Direct Observation of Cholesterol Dimers and Tetramers in Lipid Bilayers.

Cholesterol is a ubiquitous component of mammalian cell membranes and affects membrane protein function. Although cholesterol-mediated formation of ordered membrane domains has been extensively studied, molecular-level structural information about cholesterol self-association has been absent. Here, we combine solid-state nuclear magnetic resonance (NMR) spectroscopy with all-atom molecular dynamics simulations to determine the oligomeric structure of cholesterol in phospholipid bilayers. Two-dimensional 13C-13C correlation spectra of differentially labeled cholesterol indicate that cholesterol self-associates in a face-to-face fashion at membrane concentrations from 17 to 44 mol %. 2D 13C and 19F spin-counting experiments allowed us to measure the average oligomeric number of these cholesterol clusters. At low cholesterol concentrations of ∼20%, the average cluster size is centered on dimers. At a high cholesterol concentration of 44%, which is representative of virus lipid envelopes and liquid-ordered domains of cell membranes, both dimers and tetramers are observed. The cholesterol dimers are found in both phase-separated membranes that contain sphingomyelin and in disordered and miscible membranes that are free of sphingomyelin. Molecular dynamics simulations support these experimental observations and moreover provide the lifetimes, stabilities, distributions, and structures of these nanoscopic cholesterol clusters. Taken together, these NMR and MD data strongly suggest that dimers are the basic structural unit of cholesterol in phospholipid bilayers. The direct observation of cholesterol dimers and tetramers provides a revised framework for studying cholesterol interactions with membrane proteins to regulate protein functions and for understanding the pathogenic role of cholesterol in diseases.

Cholesterol Interaction with the Trimeric HIV Fusion Protein gp41 in Lipid Bilayers Investigated by Solid-State NMR Spectroscopy and Molecular Dynamics Simulations.

HIV-1 entry into cells is mediated by the fusion protein gp41. Cholesterol plays an important role in this virus-cell fusion, but molecular structural information about cholesterol-gp41 interaction is so far absent. Here, we present experimental and computational data about cholesterol complexation with gp41 in lipid bilayers. We focus on the C-terminal region of the protein, which comprises a membrane-proximal external region (MPER) and the transmembrane domain (TMD). We measured peptide-cholesterol contacts in virus-mimetic lipid bilayers using solid-state NMR spectroscopy, and augmented these experimental data with all-atom molecular dynamics simulations. 2D 19F NMR spectra show correlation peaks between MPER residues and the cholesterol isooctyl tail, indicating that cholesterol is in molecular contact with the MPER-TMD trimer. 19F-13C distance measurements between the peptide and 13C-labeled cholesterol show that C17 on the D ring and C9 at the intersection of B and C rings are ~7.0 Å from the F673 side-chain 4-19F. At high peptide concentrations in the membrane, the 19F-13C distance data indicate three cholesterol molecules bound near F673 in each trimer. Mutation of a cholesterol recognition amino acid consensus motif did not change these distances, indicating that cholesterol binding does not require this sequence motif. Molecular dynamics simulations further identify two hotspots for cholesterol interactions. Taken together, these experimental data and simulations indicate that the helix-turn-helix conformation of the MPER-TMD is responsible for sequestering cholesterol. We propose that this gp41-cholesterol interaction mediates virus-cell fusion by recruiting gp41 to the boundary of the liquid-disordered and liquid-ordered phases to incur membrane curvature.

In vitro 0N4R tau fibrils contain a monomorphic ß-sheet core enclosed by dynamically heterogeneous fuzzy coat segments.

Misfolding of the microtubule-binding protein tau into filamentous aggregates is characteristic of many neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Determining the structures and dynamics of these tau fibrils is important for designing inhibitors against tau aggregation. Tau fibrils obtained from patient brains have been found by cryo-electron microscopy to adopt disease-specific molecular conformations. However, in vitro heparin-fibrillized 2N4R tau, which contains all four microtubule-binding repeats (4R), was recently found to adopt polymorphic structures. Here we use solid-state NMR spectroscopy to investigate the global fold and dynamics of heparin-fibrillized 0N4R tau. A single set of 13C and 15N chemical shifts was observed for residues in the four repeats, indicating a single ß-sheet conformation for the fibril core. This rigid core spans the R2 and R3 repeats and adopts a hairpin-like fold that has similarities to but also clear differences from any of the polymorphic 2N4R folds. Obtaining a homogeneous fibril sample required careful purification of the protein and removal of any proteolytic fragments. A variety of experiments and polarization transfer from water and mobile side chains indicate that 0N4R tau fibrils exhibit heterogeneous dynamics: Outside the rigid R2-R3 core, the R1 and R4 repeats are semirigid even though they exhibit ß-strand character and the proline-rich domains undergo large-amplitude anisotropic motions, whereas the two termini are nearly isotropically flexible. These results have significant implications for the structure and dynamics of 4R tau fibrils in vivo.

Elucidating ligand-bound structures of membrane proteins using solid-state NMR spectroscopy.

