CRAC Motif Research Articles

Sterol interactions influence the function of Wsc sensors

The Wsc1, Wsc2, and Wsc3 proteins are essential cell surface sensors that respond to cell wall perturbation by activating the cell wall integrity pathway (CWIP). We show here that in situ production of cholesterol (in place of ergosterol) induces hyper-phosphorylation of Slt2, the MAPK of the CWIP, and upregulates cell wall biosynthesis. Deletion of all three Wsc genes in K. phaffii reverts these phenotypes. In the cholesterol-producing strain, both Wsc1 and Wsc3 accumulate in the plasma membrane. Close inspection of the transmembrane domains of all three Wsc proteins predicted by AlphaFold2 revealed the presence of CRAC sterol-binding motifs. Experiments using a photoreactive cholesterol derivative indicate intimate interaction of this sterol with the Wsc transmembrane domain, and this apparent sterol binding was abrogated in Wsc mutants with substitutions in the CRAC motif. We also observed cholesterol interaction with CRAC-like motifs in the transmembrane domains of mammalian integrins, analogs of Wsc proteins. Our results suggest that proper signaling of the Wsc sensors requires highly specific binding of the native endogenous terminal sterol, ergosterol.

SIDT2 Associates with Apolipoprotein A1 (ApoA1) and Facilitates ApoA1 Secretion in Hepatocytes.

SIDT2 is a lysosomal protein involved in the degradation of nucleic acids and the transport of cholesterol between membranes. Previous studies identified two "cholesterol recognition/interaction amino acid consensus" (CRAC) motifs in SIDT1 and SIDT2 members. We have previously shown that the first CRAC motif (CRAC-1) is essential for protein translocation to the PM upon cholesterol depletion in the cell. In the present study, we show that SIDT2 and the apolipoprotein A1 (ApoA1) form a complex which requires the second CRAC-2 motif in SIDT2 to be established. The overexpression of SIDT2 and ApoA1 results in enhanced ApoA1 secretion by HepG2 cells. This is not observed when overexpressing the SIDT2 with the CRAC-2 domain mutated to render it unfunctional. All these results provide evidence of a novel role for SIDT2 as a protein forming a complex with ApoA1 and enhancing its secretion to the extracellular space.

Interaction of Amphipathic Peptide from Influenza Virus M1 Protein with Mitochondrial Cytochrome Oxidase.

The Bile Acid Binding Site (BABS) of cytochrome oxidase (CcO) binds numerous amphipathic ligands. To determine which of the BABS-lining residues are critical for interaction, we used the peptide P4 and its derivatives A1-A4. P4 is composed of two flexibly bound modified α-helices from the M1 protein of the influenza virus, each containing a cholesterol-recognizing CRAC motif. The effect of the peptides on the activity of CcO was studied in solution and in membranes. The secondary structure of the peptides was examined by molecular dynamics, circular dichroism spectroscopy, and testing the ability to form membrane pores. P4 was found to suppress the oxidase but not the peroxidase activity of solubilized CcO. The Ki(app) is linearly dependent on the dodecyl-maltoside (DM) concentration, indicating that DM and P4 compete in a 1:1 ratio. The true Ki is 3 μM. The deoxycholate-induced increase in Ki(app) points to a competition between P4 and deoxycholate. A1 and A4 inhibit solubilized CcO with Ki(app)~20 μM at 1 mM DM. A2 and A3 hardly inhibit CcO either in solution or in membranes. The mitochondrial membrane-bound CcO retains sensitivity to P4 and A4 but acquires resistance to A1. We associate the inhibitory effect of P4 with its binding to BABS and dysfunction of the proton channel K. Trp residue is critical for inhibition. The resistance of the membrane-bound enzyme to inhibition may be due to the disordered secondary structure of the inhibitory peptide.

