The Structural Basis of Cholesterol Accessibility in Membranes

Although the majority of free cellular cholesterol is present in the plasma membrane, cholesterol homeostasis is principally regulated through sterol-sensing proteins that reside in the cholesterol-poor endoplasmic reticulum (ER). In response to acute cholesterol loading or depletion, there is rapid equilibration between the ER and plasma membrane cholesterol pools, suggesting a biophysical model in which the availability of plasma membrane cholesterol for trafficking to internal membranes modulates ER membrane behavior. Previous studies have predominantly examined cholesterol availability in terms of binding to extramembrane acceptors, but have provided limited insight into the structural changes underlying cholesterol activation. In this study, we use both molecular dynamics simulations and experimental membrane systems to examine the behavior of cholesterol in membrane bilayers. We find that cholesterol depth within the bilayer provides a reasonable structural metric for cholesterol availability and that this is correlated with cholesterol-acceptor binding. Further, the distribution of cholesterol availability in our simulations is continuous rather than divided into distinct available and unavailable pools. This data provide support for a revised cholesterol activation model in which activation is driven not by saturation of membrane-cholesterol interactions but rather by bulk membrane remodeling that reduces membrane-cholesterol affinity.     

Cholesterol Induces Specific Spatial and Orientational Order in Cholesterol/Phospholipid Membranes

Background

In lipid bilayers, cholesterol facilitates the formation of the liquid-ordered phase and enables the formation of laterally ordered structures such as lipid rafts. While these domains have an important role in a variety of cellular processes, the precise atomic-level mechanisms responsible for cholesterol''s specific ordering and packing capability have remained unresolved.

Methodology/Principal Findings

Our atomic-scale molecular dynamics simulations reveal that this ordering and the associated packing effects in membranes largely result from cholesterol''s molecular structure, which differentiates cholesterol from other sterols. We find that cholesterol molecules prefer to be located in the second coordination shell, avoiding direct cholesterol-cholesterol contacts, and form a three-fold symmetric arrangement with proximal cholesterol molecules. At larger distances, the lateral three-fold organization is broken by thermal fluctuations. For other sterols having less structural asymmetry, the three-fold arrangement is considerably lost.

Conclusions/Significance

We conclude that cholesterol molecules act collectively in lipid membranes. This is the main reason why the liquid-ordered phase only emerges for Chol concentrations well above 10 mol% where the collective self-organization of Chol molecules emerges spontaneously. The collective ordering process requires specific molecular-scale features that explain why different sterols have very different membrane ordering properties: the three-fold symmetry in the Chol-Chol organization arises from the cholesterol off-plane methyl groups allowing the identification of raft-promoting sterols from those that do not promote rafts.     

Molecular Organization of Cholesterol in Polyunsaturated Membranes: Microdomain Formation

The molecular organization of cholesterol in phospholipid bilayers composed of 1,2-diarachidonylphosphatidylcholine (20:4-20:4PC), 1-stearoyl-2-arachidonylphosphatidylcholine (18:0-20:4PC), and 20:4-20:4PC/18:0-20:4PC (1/1 mol) was investigated by solid-state ²H NMR and by low- and wide-angle x-ray diffraction (XRD). On the basis of distinct quadrupolar powder patterns arising from [3α-²H₁]cholesterol intercalated into the membrane and phase separated as solid, solubility χ_chol^NMR = 17 ± 2 mol% and tilt angle α₀ = 25 ± 1° in 20:4-20:4PC were determined. The corresponding values in 18:0-20:4PC were χ_chol^NMR ≥ 50 mol% and α₀ = 16 ± 1°. Cholesterol solubility determined by XRD was χ_chol^XRD = 15 ± 2 mol% and χ_chol^XRD = 49 ± 1 mol% for 20:4-20:4PC and 18:0-20:4PC, respectively. XRD experiments show that the solid sterol is monohydrate crystals presumably residing outside the bilayer. The ²H NMR spectrum for equimolar [3α-²H₁]cholesterol added to mixed 20:4-20:4PC/18:0-20:4PC (1/1 mol) membranes is consistent with segregation of cholesterol into 20:4-20:4PC and 18:0-20:4PC microdomains of <160 Å in size that preserve the molecular organization of sterol in the individual phospholipid constituents. Our results demonstrate unambiguously that cholesterol has low affinity to polyunsaturated fatty acids and support hypotheses of lateral phase separation of membrane constituents into sterol-poor/polyunsaturated fatty acid-rich and sterol-rich/saturated fatty acid-rich microdomains.     

