also plays a role in the scission of nascent virus particles from the plasma membrane. An amphiphilic helix in the CT (cytoplasmic tail) of M2 is supposed to insert into the lipid bilayer, thereby inducing curvature. Palmitoylation of the helix and binding to cholesterol via putative CRAC (cholesterol recognition/interaction amino acid consensus) motifs are believed to target M2 to the edge of rafts, the viral-budding site. In the present study, we tested pre-conditions of this model, i.e. that the CT interacts with membranes, and that acylation and cholesterol binding affect targeting of M2. M2-CT, purified as a glutathione transferase fusion protein, associated with [3H]photocholesterol and with liposomes. Mutation of tyrosine residues in the CRAC motifs prevented [(3)H]photocholesterol labelling and reduced liposome binding. M2-CT fused to the yellow fluorescent protein localized to the Golgi in transfected cells; membrane targeting was dependent on CRAC and (to a lesser extent) on palmitoylation. Preparation of giant plasma membrane vesicles from cells expressing full-length M2-GFP (green fluorescent protein) showed that the protein is partly present in the raft domain. Raft targeting required palmitoylation, but not the CRAC motifs. Thus palmitoylation and cholesterol binding differentially affect the intrinsic membrane binding of the amphiphilic helix.     

Peptide-induced formation of cholesterol-rich domains

The peptide N-acetyl-LWYIK-amide causes the reorganization of bilayers of phosphatidylcholine and cholesterol to produce domains enriched in cholesterol. At a cholesterol mol fraction of 0.5, addition of N-acetyl-LWYIK-amide results in the formation of cholesterol crystallites. Addition of this peptide to mixtures of 1-stearoyl-2-oleoylphosphatidylcholine with lower mol fractions of cholesterol results in an increase in the enthalpy of the chain melting transition of the phospholipid, indicating the depletion of cholesterol from a domain in the membrane. The peptide binds to membranes both with and without cholesterol. However, (1)H magic-angle spinning (MAS) nuclear Overhauser effect spectroscopy (NOESY) indicates that in the presence of cholesterol the peptide has greater penetration into the bilayer. (13)C MAS NMR indicates that the peptide has stronger interactions with the A ring of cholesterol than it does with the interior of the bilayer. These results are in contrast with those of another peptide, N-acetyl-KYWFYR-amide, which does not promote the formation of cholesterol crystallites and does not show preferential interaction with cholesterol by NMR. Therefore, cholesterol can promote the insertion of N-acetyl-LWYIK-amide into a membrane and this peptide will sequester cholesterol into domains. These properties help to explain the observation that this sequence is found to be important in causing the fusion protein of human immunodeficiency virus (HIV) to sequester into raft domains in biological membranes.     

Large changes in the CRAC segment of gp41 of HIV do not destroy fusion activity if the segment interacts with cholesterol

The membrane-proximal external region (MPER) of the gp41 fusion protein of HIV is highly conserved among isolates of this virus and is considered a target for vaccine development. This region also appears to play a role in membrane fusion as well as localization of the virus to cholesterol-rich domains in membranes. The carboxyl terminus of MPER has the sequence LWYIK and appears to have an important role in cholesterol interactions. We have tested how amino acid substitutions that would affect the conformational flexibility of this segment could alter its interaction with cholesterol. We studied a family of peptides (all peptides as N-acetyl-peptide amides) with P, G, or A substituting for W and I of the LWYIK sequence. The peptide having the greatest effect on cholesterol distribution in membranes was the most flexible one, LGYGK. The corresponding mutation in gp41 resulted in a protein retaining 72% of the fusion activity of the wild-type protein. Two other peptides were synthesized, also containing two Gly residues, GWGIK and LWGIG, and did not have the ability to sequester cholesterol as efficiently as LGYGK did. Making the corresponding mutants of gp41 showed that these other two double Gly substitutions resulted in proteins that were much less fusogenic, although they were equally well expressed at the cell surface. The study demonstrates that drastic changes can be made in the LWYIK segment with the retention of a significant fraction of the fusogenic activity, as long as the mutant proteins interact with cholesterol.     

The fusogenic tilted peptide (67-78) of α-synuclein is a cholesterol binding domain

Parkinson's disease-associated α-synuclein is an amyloidogenic protein not only expressed in the cytoplasm of neurons, but also secreted in the extracellular space and internalized into glial cells through a lipid raft-dependent process. We previously showed that α-synuclein interacts with raft glycosphingolipids through a structural motif common to various viral and amyloidogenic proteins. Here we report that α-synuclein also interacts with cholesterol, as assessed by surface pressure measurements of cholesterol-containing monolayers. Using a panel of recombinant fragments and synthetic peptides, we identified two distinct cholesterol-binding domains in α-synuclein. One of these domains, which corresponds to the tilted peptide of α-synuclein (67-78), bound cholesterol with high affinity and was toxic for cultured astrocytes. Molecular modeling suggested that cholesterol binds to this peptide with a tilt angle of 46°. α-synuclein also contains a cholesterol recognition consensus motif, which had a lower affinity for cholesterol and was devoid of toxicity. This motif is encased in the glycosphingolipid-binding domain (34-45) of α-synuclein. In raft-like model membranes containing both cholesterol and glycosphingolipids, the head groups of glycosphingolipids prevented the accessibility of cholesterol to exogenous ligands. Nevertheless, cholesterol appeared to 'signal' its presence by tuning glycosphingolipid conformation, thereby facilitating α-synuclein binding to raft-like membranes. We propose that the association of α-synuclein with lipid rafts involves both the binding of α-synuclein (34-45) to glycosphingolipids, and the interaction of the fusogenic tilted peptide (67-78) with cholesterol. Coincidentally, a similar mechanism is used by viruses (HIV-1, HTLV-I, Ebola) which display a tilted peptide and fuse with host cell membranes through a sphingolipid/cholesterol-dependent process.