INFLUENZA VIRUS AND CHOLESTEROL: TOUCH POINTS

Abstract---This review considers the observations concerning the interactions of influenza virus with cholesterol and consequences of these interactions for both the virus and a host cell. There are at least two crucial “touch points”, when IFV deals with cholesterol: first, cholesterol is required for influenza virus entry into the cell. Second, during budding, when new viral particles form their envelopes from plasma membrane of the infected cell, selectively acquiring “raft lipids”, cholesterol and sphingolipids. One possible mechanism ensuring selective accumulation of cholesterol in the viral envelope is the presence of cholesterol-binding motifs (CRAC motifs) in protein M1 and possibly other viral proteins involved in this process. These motifs could be responsible for binding of cell membrane´s cholesterol by the viral protein. The important role of cholesterol in the influenza virus life cycle raises the possibility that lowering cholesterol levels in host cells (e.g., with statins) can be useful in reducing influenza virus infectivity and productivity. However, lowering cholesterol in cell membranes below an optimal level may not be compatible with normal cell function. There is experimental evidence that CRAC-containing peptide derived from influenza virus protein M1 is indeed cytotoxic, and that extraction of membrane cholesterol with mbCD lowers the concentration of the peptide´s cytotoxic effect by an order of magnitude. In the conditions of reduced cholesterol, any infection with enveloped virus sequestering cholesterol from cell membranes may be detrimental, as further lowering cholesterol level in cell membrane during virus budding may lead to cell damage or death. Perhaps to minimize the virus infectivity and the consequences of the massive virus budding, advantageous can be alternative approaches, such as a search for and design of agents that would selectively interfere with cholesterol-dependent virus–cell interactions and inhibit them. Understanding the mechanisms and consequences of these interactions should be useful in the developing of new antiviral drugs.

Cholesterol and PIP2 Modulation of BKCa Channels.

Ca2+/voltage-gated, large conductance K+ channels (BKCa) are formed by homotetrameric association of α (slo1) subunits. Their activity, however, is suited to tissue-specific physiology largely due to their association with regulatory subunits (β and γ types), chaperone proteins, localized signaling, and the channel's lipid microenvironment. PIP2 and cholesterol can modulate BKCa activity independently of downstream signaling, yet activating Ca2+i levels and regulatory subunits control ligand action. At physiological Ca2+i and voltages, cholesterol and PIP2 reduce and increase slo1 channel activity, respectively. Moreover, slo1 proteins provide sites that seem to recognize cholesterol and PIP2: seven CRAC motifs in the slo1 cytosolic tail and a string of positively charged residues (Arg329, Lys330, Lys331) immediately after S6, respectively. A model that could explain the modulation of BKCa activity by cholesterol and/or PIP2 is hypothesized. The roles of additional sites, whether in slo1 or BKCa regulatory subunits, for PIP2 and/or cholesterol to modulate BKCa function are also discussed.

Interaction of Peptides Containing CRAC Motifs with Lipids in Membranes of Various Composition

The lateral distribution of integral and peripheral proteins, as well as lipids in the plasma membranes of mammalian cells is extremely heterogeneous. It is believed that various lipid-protein domains are formed in membranes. Domains enriched in sphingomyelin and cholesterol are called rafts. It is assumed that the distribution of proteins into rafts is largely related to the presence in their primary sequence of a specific amino acid region called the CRAC motif, which is responsible for cholesterol binding. In this work, the interaction of two peptides containing CRAC motifs in their structure with membranes of different compositions was studied by means of molecular dynamics. It has been shown that the average number of lipid molecules in contact with each peptide is proportional to the mole fraction of lipid in the membrane. The predominant interaction of peptides with cholesterol was not observed. In addition, cholesterol did not form long-lived contacts with any amino acid or amino acid sequence. We suppose that in some cases the predominant lateral distribution of peptides and proteins containing CRAC motifs into rafts may be due to amphipathicity of the CRAC motif rather than due to specific strong binding of cholesterol.

Role of Cholesterol in Transmembrane Dimerization of the ErbB2 Growth Factor Receptor.

