␣-Synuclein is known to play a causative role in Parkinson disease. Although its physiological functions are not fully understood, ␣-synuclein has been shown to interact with synaptic vesicles and modulate neurotransmitter release. However, the structure of its physiologically relevant membrane-bound state remains unknown. Here we developed a site-directed spin labeling and EPR-based approach for determining the structure of ␣-synuclein bound to a lipid bilayer. Continuous-wave EPR was used to assign local secondary structure and to determine the membrane immersion depth of lipid-exposed residues, whereas pulsed EPR was used to map long-range distances. The structure of ␣-synuclein was built and refined by using simulated annealing molecular dynamics restrained by the immersion depths and distances. We found that ␣-synuclein forms an extended, curved ␣-helical structure that is over 90 aa in length. The monomeric helix has a superhelical twist similar to that of right-handed coiled-coils which, like ␣-synuclein, contain 11-aa repeats, but which are soluble, oligomeric proteins (rmsd ‫؍‬ 0.82 Å). The ␣-synuclein helix extends parallel to the curved membrane in a manner that allows conserved Lys and Glu residues to interact with the zwitterionic headgroups, while uncharged residues penetrate into the acyl chain region. This structural arrangement is significantly different from that of ␣-synuclein in the presence of the commonly used membranemimetic detergent SDS, which induces the formation of two antiparallel helices. Our structural analysis emphasizes the importance of studying membrane protein structure in a bilayer environment.EPR ͉ Parkinson's disease ͉ fibril-forming proteins ͉ 11-aa repeats T he interaction of ␣-synuclein with membranes is thought to be important in its physiologic function in vivo, as well as in its misfolding and aggregation in the pathogenesis of Parkinson disease (1-10). Although the function of ␣-synuclein in vivo is not fully understood, it has been observed to localize to presynaptic nerve termini, where it modulates presynaptic pool size and neurotransmitter release (11-16). These functions are likely to be mediated by the interaction of ␣-synuclein with synaptic vesicles, and in vitro studies have shown that ␣-synuclein interacts strongly with highly curved vesicles that are similar in size to synaptic vesicles (17, 18). The structural characterization of membrane-bound ␣-synuclein is significant, given the importance of membrane interactions to the pathologic and physiologic roles of ␣-synuclein.Previous studies have revealed that the interaction of monomeric ␣-synuclein with negatively charged vesicles induces a predominantly ␣-helical structure located in the N-terminal region of the protein (17,19,20). This region contains seven 11-aa-repeat regions that share some sequence similarity with apolipoproteins [supporting information (SI) Fig. S1]. Sequence analysis using algorithms for apolipoproteins predicts the formation of five separate helices (17). However, no high-resolutio...

A spectrum of membrane curvatures exists within cells, and proteins have evolved different modules to detect, create, and maintain these curvatures. Here we present the crystal structure of one such module found within human FCHo2. This F-BAR (extended FCH) module consists of two F-BAR domains, forming an intrinsically curved all-helical antiparallel dimer with a Kd of 2.5 microM. The module binds liposomes via a concave face, deforming them into tubules with variable diameters of up to 130 nm. Pulse EPR studies showed the membrane-bound dimer is the same as the crystal dimer, although the N-terminal helix changed conformation on membrane binding. Mutation of a phenylalanine on this helix partially attenuated narrow tubule formation, and resulted in a gain of curvature sensitivity. This structure shows a distant relationship to curvature-sensing BAR modules, and suggests how similar coiled-coil architectures in the BAR superfamily have evolved to expand the repertoire of membrane-sculpting possibilities.

Background: Human islet amyloid polypeptide (hIAPP) fibrils of unknown structure are formed in type 2 diabetes. Results: A hIAPP fibril structure was derived from EPR data, electron microscopy, and computer modeling. Conclusion:The fibril is a left-handed helix that contains hIAPP monomers in a staggered conformation. Significance: The results provide the basis for therapeutic prevention of fibril formation and growth.