Detection of Cholesterol in Cell Membranes by Use of Bacterial Toxins

A method is described for the detection of cholesterol in membranes from erythrocytes, mycoplasmas, and bacterial cells by a ferritin-labeling technique. Membranes treated with cereolysin, a bacterial hemolysin which specifically binds to cholesterol, and then treated with ferritin-antitetanolysin, were specifically ferritin-labeled for cholesterol. A similar antigen-antibody system, streptolysin O-ferritin-antistreptolysin, was also used successfully with erythrocyte membranes. There was an uneven distribution of ferritin in erythrocyte membranes suggesting that the distribution of cholesterol may not be entirely random. Mycoplasma gallisepticum was intensely labeled, but Acholeplasma laidlawii with or without cholesterol in the membranes was not labeled, suggesting an unusual location for cholesterol in A. laidlawii membranes. As controls, two of three species of bacterial membranes lacking cholesterol were not ferritin-labeled.     

Structural Basis for Ca2+-mediated Interaction of the Perforin C2 Domain with Lipid Membranes

Natural killer cells and cytotoxic T-lymphocytes deploy perforin and granzymes to kill infected host cells. Perforin, secreted by immune cells, binds target membranes to form pores that deliver pro-apoptotic granzymes into the target cell. A crucial first step in this process is interaction of its C2 domain with target cell membranes, which is a calcium-dependent event. Some aspects of this process are understood, but many molecular details remain unclear. To address this, we investigated the mechanism of Ca²⁺ and lipid binding to the C2 domain by NMR spectroscopy and x-ray crystallography. Calcium titrations, together with dodecylphosphocholine micelle experiments, confirmed that multiple Ca²⁺ ions bind within the calcium-binding regions, activating perforin with respect to membrane binding. We have also determined the affinities of several of these binding sites and have shown that this interaction causes a significant structural rearrangement in CBR1. Thus, it is proposed that Ca²⁺ binding at the weakest affinity site triggers changes in the C2 domain that facilitate its interaction with lipid membranes.     

Ultrafast Diffusion of a Fluorescent Cholesterol Analog in Compartmentalized Plasma Membranes

Cholesterol distribution and dynamics in the plasma membrane (PM) are poorly understood. The recent development of Bodipy488‐conjugated cholesterol molecule (Bdp‐Chol) allowed us to study cholesterol behavior in the PM, using single fluorescent‐molecule imaging. Surprisingly, in the intact PM, Bdp‐Chol diffused at the fastest rate ever found for any molecules in the PM, with a median diffusion coefficient (D) of 3.4 µm²/second, which was ～10 times greater than that of non‐raft phospholipid molecules (0.33 µm²/second), despite Bdp‐Chol's probable association with raft domains. Furthermore, Bdp‐Chol exhibited no sign of entrapment in time scales longer than 0.5 milliseconds. In the blebbed PM, where actin filaments were largely depleted, Bdp‐Chol and Cy3‐conjugated dioleoylphosphatidylethanolamine (Cy3‐DOPE) diffused at comparable Ds (medians = 5.8 and 6.2 µm²/second, respectively), indicating that the actin‐based membrane skeleton reduces the D of Bdp‐Chol only by a factor of ～2 from that in the blebbed PM, whereas it reduces the D of Cy3‐DOPE by a factor of ～20. These results are consistent with the previously proposed model, in which the PM is compartmentalized by the actin‐based membrane‐skeleton fence and its associated transmembrane picket proteins for the macroscopic diffusion of all of the membrane molecules, and suggest that the probability of Bdp‐Chol passing through the compartment boundaries, once it enters the boundary, is ～10× greater than that of Cy3‐DOPE. Since the compartment sizes are greater than those of the putative raft domains, we conclude that raft domains coexist with membrane‐skeleton‐induced compartments and are contained within them.      

Phospholipid Chain Interactions with Cholesterol Drive Domain Formation in Lipid Membranes

Cholesterol is a key component of eukaryotic membranes, but its role in cellular biology in general and in lipid rafts in particular remains controversial. Model membranes are used extensively to determine the phase behavior of ternary mixtures of cholesterol, a saturated lipid, and an unsaturated lipid with liquid-ordered and liquid-disordered phase coexistence. Despite many different experiments that determine lipid-phase diagrams, we lack an understanding of the molecular-level driving forces for liquid phase coexistence in bilayers with cholesterol. Here, we use atomistic molecular dynamics computer simulations to address the driving forces for phase coexistence in ternary lipid mixtures. Domain formation is directly observed in a long-timescale simulation of a mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine, unsaturated 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, and cholesterol. Free-energy calculations for the exchange of the saturated and unsaturated lipids between the ordered and disordered phases give insight into the mixing behavior. We show that a large energetic contribution to domain formation is favorable enthalpic interactions of the saturated lipid in the ordered phase. This favorable energy for forming an ordered, cholesterol-rich phase is opposed by a large unfavorable entropy. Martini coarse-grained simulations capture the unfavorable free energy of mixing but do not reproduce the entropic contribution because of the reduced representation of the phospholipid tails. Phospholipid tails and their degree of unsaturation are key energetic contributors to lipid phase separation.     