The association of ErbB2 growth factor receptors is critical for cell growth and potentiates tumor proliferation in several cancer types. An important aspect in ErbB2 association is the role of lipids such as cholesterol, especially since their metabolism is often reprogrammed in cancer cells. Here, we have coupled metadynamics with coarse-grain simulations to identify cholesterol effects in the transmembrane dimerization of ErbB2 receptors. Overall, cholesterol interactions are observed with the receptor that directly tunes the association energetics. Several dimer conformations are identified both in the presence and absence of cholesterol, although the dimer regime appears to be more favorable in the presence of cholesterol. We observe an overall modulation of the underlying energy profile and the symmetric active and inactive conformational states are not distinguished in the presence of cholesterol. We show that cholesterol binds to the receptor transmembrane domain at a site (CRAC motif) that overlaps with the dimer interface (SmXXXSm motif). The competition between the transmembrane interactions and cholesterol interactions decides the final conformational landscape. Our work is an important step toward characterizing cholesterol effects in ErbB2 membrane receptor function.

Prohibitin‐1 plays a multifaceted role in mediating luteinizing hormone‐induced steroidogenesis in Leydig cells.

Leydig cells (LCs) present in the interstitium between seminiferous tubules in the testis are responsible for the biosynthesis of testosterone from cholesterol in response to luteinizing hormone (LH) from the pituitary. The principal pathways of testosterone biosynthesis, the identities of the main steroidogenic enzymes, and genetic disorders of most of these enzymes have been elucidated. However, many important questions related to LH signaling and cholesterol transport remain unknown. For example, the mechanisms of StAR‐mediated cholesterol transport from the outer mitochondrial membrane to cytochrome P450 side chain cleavage (P450scc or Cyp11a1) enzyme in the inner mitochondrial membrane, which is a critical step in steroidogenesis, remain elusive. It is conceivable that we are missing some important players involved in this process.Prohibitin‐1 (PHB1) is a pleiotropic protein with cell compartment‐specific functions, including the phosphorylation‐dependent membrane signaling and mitochondrial chaperone, which appears to require heterodimerization with its homologous protein prohibitin‐2 (PHB2) in the inner mitochondrial membrane. Previously, we have reported transgenic mouse models expressing PHB1 (PHB‐Tg) or Y114F‐PHB1 (mPHB‐Tg) from the fatty acid binding protein‐4 (Fabp4) gene promoter. During their phenotypic characterization, unexpectedly, we found that male mPHB‐Tg mice, but not PHB‐Tg mice, have high testosterone levels. Subsequent analysis revealed that it was due to leaky overexpression of m/PHB1 in LCs. This prompted us to investigate whether LH regulates PHB1 in LCs. A dose and time‐dependent effect of human chorionic gonadotropin, hCG (pituitary analog of LH) was found on PHB1 expression levels in LCs. To define the cell compartment‐specific role of PHB1 in LCs, we investigated the functional status of LH signaling in testes/LCs and ultrastructure of LCs from transgenic mice. A difference in phospho‐ERK levels was found between PHB‐Tg and mPHB‐Tg, which was higher in mPHB1 expressing cells. A similar effect was found in PHB1 manipulated MA‐10 cells (a model LC line). A parallel change in ultrastructural features of LCs (mitochondrial structure and lipid droplets) was found in PHB and mPHB overexpressing cells. Moreover, co‐immunoprecipitation experiments showed that PHB1 and PHB2 interact with P450scc and/or StAR. Analysis of PHBs protein sequences revealed the presence of multiple CRAC and/or CARC motifs (cholesterol recognition motifs) in both proteins. In aggregate, this finding suggests that PHB1 is a LH regulated protein in LCs and plays a multifaceted role in LH‐induced steroidogenesis, including ERK activation and potentially in the functional coupling of StAR and P450scc across mitochondrial membrane for steroidogenesis. The scope of our findings is broad because the basics of cholesterol transport across mitochondrial membranes are similar in different steroidogenic tissues.

Structural Determinants of Cholesterol Recognition in Helical Membrane Proteins

Cholesterol is an integral component of mammalian membranes. It has been shown to modulate membrane dynamics and alter protein function. However, understanding the molecular mechanisms of how cholesterol alters protein function through specific interactions is complicated by our inability to differentiate cholesterol as a ligand and as a lipid in the membrane. Specifically, it is difficult to discern if a cholesterol-membrane protein interaction observed in an experimental structure is specific, unspecific, or a crystallization artefact. The only widely recognized search algorithm for cholesterol binding pockets is sequence-based, i.e. the CRAC and CARC motifs. While, both motifs have been identified on multiple proteins, there is inconclusive evidence on the necessity and sufficiency of its role in identifying cholesterol functional modifiers on its own. To elucidate cholesterol's role on the functional changes of an array of protein types, we characterized the rate of evolution of interacting residues, binding pocket residue types, and binding pocket spatial arrangement of high-resolution crystal structures of cholesterol-membrane protein complexes in the protein data bank (PDB). We compare the predictive power of this multi-layer analysis to the outcomes of the sequence-based CRAC and CARC motifs. Information gleaned from our characterization will eventually enable a structure-based approach for prediction and design of cholesterol binding sites.

Cholesterol-Recognition Motifs in Membrane Proteins.

The impact of cholesterol on the structure and function of membrane proteins was recognized several decades ago, but the molecular mechanisms underlying these effects have remained elusive. There appear to be multiple mechanisms by which cholesterol interacts with proteins. A complete understanding of cholesterol-sensing motifs is still undergoing refinement. Initially, cholesterol was thought to exert only non-specific effects on membrane fluidity. It was later shown that this lipid could specifically interact with membrane proteins and affect both their structure and function. In this article, we have summarized and critically analyzed our evolving understanding of the affinity, specificity and stereoselectivity of the interactions of cholesterol with membrane proteins. We review the different computational approaches that are currently used to identify cholesterol binding sites in membrane proteins and the biochemical logic that governs each type of site, including CRAC, CARC, SSD and amphipathic helix motifs. There are physiological implications of these cholesterol-recognition motifs for G-protein coupled receptors (GPCR) and ion channels, in membrane trafficking and membrane fusion (SNARE) proteins. There are also pathological implications of cholesterol binding to proteins involved in neurological disorders (Alzheimer, Parkinson, Creutzfeldt-Jakob) and HIV fusion. In each case, our discussion is focused on the key molecular aspects of the cholesterol and amino acid motifs in membrane-embedded regions of membrane proteins that define the physiologically relevant crosstalk between the two. Our understanding of the factors that determine if these motifs are functional in cholesterol binding will allow us enhanced predictive capabilities.

The Structure of Melanoregulin Reveals a Role for Cholesterol Recognition in the Protein's Ability to Promote Dynein Function

Melanoregulin (Mreg) is a small, highly charged, multiply palmitoylated protein present on the membrane of melanosomes. Mreg is implicated in the transfer of melanosomes from melanocytes to keratinocytes, and in promoting the microtubule minus end-directed transport of these organelles. The possible molecular function of Mreg was identified by solving its structure using nuclear magnetic resonance (NMR) spectroscopy. Mreg contains six α helices forming a fishhook-like fold in which positive and negative charges occupy opposite sides of the protein's surface and sandwich a putative, cholesterol recognition sequence (CRAC motif). Mreg containing a point mutation within its CRAC motif still targets to late endosomes/lysosomes, but no longer promotes their microtubule minus end-directed transport. Moreover, wild-type Mreg does not promote the microtubule minus end-directed transport of late endosomes/lysosomes in cells transiently depleted of cholesterol. Finally, reversing the charge of three clustered acidic residues partially inhibits Mreg's ability to drive these organelles to microtubule minus ends.

