The role of cholesterol binding in the control of cholesterol by the Scap-Insig system.

Scap and Insig, two proteins embedded in the membrane of the endoplasmic reticulum (ER), regulate the synthesis of cholesterol in animal cells by forming a dimer in the presence of high concentrations of cholesterol. Cryo-electron microscopic structures for the Scap-Insig dimer show a sterol-binding site at the dimer interface, but none of the structures include cholesterol itself. Here, a molecular docking approach developed to characterise cholesterol binding to the transmembrane (TM) regions of membrane proteins is used to characterise cholesterol binding to sites on the TM surface of the dimer and to the interfacial binding site. Binding of cholesterol is also observed at sites on the extra-membranous luminal domains of Scap, but the properties of these sites suggest that they will be unoccupied in vivo. Comparing the structure of Scap in the dimer with that predicted by AlphaFold for monomeric Scap suggests that dimer formation could result in relocation of TM helix 7 of Scap and of the loop between TM6 and 7, and that this could be the key change on Scap that signals that there is a high concentration of cholesterol in the ER.

Interfacial binding sites for cholesterol on GABAA receptors and competition with neurosteroids.

γ-Aminobutyric acid type A (GABAA) receptors in the brain are located in the outer membranes of brain cells where the concentration of cholesterol is high. Of the 25 available high-resolution structures available for GABAA receptors, none were determined in the presence of cholesterol, but four include resolved molecules of cholesterol hemisuccinate (CHS). Here, a molecular docking procedure is used to sweep the transmembrane (TM) surfaces of the receptors for cholesterol binding sites. Cholesterol docking poses determined in this way match 89% of the resolved CHS when CHS molecules deemed unlikely to represent typical bound cholesterols are excluded. The receptors are pentameric, and their TM surfaces consist of a set of five facets, each including pairs of TM helices from two adjacent subunits. Each facet contains hydrophobic hollows running from one side of the membrane to the other, within which are six potential binding sites for cholesterol, three on each side of the membrane. High-resolution structures of GABAA receptors with bound neurosteroids show that neurosteroids bind in these cholesterol binding sites, so the binding of neurosteroids and cholesterol will be competitive.

Interfacial Binding Sites for Cholesterol on Kir, Kv, K2P, and Related Potassium Channels.

Inwardly rectifying, voltage-gated, two-pore domain, and related K+ channels are located in eukaryotic membranes rich in cholesterol. Here, molecular docking is used to detect specific binding sites ("hot spots") for cholesterol on K+ channels with characteristics that match those of known cholesterol binding sites. The transmembrane surfaces of all available high-resolution structures for K+ channels were swept for potential binding sites. Cholesterol poses were found to be located largely in hollows between protein ridges. A comparison between cholesterol poses and resolved phospholipids suggests that not all cholesterol molecules binding to the transmembrane surface of a K+ channel will result in displacement of a phospholipid molecule from the surface. Competition between cholesterol binding and binding of anionic phospholipids essential for activity could explain some of the effects of cholesterol on channel function.

Interfacial Binding Sites for Cholesterol on TRP Ion Channels.

Transient receptor potential (TRP) channels are members of a large family of ion channels located in membranes rich in cholesterol, some of whose functions are affected by the cholesterol content of the membrane. Here, cholesterol binding to TRPs is studied using a docking procedure that allows the transmembrane surface of a TRP to be swept rapidly for potential binding sites at the interfaces on the two sides of the membrane. Cholesterol docking poses determined in this way match 89% of the cholesterol hemisuccinate molecules in published TRP structures when cholesterol hemisuccinate molecules unlikely to represent typical bound cholesterols are excluded. TRPs are tetrameric, with large clefts at the interfaces between subunits; cholesterol poses are located in hollows, largely within these clefts. Comparison of cholesterol poses with phospholipid binding sites suggests that binding of cholesterol to a TRP need not result in displacement of phospholipid molecules from the TRP surface.

Interfacial Binding Sites for Cholesterol on G Protein-Coupled Receptors.