Magic-angle-spinning (MAS) solid-state NMR spectroscopy is a versatile technique to elucidate functionally important protein-ligand interactions in lipid membranes. Here, we review recent solid-state NMR studies of membrane protein interactions with cholesterol, lipids, transported substrates, and peptide ligands. These studies are conducted in synthetic or native lipid bilayers to provide an accurate environment for ligand binding. The solid-state NMR approaches include multinuclear detection to gain comprehensive structural information, distance measurements to locate ligand-binding sites, and dynamic nuclear polarization and 1H detection to enhance spectral sensitivity. These studies provide novel insights into the mechanisms of virus budding, virus entry into cells, transmembrane signaling, substrate transport, antibacterial action, and many other biological processes.

Determining Cholesterol Binding to Membrane Proteins by Cholesterol 13C Labeling in Yeast and Dynamic Nuclear Polarization NMR.

We present a general strategy for determining the cholesterol-binding site of eukaryotic membrane proteins in native-like lipid membranes by NMR spectroscopy. The strategy combines yeast biosynthetic 13C enrichment of cholesterol with detection of protein-cholesterol 13C-13C cross peaks in 2D correlation NMR spectra under the dynamic nuclear polarization (DNP) condition. Low-temperature DNP not only allows high-sensitivity detection of weak protein-cholesterol cross peaks in 2D spectra but also immobilizes cholesterol and protein to enable intermolecular distance measurements. We demonstrate this approach on the influenza M2 protein, which utilizes cholesterol to conduct membrane scission in the last step of virus budding and release from the host cell plasma membrane. A 13C-13C double-quantum filter was employed to significantly simplify the 2D 13C-13C correlation spectra and facilitate the identification of protein-cholesterol cross peaks. A number of cross peaks between the M2 transmembrane residues' side chains and the cholesterol sterol group were detected, which complement recently measured protein contacts to the isooctyl tail of cholesterol to define an extended binding interface. These results provide atomic-level evidence of M2-cholesterol interaction to cause membrane curvature and scission, and the approach is generally applicable to other eukaryotic membrane proteins for understanding the influence of cholesterol on membrane protein function.

Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR.

The influenza M2 protein not only forms a proton channel but also mediates membrane scission in a cholesterol-dependent manner to cause virus budding and release. The atomic interaction of cholesterol with M2, as with most eukaryotic membrane proteins, has long been elusive. We have now determined the cholesterol-binding site of the M2 protein in phospholipid bilayers using solid-state NMR spectroscopy. Chain-fluorinated cholesterol was used to measure cholesterol proximity to M2 while sterol-deuterated cholesterol was used to measure bound-cholesterol orientation in lipid bilayers. Carbon-fluorine distance measurements show that at a cholesterol concentration of 17 mol%, two cholesterol molecules bind each M2 tetramer. Cholesterol binds the C-terminal transmembrane (TM) residues, near an amphipathic helix, without requiring a cholesterol recognition sequence motif. Deuterium NMR spectra indicate that bound cholesterol is approximately parallel to the bilayer normal, with the rough face of the sterol rings apposed to methyl-rich TM residues. The distance- and orientation-restrained cholesterol-binding site structure shows that cholesterol is stabilized by hydrophobic interactions with the TM helix and polar and aromatic interactions with neighboring amphipathic helices. At the 1:2 binding stoichiometry, lipid 31P spectra show an isotropic peak indicative of high membrane curvature. This M2-cholesterol complex structure, together with previously observed M2 localization at phase boundaries, suggests that cholesterol mediates M2 clustering to the neck of the budding virus to cause the necessary curvature for membrane scission. The solid-state NMR approach developed here is generally applicable for elucidating the structural basis of cholesterol's effects on membrane protein function.

Structural Polymorphism of Alzheimer's ß-Amyloid Fibrils as Controlled by an E22 Switch: A Solid-State NMR Study.

The amyloid-ß (Aß) peptide of Alzheimer's disease (AD) forms polymorphic fibrils on the micrometer and molecular scales. Various fibril growth conditions have been identified to cause polymorphism, but the intrinsic amino acid sequence basis for this polymorphism has been unclear. Several single-site mutations in the center of the Aß sequence cause different disease phenotypes and fibrillization properties. The E22G (Arctic) mutant is found in familial AD and forms protofibrils more rapidly than wild-type Aß. Here, we use solid-state NMR spectroscopy to investigate the structure, dynamics, hydration and morphology of Arctic E22G Aß40 fibrils. (13)C, (15)N-labeled synthetic E22G Aß40 peptides are studied and compared with wild-type and Osaka E22Δ Aß40 fibrils. Under the same fibrillization conditions, Arctic Aß40 exhibits a high degree of polymorphism, showing at least four sets of NMR chemical shifts for various residues, while the Osaka and wild-type Aß40 fibrils show a single or a predominant set of chemical shifts. Thus, structural polymorphism is intrinsic to the Arctic E22G Aß40 sequence. Chemical shifts and inter-residue contacts obtained from 2D correlation spectra indicate that one of the major Arctic conformers has surprisingly high structural similarity with wild-type Aß42. (13)C-(1)H dipolar order parameters, (1)H rotating-frame spin-lattice relaxation times and water-to-protein spin diffusion experiments reveal substantial differences in the dynamics and hydration of Arctic, Osaka and wild-type Aß40 fibrils. Together, these results strongly suggest that electrostatic interactions in the center of the Aß peptide sequence play a crucial role in the three-dimensional fold of the fibrils, and by inference, fibril-induced neuronal toxicity and AD pathogenesis.