Molecular Dynamics Simulations Reveal the Role of Membrane Cholesterol during Pore Forming Pathway of Cytolysin A

Cytolysin A (ClyA), an α pore-forming toxin expressed by E. Coli as a water-soluble monomer, undergoes a large conformational change during pore formation. Although cholesterol is known to enhance the lytic activity of ClyA, molecular aspects of the interactions between cholesterol and ClyA during pore formation are not well understood. Using all-atom molecular dynamics simulations ranging from 0.5 - 0.9 µs, we study a single membrane-inserted protomer, a dimer (two protomers) and the dodecameric ClyA pore embedded in a DOPC+30% cholesterol bilayer. In the single membrane-inserted protomer, high cholesterol occupancy was observed around the transmembrane residues of N-terminus which form part of a CRAC motif and also around residues of the β-tongue. Although high cholesterol occupancy sites were not observed near the N-terminus in the dimer simulations, a cholesterol molecule was preferentially located in the pocket formed between two adjacent β-tongues of the dimer. Cholesterol spent 97% of the simulation time (600 ns) inside this pocket sampling two major orientations. Energies of two conformations were reported from docking simulations. Formation of transmembrane water channels were observed in both single membrane inserted and dimer ClyA simulations. From the dodecameric pore simulations, density map showed regions of high cholesterol population between the β-tongue pockets and mobility map indicated slower cholesterol in the vicinity of the pore as compared to bulk. Our simulations elucidate specific interactions with cholesterol that could stabilize both the single membrane inserted protomeric state as well as the dodecameric pore.

Computational Analysis of Cholesterol Binding and Pore-Lining Regions in Alpha-Synuclein: Role in Mitochondrial Function

Alpha-synuclein (α-syn) protein is the major component of Lewy bodies, which are characteristic pathological trademarks for neurodegenerative diseases (e.g. Parkinson’s and Alzheimer’s diseases). It is primarily expressed in neural tissue, with smaller amounts found in heart, muscle and other tissues. The canonical form found in Homo sapiens (α-syn-1) contains 140 residues and interacts with neuronal mitochondria via an N-terminal 32 residue mitochondrial-targeting signal. All isoforms (there are 3) have multiple highly conserved lipid binding (KTKE(Q)G(Q) V) motifs, thought to mediate binding to phospholipid membranes. Two isoforms also contain an EF-hand-like (helixloop- helix) sequence found in a large family of calcium-binding proteins, as well as three copper binding sites. We investigate protein topology using computational analysis and find that each isoform contains a pore-lining region, two cholesterol-binding (CRAC/CARC) and three or four lipid binding motifs, with one cholesterol motif overlapping the pore-lining region. Two lipid-binding motifs also overlap the N-terminal mitochondrial-targeting region consistent with evidence that α-syn inserts into mitochondrial inner membrane. α-Syn-1 reportedly occurs physiologically as a helically folded tetramer that requires N-terminal acetylation. Thus, each α-syn-1 tetramer could contain 4 mitochondrial targeting regions, up to 4 pore-lining regions, 4 EF-hand domains, 8 bound cholesterol molecules and 16 lipid binding motifs with pore-lining regions merging to form a membrane channel. Cholesterol binding to CRAC motifs may in turn facilitate protein folding, Ca2+-channel formation, as well as mitochondrial membrane lipid-protein interactions, altering mitochondrial bioenergetics. Disruption of mitochondrial bioenergetics may be involved in the pathogenesis of Alzheimer’s disease and Parkinsonism.

Mapping Cholesterol Interaction Sites on Serotonin Transporter through Coarse-Grained Molecular Dynamics.

Serotonin transporter (SERT) modulates serotonergic signaling via re-uptake of serotonin in pre-synaptic cells. The inclusion in cholesterol-enriched membrane domains is crucial for SERT activity, suggesting a cross-talk between the protein and the sterol. Here, we develop a protocol to identify potential cholesterol interaction sites coupling statistical analysis to multi-microsecond coarse-grained molecular dynamics simulations of SERT in a previously validated raft-like membrane model. Six putative sites were found, including a putative CRAC motif on TM4 and a CARC motif on TM10. Among them, four hot-spots near regions related to ion binding, transport, and inhibition were detected. Our results encourage prospective studies to unravel mechanistic features of the transporter and related drug discovery implications.

Cholesterol and Its Derivatives Reversibly Inhibit Proteinase K.