A docking procedure is described that allows the transmembrane surface of a G protein-coupled receptor (GPCR) to be swept rapidly for potential binding sites for cholesterol at the bilayer interfaces on the two sides of the membrane. The procedure matches 89% of the cholesterols resolved in published GPCR crystal structures, when cholesterols likely to be crystal packing artifacts are excluded. Docking poses are shown to form distinct clusters on the protein surface, the clusters corresponding to "greasy hollows" between protein ridges. Docking poses depend on the angle of tilt of the GPCR in the surrounding lipid bilayer. It is suggested that thermal motion could alter the optimal binding pose for a cholesterol molecule, with the range of binding poses within a cluster providing a guide to the range of thermal motions likely for a cholesterol within a binding site.

A Database of Predicted Binding Sites for Cholesterol on Membrane Proteins, Deep in the Membrane.

The outer membranes of animal cells contain high concentrations of cholesterol, of which a small proportion is located deep within the hydrophobic core of the membrane. An automated docking procedure is described that allows the characterization of binding sites for these deep cholesterol molecules on the membrane-spanning surfaces of membrane proteins and in protein cavities or pores, driven by hydrogen bond formation. A database of this class of predicted binding site is described, covering 397 high-resolution structures. The database includes sites on the transmembrane surfaces of many G-protein coupled receptors; within the fenestrations of two-pore K+ channels and ATP-gated P2X3 channels; in the central cavities of a number of transporters, including Glut1, Glut5, and P-glycoprotein; and in deep clefts in mitochondrial complexes III and IV.

G protein coupled receptor interactions with cholesterol deep in the membrane.

G protein coupled receptors (GPCRs) are located in membranes rich in cholesterol. The membrane spanning surfaces of GPCRs contain exposed backbone carbonyl groups and residue side chains potentially capable of forming hydrogen bonds to cholesterol molecules buried deep within the hydrophobic core of the lipid bilayer. Coarse-grained molecular dynamics (CGMD) simulations allow the observation of GPCRs in cholesterol-containing lipid bilayers for long times (50µs), sufficient to ensure equilibration of the system. We have detected a number of deep cholesterol binding sites on ß2 adrenergic and A2A adenosine receptors, and shown changes in these sites on agonist binding. The requirements for binding are modest, just a potential hydrogen bond partner close to a cleft or hole in the surface. This makes it likely that similar binding sites for cholesterol will exist on other classes of membrane protein.

Biological membranes: the importance of molecular detail.

Are lipid interactions with membrane proteins best described in terms of the physical properties of the lipid bilayer or in terms of direct molecular interactions between particular lipid molecules and particular sites on a protein? A molecular interpretation is more challenging because it requires detailed knowledge of the 3D structure of a membrane protein, but recent studies have suggested that a molecular interpretation is necessary. Here, the idea is explored that lipid molecules modify the ways that transmembrane α-helices pack into bundles, by penetrating between the helices and by binding into clefts between the helices, and that these effects on helix packing will modulate the activity of a membrane protein.

The effects of sarcolipin over-expression in mouse skeletal muscle on metabolic activity.

Studies in sarcolipin knockout mice have led to the suggestion that skeletal muscle sarcolipin plays a role in thermogenesis. The mechanism proposed is uncoupling of the sarcoplasmic reticulum calcium pump. However, in other work sarcolipin was not detected in mouse skeletal tissue. We have therefore measured sarcolipin levels in mouse skeletal muscle using semi-quantitative western blotting and synthetic mouse sarcolipin. Sarcolipin levels were so low that it is unlikely that knocking out sarcolipin would have a measurable effect on thermogenesis by SERCA. In addition, overexpression of neither wild type nor FLAG-tagged variants of mouse sarcolipin in transgenic mice had any major significant effects on body mass, energy expenditure, even when mice were fed on a high fat diet.

Effects of lipid structure on the state of aggregation of potassium channel KcsA.