Structural Basis of Drug Binding to CYP46A1, an Enzyme That Controls Cholesterol Turnover in the Brain

Cytochrome P450 46A1 (CYP46A1) initiates the major pathway of cholesterol elimination from the brain and thereby controls cholesterol turnover in this organ. We determined x-ray crystal structures of CYP46A1 in complex with four structurally distinct pharmaceuticals; antidepressant tranylcypromine (2.15 Å), anticonvulsant thioperamide (1.65 Å), antifungal voriconazole (2.35 Å), and antifungal clotrimazole (2.50 Å). All four drugs are nitrogen-containing compounds that have nanomolar affinity for CYP46A1 in vitro yet differ in size, shape, hydrophobicity, and type of the nitrogen ligand. Structures of the co-complexes demonstrate that each drug binds in a single orientation to the active site with tranylcypromine, thioperamide, and voriconazole coordinating the heme iron via their nitrogen atoms and clotrimazole being at a 4 Å distance from the heme iron. We show here that clotrimazole is also a substrate for CYP46A1. High affinity for CYP46A1 is determined by a set of specific interactions, some of which were further investigated by solution studies using structural analogs of the drugs and the T306A CYP46A1 mutant. Collectively, our results reveal how diverse inhibitors can be accommodated in the CYP46A1 active site and provide an explanation for the observed differences in the drug-induced spectral response. Co-complexes with tranylcypromine, thioperamide, and voriconazole represent the first structural characterization of the drug binding to a P450 enzyme.     

The Interaction of the Polyene Antibiotic Lucensomycin with Cholesterol in Erythrocyte Membranes and in Model Systems: II. Cooperative Effects in Erythrocyte Membranes

Fluorometric titration curves of erythrocyte membranes with increasing lucensomycin have a sigmoid shape. This behavior, which was not present when colloidal cholesterol suspensions were used, is, however, not peculiar to the membrane structure, being also present in cholesterol-containing phospholipid micelles. Addition of acetic acid induced or increased sigmoidicity. This behavior can either be due to a true cooperativity in binding or to different fluorescence yields of the various lucensomycin-membrane complexes. The latter hypothesis appears to be slightly favored.     

蛋白质磷酸化作用的结构基础

蛋白质可逆磷酸化涉及到几乎所有细胞活动的调节.着重探讨了影响蛋白激酶作用专一性的几个因素和磷酸化影响蛋白质功能的结构基础及作用机制.     

来源于红球菌胆固醇氧化酶ChOG的异源表达、纯化及催化反应结构分析

胆固醇氧化酶是胆固醇代谢过程中的关键酶,临床上用胆固醇氧化酶作为检测血清胆固醇含量的应用潜力巨大。将来源于红球菌Rhodococcus ruber的胆固醇氧化酶Ch OG,分别转化到大肠杆菌宿主BL21(DE3)和Rosetta(DE3)中,在不同条件下进行诱导表达,结果表明:BL21(DE3)菌株在诱导温度为16℃、IPTG浓度为0.1 mmol/L时,Ch OG可溶性表达量最高(0.49 mg/ml)。Ch OG的最适反应温度为30℃,最适反应p H为7.5。最适反应条件下,酶活性达到8.0 U/mg。利用TLC、HPLC对Ch OG催化产物胆甾-4-烯-3-酮进行了鉴定分析。三维结构及定点突变分析表明Glu406及Arg408、Glu261在进行胆固醇C3羟基的脱氢、质子传递,以及底物异构化方面发挥着重要作用。     

Noninvasive Neutron Scattering Measurements Reveal Slower Cholesterol Transport in Model Lipid Membranes

Proper cholesterol transport is essential to healthy cellular activity and any abnormality can lead to several fatal diseases. However, complete understandings of cholesterol homeostasis in the cell remains elusive, partly due to the wide variability in reported values for intra- and intermembrane cholesterol transport rates. Here, we used time-resolved small-angle neutron scattering to measure cholesterol intermembrane exchange and intramembrane flipping rates, in situ, without recourse to any external fields or compounds. We found significantly slower transport kinetics than reported by previous studies, particularly for intramembrane flipping where our measured rates are several orders of magnitude slower. We unambiguously demonstrate that the presence of chemical tags and extraneous compounds employed in traditional kinetic measurements dramatically affect the system thermodynamics, accelerating cholesterol transport rates by an order of magnitude. To our knowledge, this work provides new insights into cholesterol transport process disorders, and challenges many of the underlying assumptions used in most cholesterol transport studies to date.     

The Structural Basis of ATP as an Allosteric Modulator

Adenosine-5’-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems.