Philip T. F. Williamson

The conformation of acetylcholine at its target site in the membrane-embedded nicotinic acetylcholine receptor

The conformation of the neurotransmitter acetylcholine bound to the fully functional nicotinic acetylcholine receptor embedded in its native membrane environment has been characterized by using frequency-selective recoupling solid-state NMR. Six dipolar couplings among five resolved 13C-labeled atoms of acetylcholine were measured. Bound acetylcholine adopts a bent conformation characterized with a quaternary ammonium-to-carbonyl distance of 5.1 Å. In this conformation, and with its orientation constrained to that previously determined by us, the acetylcholine could be docked satisfactorily in the agonist pocket of the agonist-bound, but not the agonist-free, crystal structure of a soluble acetylcholine-binding protein from Lymnaea stagnali. The quaternary ammonium group of the acetylcholine was determined to be within 3.9 Å of five aromatic residues and its acetyl group close to residues C187/188 of the principle and residue L112 of the complementary subunit. The observed >CO chemical shift is consistent with H bonding to the nicotinic acetylcholine receptor residues γY116 and δT119 that are homologous to L112 in the soluble acetylcholine-binding protein.

NMR of bicelles: orientation and mosaic spread of the liquid-crystal director under sample rotation

Model-membrane systems composed of liquid-crystalline bicellar phases can be uniaxially oriented with respect to a magnetic field, thereby facilitating structural and dynamics studies of membrane-associated proteins. Here we quantitatively characterize a method that allows the manipulation of the direction of this uniaxial orientation. Bicelles formed from DMPC/DHPC are examined by 31P NMR under variable-angle sample-spinning (VAS) conditions, confirming that the orientation of the liquid-crystalline director can be influenced by sample spinning. The director is perpendicular to the rotation axis when Θ (the angle between the sample-spinning axis and the magnetic field direction) is smaller than the magic angle, and is parallel to the rotation axis when Θ is larger than the magic angle. The new 31P NMR VAS data presented are considerably more sensitive to the orientation of the bicelle than earlier 2H studies and the analysis of the sideband pattern allows the determination of the orientation of the liquid-crystal director and its variation over the sample, i.e., the mosaic spread. Under VAS, the mosaic spread is small if Θ deviates significantly from the magic angle but becomes very large at the magic angle.

Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers

A structural look at α-synuclein oligomers Fibrillar aggregates of the protein α-synuclein (αS) are the major constituents of Lewy bodies in Parkinsons disease. However, small oligomers that accumulate during the process of fibril formation are thought to cause the neuronal toxicity associated with the onset and progression of Parkinsons disease. Little is known about the detailed structural properties of αS oligomers and the molecular mechanisms that lead to their toxicity. Fusco et al. report the structural characterization of two forms of αS oligomers, which elucidates the fundamental structural elements giving rise to neuronal toxicity. Science, this issue p. 1440 Oligomers of α-synuclein generate neuronal damage when insertion of a highly structured core disrupts membrane integrity. Oligomeric species populated during the aggregation process of α-synuclein have been linked to neuronal impairment in Parkinson’s disease and related neurodegenerative disorders. By using solution and solid-state nuclear magnetic resonance techniques in conjunction with other structural methods, we identified the fundamental characteristics that enable toxic α-synuclein oligomers to perturb biological membranes and disrupt cellular function; these include a highly lipophilic element that promotes strong membrane interactions and a structured region that inserts into lipid bilayers and disrupts their integrity. In support of these conclusions, mutations that target the region that promotes strong membrane interactions by α-synuclein oligomers suppressed their toxicity in neuroblastoma cells and primary cortical neurons.