The state of aggregation of potassium channel KcsA was determined as a function of lipid:protein molar ratio in bilayer membranes of the zwitterionic lipid phosphatidylcholine (PC) and of the anionic lipid phosphatidylglycerol (PG). EPR (electron paramagnetic resonance) with spin-labeled phospholipids was used to determine the number of motionally restricted lipids per KcsA tetramer. Unexpectedly, this number decreased with a decreasing lipid:KcsA tetramer molar ratio in the range of 88:1 to 30:1, consistent with sharing of annular lipid shells and KcsA-KcsA contact at high mole fractions of protein. Fluorescence quenching experiments with brominated phospholipids showed a decrease in fluorescence quenching at low lipid:KcsA tetramer mole ratios, also consistent with KcsA-KcsA contact at high mole fractions of protein. The effects of low mole ratios of lipid seen in EPR and fluorescence quenching experiments were more marked in bilayers of PC than in bilayers of PG, suggesting stronger association of PG than PC with KcsA. This was confirmed by direct measurement of lipid association constants using spin-labeled phospholipids, showing higher association constants for all anionic lipids than for PC. The results show that the probability of contacts between KcsA tetramers will be very low at lipid:protein molar ratios that are typical of native biological membranes.

Characterizing the fatty acid binding site in the cavity of potassium channel KcsA.

We show that interactions of fatty acids with the central cavity of potassium channel KcsA can be characterized using the fluorescence probe 11-dansylaminoundecanoic acid (Dauda). The fluorescence emission spectrum of Dauda bound to KcsA in bilayers of dioleoylphosphatidylcholine contains three components, which can be attributed to KcsA-bound and lipid-bound Dauda together with unbound Dauda. The binding of Dauda to KcsA was characterized by a dissociation constant of 0.47 ± 0.10 µM with 0.94 ± 0.06 binding site per KcsA tetramer. Displacement of KcsA-bound Dauda by the tetrabutylammonium (TBA) ion confirmed that the Dauda binding site was in the central cavity of KcsA. Dissociation constants for a range of fatty acids were determined by displacement of Dauda: binding of fatty acids increased in strength with an increasing chain length from C14 to C20 but then decreased in strength from C20 to C22. Increasing the number of double bonds in the chain from one to four had little effect on binding, dissociation constants for oleic acid and arachidonic acid, for example, being 2.9 ± 0.2 and 3.0 ± 0.4 µM, respectively. Binding of TBA to KcsA was very slow, whereas binding of Dauda was fast, suggesting that TBA can enter the cavity only through an open channel whereas Dauda can bind to the closed channel, presumably entering the cavity via the lipid bilayer.

Multiple binding sites for fatty acids on the potassium channel KcsA.

Interactions of fatty acids with the potassium channel KcsA were studied using Trp fluorescence quenching and electron paramagnetic resonance (EPR) techniques. The brominated analogue of oleic acid was shown to bind to annular sites on KcsA and to the nonannular sites at each protein-protein interface in the homotetrameric structure with binding constants relative to dioleoylphosphatidylcholine of 0.67 ± 0.04 and 0.87 ± 0.08, respectively. Mutation of the two Arg residues close to the nonannular binding sites had no effect on fatty acid binding. EPR studies with a spin-labeled analogue of stearic acid detected a high-affinity binding site for the fatty acid with strong immobilization. Fluorescence quenching studies with the spin-labeled analogue showed that the binding site detected in the EPR experiments could not be one of the annular or nonannular binding sites. Instead, it is proposed that the EPR studies detect binding to the central hydrophobic cavity of the channel, with a binding constant in the range of ~0.1-1 µM.

The localization of the ER retrieval sequence for the calcium pump SERCA1.

A number of studies using chimeric constructs made by fusing endoplasmic/sarcoplasmic reticulum calcium pump (SERCA) sequences with those of the plasma membrane located calcium pump (PMCA) have suggested that the retention/retrieval signal responsible for maintaining SERCA in the endoplasmic reticulum (ER) is located within the N-terminus of these pumps. Because of the difficulties in identifying the presence of constructs at the plasma membrane we have used a trans-Golgi network (TGN) marker to evaluate whether chimeric proteins are retained by the ER or have lost their retention/retrieval sequences and are able to enter the wider endomembrane system and reach the TGN. In this study, attempts to locate this retention/retrieval sequence demonstrate that the retention sequences are located not in the N-terminus, as previously suggested, but in the largely transmembranous C-terminal domain of SERCA. Further attempts to identify the precise retention/retrieval motif using SERCA1/PMCA3 chimeras were unsuccessful. This may be due to the fact that introducing SERCA1 sequences into the C-terminal PMCA3 sequence and vice versa disrupts the organization of the closely packed transmembrane helices leading to retention of such constructs by the quality control mechanisms of the ER. An alternative explanation is that SERCAs have targeting motifs that are non-linear, being made up of several segments of sequence to form a patch that interacts with the retrieval machinery.