Partially folded proteins, characterized as exhibiting secondary structure elements with loose or absent tertiary contacts, represent important intermediates in both physiological protein folding and pathological protein misfolding. To aid in the characterization of the structural state(s) of such proteins, a novel structure calculation scheme is presented that combines structural restraints derived from pulsed EPR and NMR spectroscopy. The methodology is established for the protein α-synuclein (αS), which exhibits characteristics of a partially folded protein when bound to a micelle of the detergent sodium lauroyl sarcosinate (SLAS). By combining 18 EPR-derived interelectron spin label distance distributions with NMR-based secondary structure definitions and bond vector restraints, interelectron distances were correlated and a set of theoretical ensemble basis populations was calculated. A minimal set of basis structures, representing the partially folded state of SLAS-bound αS, was subsequently derived by back-calculating correlated distance distributions. A surprising variety of well-defined protein-micelle interactions was thus revealed in which the micelle is engulfed by two differently arranged anti-parallel αS helices. The methodology further provided the population ratios between dominant ensemble structural states, whereas limitation in obtainable structural resolution arose from spin label flexibility and residual uncertainties in secondary structure definitions. To advance the understanding of protein-micelle interactions, the present study concludes by showing that, in marked contrast to secondary structure stability, helix dynamics of SLAS-bound αS correlate with the degree of protein-induced departures from free micelle dimensions.

Membrane remodeling is controlled by proteins that can promote the formation of highly curved spherical or cylindrical membranes. How a protein induces these different types of membrane curvature and how cells regulate this process is still unclear. Endophilin A1 is a protein involved in generating endocytotic necks and vesicles during synaptic endocytosis and can transform large vesicles into lipid tubes or small and highly curved vesicles in vitro. By using EM and electron paramagnetic resonance of endophilin A1, we find that tubes are formed by a close interaction with endophilin A1's BIN/amphiphysin/Rvs (BAR) domain and deep insertion of its amphipathic helices. In contrast, vesicles are predominantly stabilized by the shallow insertion of the amphipathic helical wedges with the BAR domain removed from the membrane. By showing that the mechanism of membrane curvature induction is different for vesiculation and tubulation, these data also explain why previous studies arrived at different conclusions with respect to the importance of scaffolding and wedging in the membrane curvature generation of BAR proteins. The Parkinson disease-associated kinase LRRK2 phosphorylates S75 of endophilin A1, a position located in the acyl chain region on tubes and the aqueous environment on vesicles. We find that the phosphomimetic mutation S75D favors vesicle formation by inhibiting this conformational switch, acting to regulate endophilin A1-mediated curvature. As endophilin A1 is part of a protein superfamily, we expect these mechanisms and their regulation by posttranslational modifications to be a general means for controlling different types of membrane curvature in a wide range of processes in vivo.site-directed spin labeling | double electron-electron resonance N umerous cellular remodeling events are controlled by proteins that can regulate membrane shape (1). In the example of synaptic endocytosis, proteins must engender invagination, drive pit formation, stabilize neck structures, and ultimately cause fission. These steps are executed through spatial and temporal application of membrane-altering proteins that contribute a range of curvatures (2, 3). Moreover, misregulated expression or posttranslational modification of these proteins is implicated in a number of diseases (4-7).An increasing body of evidence suggests that endophilin plays an essential role in synaptic endocytosis by recruiting cofactors such as dynamin (8-10) and synaptojanin (11-14) as well as by inducing membrane curvature (15-18). It has been observed to localize to the synaptic vesicle pool and endocytotic neck regions in vivo and generate small highly curved vesicles and lipid tubes from large vesicles in vitro (8,9,11,19). It has thus been proposed that a main component of the function of endophilin in vivo is its ability to regulate membrane curvature.In general, proteins generate curvature through several mechanisms: by forcing membranes to conform to their own intrinsic protein shape (i.e., scaffolding) (3), inserting amphipathic segments in...