Microorganisms express a variety of proteases that degrade many proteins of the host body and subvert host immune response. While elucidating the mechanism/s of an immune stimulatory drug that contains bile lipid, regulation of proteolytic activity was investigated. The drug and bile lipids both stabilize Proteinase K, an aggressive protease of fungal origin against auto-digestion. Among the components of bile lipids, only cholesterol and its derivatives stabilize the enzyme. Biophysical evidences such as scattering of light, intrinsic and extrinsic fluorescence emission spectra, circular dichroism spectra, atomic force microscopy, and transmission electron microscopy images indicated that cholesterol and its derivatives interact with Proteinase K. Inhibition kinetics using esterolysis of ATEE revealed non-competitive inhibition by cholesterol. Surface Plasmon Resonance and mass spectrometric analysis indicated 1:1 stoichiometry of binding and with dissociation constant in the μM range. Further, the presence of four cholesterol recognition amino acid consensus motifs (CRAC motifs I-IV) was identified in Proteinase K. Bioinformatics analysis revealed that the whole stretch of cholesterol interacts very well with the hydrophobic groove of motif II only among the four CRAC motifs. Variation of cholesterol content of HepG2 human liver carcinoma cells showed positive correlation with binding of fluorescence tagged Proteinase K. Under these conditions, binding of Proteinase K to the cells did not affect their morphology, viability and growth kinetics. Cell bound Proteinase K could be released by an excess of its substrate, thereby restoring reversibly its proteolytic activity. J. Cell. Physiol. 232: 596-609, 2017. © 2016 Wiley Periodicals, Inc.

Cholesterol Interaction with the MAGUK Protein Family Member, MPP1, via CRAC and CRAC-Like Motifs: An In Silico Docking Analysis.

Cholesterol is essential for the proper organization of the biological membrane. Therefore, predicting which proteins can bind cholesterol is important in understanding how proteins participate in lateral membrane organization. In this study, a simple bioinformatics approach was used to establish whether MPP1, a member of the MAGUK protein family, is capable of binding cholesterol. Modelled and experimentally-validated fragment structures were mined from online resources and searched for CRAC and CRAC-like motifs. Several of these motifs were found in the primary structure of MPP1, and these were structurally visualized to see whether they localized to the protein surface. Since all of the CRAC and CRAC-like motifs were found at the surface of MPP1 domains, in silico docking experiments were performed to assess the possibility of interaction between CRAC motifs and cholesterol. The results obtained show that MPP1 can bind cholesterol via CRAC and CRAC-like motifs with moderate to high affinity (KI in the nano- to micro-molar range). It was also found that palmitoylation-mimicking mutations (C/F or C/M) did not affect the affinity of MPP1 towards cholesterol. Data presented here may help to understand at least one of the molecular mechanisms via which MPP1 affects lateral organization of the membrane.

Multiple Cholesterol Recognition/Interaction Amino Acid Consensus (CRAC) Motifs in Cytosolic C Tail of Slo1 Subunit Determine Cholesterol Sensitivity of Ca2+- and Voltage-gated K+ (BK) Channels

Large conductance, Ca(2+)- and voltage-gated K(+) (BK) channel proteins are ubiquitously expressed in cell membranes and control a wide variety of biological processes. Membrane cholesterol regulates the activity of membrane-associated proteins, including BK channels. Cholesterol modulation of BK channels alters action potential firing, colonic ion transport, smooth muscle contractility, endothelial function, and the channel alcohol response. The structural bases underlying cholesterol-BK channel interaction are unknown. Such interaction is determined by strict chemical requirements for the sterol molecule, suggesting cholesterol recognition by a protein surface. Here, we demonstrate that cholesterol action on BK channel-forming Cbv1 proteins is mediated by their cytosolic C tail domain, where we identified seven cholesterol recognition/interaction amino acid consensus motifs (CRAC4 to 10), a distinct feature of BK proteins. Cholesterol sensitivity is provided by the membrane-adjacent CRAC4, where Val-444, Tyr-450, and Lys-453 are required for cholesterol sensing, with hydrogen bonding and hydrophobic interactions participating in cholesterol location and recognition. However, cumulative truncations or Tyr-to-Phe substitutions in CRAC5 to 10 progressively blunt cholesterol sensitivity, documenting involvement of multiple CRACs in cholesterol-BK channel interaction. In conclusion, our study provides for the first time the structural bases of BK channel cholesterol sensitivity; the presence of membrane-adjacent CRAC4 and the long cytosolic C tail domain with several other CRAC motifs, which are not found in other members of the TM6 superfamily of ion channels, very likely explains the unique cholesterol sensitivity of BK channels.

