We apply pulsed dipolar ESR spectroscopy (Ku-band DEER) to elucidate the global conformation of the Parkinson's disease-associated protein, alpha-synuclein (αS) bound to small unilamellar phospholipid vesicles, rod-like SDS micelles, or lipid bicelles. By measuring distances as long as ~7 nm between introduced pairs of nitroxide spin labels, we show that distances are close to the expectations for a single continuous helix in all cases studied. In particular, we find distances of 7.5 nm between sites 24 and 72; 5.5 nm between sites 24 and 61; and 2 nm between sites 35 and 50. We conclude that αS does not retain a 'hairpin' structure with two antiparallel helices, as is known to occur with spheroidal micelles, in agreement with our earlier finding that the protein's geometry is determined by the surface topology rather than being constrained by the inter-helix linker. While the possibility of local helix discontinuities in the structure of membrane-bound αS remains, our data are more consistent with one intact helix. Importantly, we demonstrate that bicelles produce very similar results to liposomes, while offering a major improvement in experimentally accessible distance range and resolution, and thus are an excellent lipid membrane mimetic for the purpose of pulse dipolar ESR spectroscopy.Alpha-synuclein (αS) was originally discovered as a protein highly enriched in synaptosome preparations from the electric ray T. californica 1 and was later linked to both familial and sporadic Parkinson's disease (PD) through the discovery that αS point mutations or gene duplication/triplication cause familial PD and through the identification of αS as the major component of amyloid fibril aggregates present in the Lewy body deposits that are a diagnostic hallmark of PD. Both the normal function of αS and the precise relation between its aggregation and deposition in Lewy bodies and PD remain unclear. When isolated in solution, the protein is intrinsically disordered, but in the presence of lipid surfaces αS adopts a highly helical structure 2 that is believed to mediate its normal function(s). NMR-based characterization of this helical structure using detergent micelles as a membrane mimetic has shown that the protein E-mail: dae2005@med.cornell.edu; jhf@cornell.edu. adopts two extended surface-bound helices separated by a non-helical linker, that the helices are oriented in an antiparallel fashion, and that no inter-helical contacts are formed. 3-7 The slow tumbling rate of intact phospholipid vesicles precluded direct studies of the vesicle-bound conformation of αS using solution NMR methods, but it was proposed 3,8 that in the vesiclebound state, the two helices may become collinear and fuse into a single long surface-bound helix. Support for this possibility was provided by pulsed dipolar ESR (PDS) distance measurements of αS bound to different sized micelles, which showed that the helices splay further apart on the surface of larger micelles. 9 NIH Public AccessHere we use PDS, 10-13 namely 17.3 GHz DEER (cf. Su...

Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signaling and preventing glutamate-mediated excitotoxicity. Crystallographic studies on an archaeal homologue, GltPh, showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold. Here, we report direct observations of these 'elevator-like' transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that GltPh bearing two “humanizing” mutations exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motions and substrate uptake. Crystallographic and computational investigations reveal that these mutations favor structurally “unlocked” states with increased solvent occupancy at the interface between the transport domain and the trimeric scaffold.

Sodium and aspartate symporter from Pyrococcus horikoshii, GltPh, is a homologue of the mammalian glutamate transporters, homotrimeric integral membrane proteins controlling the neurotransmitter levels in brain synapses. These transporters function by alternating between outward and inward facing states, in which the substrate binding site is oriented toward the extracellular space and the cytoplasm, respectively. Here we employ double electron-electron resonance (DEER) spectroscopy to probe the structure and the state distribution of the subunits in the trimer within distinct hydrophobic environments of detergent micelles and lipid bilayers. Our experiments reveal a conformational ensemble of protomers sampling the outward and inward facing states with nearly equal probabilities, indicative of comparable energies, and independently of each other. On average, the distributions vary only modestly in detergent and in bilayers, but in several mutants unique conformations are stabilized by the latter.

Tau is a microtubule-associated protein that is genetically linked to dementia and linked to Alzheimer's disease via its presence in intraneuronal neurofibrillary tangle deposits, where it takes the form of aggregated paired helical and straight filaments. Although the precise mechanisms by which tau contributes to neurodegeneration remain unclear, tau aggregation is commonly considered to be a critical component of tau-mediated pathogenicity. Nevertheless, the context in which tau aggregation begins in vivo is unknown. Tau is enriched in membrane-rich neuronal structures such as axons and growth cones, and can interact with membranes both via intermediary proteins and directly via its microtubule-binding domain (MBD). Membranes efficiently facilitate tau aggregation in vitro, and may therefore provide a physiologically relevant context for nucleating tau aggregation in vivo. Furthermore, tau-membrane interactions may potentially play a role in tau's poorly understood normal physiological functions. Despite the potential importance of direct tau-membrane interactions for tau pathology and physiology, the structural mechanisms that underlie such interactions remain to be elucidated. Here, we employ electron spin resonance spectroscopy to investigate the secondary and long-range structural properties of the MBD of three-repeat tau isoforms when bound to lipid vesicles and membrane mimetics. We show that the membrane interactions of the tau MBD are mediated by short amphipathic helices formed within each of the MBD repeats in the membrane-bound state. To our knowledge, this is the first detailed elucidation of helical tau structure in the context of intact lipid bilayers. We further show, for the first time (to our knowledge), that these individual helical regions behave as independent membrane-binding sites linked by flexible connecting regions. These results represent the first (to our knowledge) detailed structural view of membrane-bound tau and provide insights into potential mechanisms for membrane-mediated tau aggregation. Furthermore, the results may have implications for the structural basis of tau-microtubule interactions and microtubule-mediated tau aggregation.

Viral fusogens merge viral and cell membranes during cell penetration. Their ectodomains drive fusion by undergoing large-scale refolding, but little is known about the functionally important regions located within or near the membrane. Here, we report the crystal structure of the full-length glycoprotein B, the fusogen from Herpes Simplex Virus, complemented by electron spin resonance measurements. The membrane-proximal (MPR), transmembrane (TMD), and cytoplasmic (CTD) domains form a uniquely folded trimeric pedestal beneath the ectodomain, which balances dynamic flexibility with extensive, stabilizing membrane interactions. Hyperfusogenic mutations within the CTD destabilize it, targeting trimeric interfaces, structural motifs, and membrane-interacting elements. Thus, we propose that the CTD trimer observed in the structure stabilizes gB in its prefusion state despite being appended to the postfusion ectodomain. Our data suggest a model for how this dynamic, membrane-dependent “clamp” controls the fusogenic refolding of gB.