Retrieval from the ER-golgi intermediate compartment is key to the targeting of c-terminally anchored ER-resident proteins.

Endoplasmic reticulum (ER) resident proteins may be maintained in the ER by retention, where the leak into post-ER compartments is absent or slow, or retrieval, where a significant leak is countered by retrieval from post-ER compartments. Here the targeting of the C-terminally anchored protein ER-resident protein, cytochrome b5a (cytb5a), considered to be maintained in the ER mainly by the process of retention, is compared with that of sarcolipin (SLN) and phospholamban (PLB); also C-terminally anchored ER-residents. Laser confocal microscopy, and cell fractionation of green fluorescent protein-tagged constructs expressed in COS 7 cells indicate that while calnexin appears to be retained in the ER with no evidence of leak into the ER-Golgi intermediate compartment (ERGIC), significant amounts of cytb5a, SLN, and PLB are detectable in the ERGIC, indicating that there is considerable leak from the ER. This is supported by an in vitro budding assay that shows that while small amounts of calnexin appear in the transport vesicles budding off from the ER, significant amounts of cytb5a and SLN are found in such vesicles. These data support the hypothesis that retrieval plays a major role in ensuring that C-terminally anchored proteins are maintained in the ER.

Lipid-protein interactions.

Intrinsic membrane proteins are solvated by a shell of lipid molecules interacting with the membrane-penetrating surface of the protein; these lipid molecules are referred to as annular lipids. Lipid molecules are also found bound between transmembrane α-helices; these are referred to as non-annular lipids. Annular lipid binding constants depend on fatty acyl chain length, but the dependence is less than expected from models based on distortion of the lipid bilayer alone. This suggests that hydrophobic matching between a membrane protein and the surrounding lipid bilayer involves some distortion of the transmembrane α-helical bundle found in most membrane proteins, explaining the importance of bilayer thickness for membrane protein function. Annular lipid binding constants also depend on the structure of the polar headgroup region of the lipid, and hotspots for binding anionic lipids have been detected on some membrane proteins; binding of anionic lipid molecules to these hotspots can be functionally important. Binding of anionic lipids to non-annular sites on membrane proteins such as the potassium channel KcsA can also be important for function. It is argued that the packing preferences of the membrane-spanning α-helices in a membrane protein result in a structure that matches nicely with that of the surrounding lipid bilayer, so that lipid and protein can meet without either having to change very much.

Binding of anionic lipids to at least three nonannular sites on the potassium channel KcsA is required for channel opening.

In addition to the annular or boundary lipids that surround the transmembrane surface of the potassium channel KcsA from Streptomyces lividans, x-ray crystallographic studies have detected one anionic lipid molecule bound at each protein-protein interface in the homotetrameric structure, at sites referred to as nonannular sites. The binding constant for phosphatidylglycerol at the nonannular sites has been determined using fluorescence quenching methods with a mutant of KcsA lacking the normal three lipid-exposed Trp residues. Binding is weak, with a binding constant of 0.42 +/- 0.06 in units of mol fraction, implying that the nonannular sites will only be approximately 70% occupied in bilayers of 100% phosphatidylglycerol. However, the nonannular sites show high selectivity for anionic lipids over zwitterionic lipids, and it is suggested that a change in packing at the protein-protein interface leads to a closing of the nonannular binding site in the unbound state. Increasing the anionic lipid content of the membrane leads to a large increase in open channel probability, from approximately 2.5% in the presence of 25 mol % phosphatidylglycerol to approximately 62% in 100 mol % phosphatidylglycerol. The relationship between open channel probability and phosphatidylglycerol content shows cooperativity. The data are consistent with a model in which three or four of the four nonannular sites in the KcsA homotetramer have to be occupied by anionic lipid for the channel to open. The conductance of the open channel increases with increasing concentration of anionic lipid, an effect possibly due to effects of anionic lipid on the concentration of K(+) close to the membrane surface.