Switched-angle spinning applied to bicelles containing phospholipid-associated peptides

In a model study, the proton NMR spectrum of the opioid pentapeptide leucine-enkephalin associated with bicelles is investigated. The spectral resolution for a static sample is limited due to the large number of anisotropic interactions, in particular strong proton–proton couplings, but resolution is greatly improved by magic-angle sample spinning. Here we present two-dimensional switched-angle spinning NMR experiments, which correlate the high-resolution spectrum of the membrane-bound peptide under magic-angle spinning with its anisotropic spectrum, leading to well-resolved spectra. The two-dimensional spectrum allows the exploitation of the high resolution of the isotropic spectrum, while retaining the structural information imparted by the anisotropic interactions in the static spectrum. Furthermore, switched-angle spinning techniques are demonstrated that allow one to record the proton spectrum of ordered bicellar phases as a function of the angle between the rotor axis and the magnetic field direction, thereby scaling the dipolar interactions by a predefined factor.

A mirror code for protein-cholesterol interactions in the two leaflets of biological membranes

Cholesterol controls the activity of a wide range of membrane receptors through specific interactions and identifying cholesterol recognition motifs is therefore critical for understanding signaling receptor function. The membrane-spanning domains of the paradigm neurotransmitter receptor for acetylcholine (AChR) display a series of cholesterol consensus domains (referred to as “CARC”). Here we use a combination of molecular modeling, lipid monolayer/mutational approaches and NMR spectroscopy to study the binding of cholesterol to a synthetic CARC peptide. The CARC-cholesterol interaction is of high affinity, lipid-specific, concentration-dependent, and sensitive to single-point mutations. The CARC motif is generally located in the outer membrane leaflet and its reverse sequence CRAC in the inner one. Their simultaneous presence within the same transmembrane domain obeys a “mirror code” controlling protein-cholesterol interactions in the outer and inner membrane leaflets. Deciphering this code enabled us to elaborate guidelines for the detection of cholesterol-binding motifs in any membrane protein. Several representative examples of neurotransmitter receptors and ABC transporters with the dual CARC/CRAC motifs are presented. The biological significance and potential clinical applications of the mirror code are discussed.

Lipid Concentration and Molar Ratio Boundaries for the Use of Isotropic Bicelles

Bicelles are model membranes generally made of long-chain dimyristoylphosphatidylcholine (DMPC) and short-chain dihexanoyl-PC (DHPC). They are extensively used in the study of membrane interactions and structure determination of membrane-associated peptides, since their composition and morphology mimic the widespread PC-rich natural eukaryotic membranes. At low DMPC/DHPC (q) molar ratios, fast-tumbling bicelles are formed in which the DMPC bilayer is stabilized by DHPC molecules in the high-curvature rim region. Experimental constraints imposed by techniques such as circular dichroism, dynamic light scattering, or microscopy may require the use of bicelles at high dilutions. Studies have shown that such conditions induce the formation of small aggregates and alter the lipid-to-detergent ratio of the bicelle assemblies. The objectives of this work were to determine the exact composition of those DMPC/DHPC isotropic bicelles and study the lipid miscibility. This was done using 31P nuclear magnetic resonance (NMR) and exploring a wide range of lipid concentrations (2–400 mM) and q ratios (0.15–2). Our data demonstrate how dilution modifies the actual DMPC/DHPC molar ratio in the bicelles. Care must be taken for samples with a total lipid concentration ≤250 mM and especially at q ∼ 1.5–2, since moderate dilutions could lead to the formation of large and slow-tumbling lipid structures that could hinder the use of solution NMR methods, circular dichroism or dynamic light scattering studies. Our results, supported by infrared spectroscopy and molecular dynamics simulations, also show that phospholipids in bicelles are largely segregated only when q > 1. Boundaries are presented within which control of the bicelles’ q ratio is possible. This work, thus, intends to guide the choice of q ratio and total phospholipid concentration when using isotropic bicelles.