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.

Structural Bases Supporting the Critical Role of the CRAC Motif Nearest to the N-End of Slo1 Cytosolic C-Tail in Determining the BK Channel Sensitivity to Membrane Cholesterol

We demonstrated that the CRAC domain nearest the N-end of BK channel-forming cbv1 subunits (CRAC4) is the main region that provides cholesterol (CLR) sensitivity to cbv1, the signature CRAC4 Y450 being critical. Here we combined amino acid substitutions in CRAC4, bilayer electrophysiology, and computational dynamics to advance structural bases of CRAC4-CLR interactions. First, trcbv1-CRAC4 constructs including V444A or K453A were insensitive to CLR. Second, trcbv1-CRAC4 K453R retained CLR sensitivity. Third, trcbv1-CRAC4 Y450F was CLR-resistant. However, Y429F in the S6-CRAC4 linker rendered CLR-sensitive channels. Therefore, CLR sensitivity of trcbv1-CRAC4 is determined by the key residues that define a CRAC motif (Epand, 2006). Moreover, the substitution Y450F in CRAC4 was sufficient to blunt the CLR sensitivity of full-length BK channels. Computational simulations of full-length cbv1 CTD and trcbv1 CTD-CRAC4 showed CLR remaining in CRAC4 for the majority of the simulation with CLR D ring near Y450 for over one-third of the simulation. CLR A ring resided predominantly near E417, which hydrogen bonded with CRAC4 through the Y450 hydroxyl, also indicating that CLR stayed close to CRAC4. Conversely, the highest residence for trcbv1 CTD-CRAC4 Y450F was for CLR D ring and tail, which remained near H409 and Y336, respectively, neither of which are part of CRAC4. Notably higher mobility of positions 450 and K453 in CRAC4 were also noted in the simulations of trcbv1-CRAC4 Y450F, indicating that a stable interaction site for CLR is lacking when the Y450 hydroxyl is not present to hydrogen bond to E417. Data provide a structural interpretation for the prominence of CRAC4 and its Y450 in providing CLR-sensitivity to trcbv1-CRAC4 and full-length BK channels. Support: R01-HL104631;R37-AA011560 (AMD); UTHSC NI Fellowship (AKS).

A CRAC-like motif in BAX sequence: Relationship with protein insertion and pore activity in liposomes

Several proteins that interact with cholesterol have a highly conserved sequence, corresponding to the cholesterol recognition/interaction amino acid consensus. Since cholesterol has been proposed to modulate both oligomerization and insertion of the pro-apoptotic protein BAX, we investigated the existence of such a motif in the BAX sequence. Residues 113 to 119 of the recombinant BAX α5-helix, LFYFASK, correspond with the sequence motif described for the consensus pattern, –L/V-(X)(1–5)-Y-(X)(1–5)-R/K. Functional characterization of the point mutations, K119A, Y115F, and L113A in BAX, was performed in liposomes supplemented with cholesterol, comparing binding, integration, and pore forming activities. Our results show that the mutations Y115F and L113A changed the cholesterol-dependent insertion observed in the wild type protein. In addition, substitutions in the BAX sequence modified the concentration dependency of carboxyfluorescein release in liposomes, although neither pore activity of the wild type or of any of the mutants significantly increased in cholesterol-enriched liposomes. Thus, while it is likely that the putative CRAC motif in BAX accounts for its enhanced insertion in cholesterol-enriched liposomes; the pore forming properties of BAX did not depend on cholesterol content in the membranes, albeit those mutations changed the pore channeling activity of the protein.