Importance of direct interactions with lipids for the function of the mechanosensitive channel MscL.

We have studied the effects of lipid structure on the function of the mechanosensitive channel of large conductance (MscL) from Escherichia coli to determine whether effects follow from direct interaction between the lipids and protein or whether they follow indirectly from changes in the curvature stress in the membrane. The G22C mutant of MscL was reconstituted into sealed vesicles containing the fluorescent molecule calcein, and the release of calcein from the vesicles was measured following opening of the channel by reaction with [2-(triethylammonium)ethyl] methanethiosulfonate (MTSET), which introduces five positive charges into the region of the pore constriction. The presence of anionic lipids in the vesicle membrane changed the rates and amplitudes of calcein release, the effects not correlating with calculated changes in lipid spontaneous curvature. Mutation of charged residues in the Arg-104, Lys-105, Lys-106 cluster removed high-affinity binding of anionic lipids to MscL, and the presence of anionic lipid no longer affected calcein flux through MscL. Changing the zwitterionic lipid from phosphatidylcholine to phosphatidylethanolamine resulted in a large decrease in the rate of calcein release, the change in rate varying linearly with lipid composition, as expected if spontaneous curvature affected the rate of release. However, rates of release of calcein measured in the presence of phosphatidylethanolamine- N-methyl and phosphatidylethanolamine- N, N-dimethyl did not fit the correlation between rate and curvature established for the phosphatidylcholine/phosphatidylethanolamine mixtures. Rather, the effects of zwitterionic lipid headgroup on calcein flux suggested that what was important was the presence of a proton in the headgroup, able to take part in hydrogen bonding to MscL. We conclude that the function of MscL is likely to be modulated by direct interaction with the surrounding, annular phospholipids that contact the protein in the membrane.

Lipid sorting: lipids do it on their own.

How are lipid molecules sorted between organelles in eukaryotic cells? A recent paper shows that the work needed to bend a membrane and form a vesicle is sufficient to sort lipid molecules.

Phospholamban and sarcolipin are maintained in the endoplasmic reticulum by retrieval from the ER-Golgi intermediate compartment.

OBJECTIVE: Phospholamban and sarcolipin are small transmembrane proteins that modulate cardiac contractility through their interaction with the sarcoplasmic reticulum (SR) calcium pumps (SERCAs). We have examined the hypothesis that phospholamban and sarcolipin are maintained in the SR by a process of retrieval from post-SR compartments and the role of their transmembrane domains in targeting. METHODS: Antibodies directed against phospholamban and protein markers of the endoplasmic reticulum/Golgi intermediate compartment (ERGIC) and the trans-Golgi were used in fluorescence microscopy studies of cultured human fetal cardiac myocytes. In addition, sarcolipin and phospholamban were tagged at the N-terminus with enhanced-green-fluorescent protein (EGFP) and expressed in COS 7 cells. The EGFP-tagged constructs were localised using fluorescence microscopy and cell fractionation. The length of the transmembrane domains of phospholamban and sarcolipin were extended and the effect on cellular location was also examined. RESULTS: In fetal cardiac myocytes phospholamban was located in the SR and the ERGIC, but did not migrate to the trans-Golgi network. Tagged-phospholamban and sarcolipin were located in the endoplasmic reticulum (ER) of COS 7 cells indicating that their targeting was unaffected by the EGFP tag. Significant proportions of the tagged phospholamban and sarcolipin were also located in the ERGIC but not in the trans-Golgi. Increasing the length of the transmembranous domains of EGFP-tagged phospholamban and sarcolipin resulted in their mis-targeting to the plasma membrane. CONCLUSIONS: Phospholamban and sarcolipin are maintained in the SR/ER by a process that includes their retrieval from the ERGIC following their passage from the SR/ER into the ERGIC. The transmembrane domains of phospholamban and sarcolipin are involved in the retrieval process.