Probing the environment of neurotensin whilst bound to the neurotensin receptor by solid state NMR

A functionally active analogue of neurotensin, neurotensin(8–13), has been observed whilst bound to the agonist‐binding site of the rat neurotensin receptor by nuclear magnetic resonance (NMR). Through the application of slow magic angle sample spinning and high‐power proton decoupling, sufficient resolution and sensitivity were obtained in the carbon‐13 spectrum to allow an assignment of many of the side chain resonances arising from uniformly carbon‐13/nitrogen‐15‐labelled neurotensin(8–13) whilst bound to the neurotensin receptor. Significant perturbations in carbon‐13 chemical shift were observed upon the binding of the neurotensin(8–13) to the receptor. Most importantly significant shifts were observed in both the carboxy terminus and tyrosine side chain of the neurotensin(8–13), suggesting that these sites are important in the interaction of the neurotensin with the agonist‐binding site on the neurotensin receptor. Conversely, no perturbations were observed for the carbon‐13 sites within the guanidinium groups of the arginine side chains, indicating little interaction with the receptor‐binding site, or a shielding of the local environment by the surrounding nitrogen atoms. These NMR observations lend further support to previous structure–activity studies, site‐directed mutagenesis and modelling studies of the agonist‐binding site of the neurotensin receptor, from which the same specific residues for which NMR perturbations were observed are important for neurotensin receptor activation by neurotensin.

Dynamics and orientation of N+(CD3)(3)-bromoacetylcholine bound to its binding site on the nicotinic acetylcholine receptor

Dynamic and structural information has been obtained for an analogue of acetylcholine while bound to the agonist binding site on the nicotinic acetylcholine receptor (nAcChoR), using wide-line deuterium solid-state NMR. Analysis of the deuterium lineshape obtained at various temperatures from unoriented nAcChoR membranes labeled with deuterated bromoacetylcholine (BAC) showed that the quaternary ammonium group of the ligand is well constrained within the agonist binding site when compared with the dynamics observed in the crystalline solids. This motional restriction would suggest that a high degree of complementarity exists between the quaternary ammonium group of the ligand and the protein within the agonist binding site. nAcChoR membranes were uniaxially oriented by isopotential centrifugation as determined by phosphorous NMR of the membrane phospholipids. Analysis of the deuterium NMR lineshape of these oriented membranes enriched with the nAcChoR labeled with N+(CD3)3-BAC has enabled us to determine that the angle formed between the quaternary ammonium group of the BAC and the membrane normal is 42° in the desensitized form of the receptor. This measurement allows us to orient in part the bound ligand within the proposed receptor binding site.

Probing the interaction of lipids with the non-annular binding sites of the potassium channel KcsA by magic-angle spinning NMR

The activity of the potassium channel KcsA is tightly regulated through the interactions of anionic lipids with high-affinity non-annular lipid binding sites located at the interface between the channels subunits. Here we present solid-state phosphorous NMR studies that resolve the negatively charged lipid phosphatidylglycerol within the non-annular lipid-binding site. Perturbations in chemical shift observed upon the binding of phosphatidylglycerol are indicative of the interaction of positively charged sidechains within the non-annular binding site and the negatively charged lipid headgroup. Site directed mutagenesis studies have attributed these charge interactions to R64 and R89. Functionally the removal of the positive charges from R64 and R89 appears to act synergistically to reduce the probability of channel opening.

Stability and membrane orientation of the fukutin transmembrane domain: a combined multiscale molecular dynamics and circular dichroism study.

The N-terminal domain of fukutin-I has been implicated in the localization of the protein in the endoplasmic reticulum and Golgi Apparatus. It has been proposed to mediate this through its interaction with the thinner lipid bilayers found in these compartments. Here we have employed multiscale molecular dynamics simulations and circular dichroism spectroscopy to explore the structure, stability, and orientation of the short 36-residue N-terminus of fukutin-I (FK1TMD) in lipids with differing tail lengths. Our results show that FK1TMD adopts a stable helical conformation in phosphatidylcholine lipids when oriented with its principal axis perpendicular to the bilayer plane. The stability of the helix is largely insensitive to the lipid tail length, preventing hydrophobic mismatch by virtue of its mobility and ability to tilt within the lipid bilayers. This suggests that changes in FK1TMD tilt in response to bilayer properties may be implicated in the regulation of its trafficking. Coarse-grained simulations of the complex Golgi membrane suggest the N-terminal domain may induce the formation of microdomains in the surrounding membrane through its preferential interaction with 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol 4,5-bisphosphate lipids.