Endophilin-A1 is a BAR domain-containing protein enriched at synapses and is implicated in synaptic vesicle endocytosis. It binds to dynamin and synaptojanin via a C-terminal SH3 domain. We examine the mechanism by which the BAR domain and an N-terminal amphipathic helix, which folds upon membrane binding, work as a functional unit (the N-BAR domain) to promote dimerisation and membrane curvature generation. By electron paramagnetic resonance spectroscopy, we show that this amphipathic helix is peripherally bound in the plane of the membrane, with the midpoint of insertion aligned with the phosphate level of headgroups. This places the helix in an optimal position to effect membrane curvature generation. We solved the crystal structure of rat endophilin-A1 BAR domain and examined a distinctive insert protruding from the membrane interaction face. This insert is predicted to form an additional amphipathic helix and is important for curvature generation. Its presence defines an endophilin/nadrin subclass of BAR domains. We propose that N-BAR domains function as low-affinity dimers regulating binding partner recruitment to areas of high membrane curvature.

␣-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...

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Many of the proposed physiological functions of ␣-synuclein, a protein involved in the pathogenesis of Parkinson's disease, are related to its ability to interact with phospholipids. To better understand the conformational changes that occur upon membrane binding of monomeric ␣-synuclein, we performed EPR analysis of 47 singly labeled ␣-synuclein derivatives. We show that membrane interaction is mediated by major conformational changes within seven N-terminal 11-aa repeats, which reorganize from a highly dynamic structure into an elongated helical structure devoid of significant tertiary packing. Furthermore, we find that analogous positions from different repeats are in equivalent locations with respect to membrane proximity. These and other findings suggest a curved membrane-dependent ␣-helical structure, wherein each 11-aa repeat takes up three helical turns. Similar helical structures could also apply to apolipoproteins and other lipid-interacting proteins with related 11-aa repeats.T he protein ␣-synuclein is the main component of Lewy bodies, a class of intracellular inclusions that is highly characteristic in Parkinson's disease (PD) (1). A causative role of ␣-synuclein in PD has been supported by genetic studies of familial forms of this disease (2-4) as well as by various animal models (5). In addition to its involvement in PD, ␣-synuclein may also play important roles in Alzheimer's disease, dementia with Lewy bodies, multiple system atrophy, and Hallervorden-Spatz syndrome (6, 7).Although not yet fully understood, the physiological function of ␣-synuclein is likely to involve a role in modulating synaptic plasticity (8), presynaptic vesicle pool size, and neurotransmitter release (9-11), as well as vesicle recycling (12). In agreement with these membrane-related functions, ␣-synuclein has been shown to interact with liposomes in vitro (13-16). According to circular dichroism analysis, this interaction causes ␣-synuclein to undergo a conformational change from an unstructured monomer in solution (13,17,18) to an ␣-helical, membrane-bound protein. Based upon sequence analysis, it was recognized early on that the N-terminal portion of ␣-synuclein was likely to mediate lipid interaction (8,13). The N terminus of ␣-synuclein contains seven repeats, each of which is made up of 11 aa (Fig. 1). These repeats are similar to those found in apolipoproteins, and it was proposed that the lipid interaction of ␣-synuclein could be similar to that of the apolipoproteins (8, 13). The involvement of the N terminus in membrane interaction was subsequently confirmed experimentally by analysis of ␣-synuclein deletion mutants (15) and NMR studies of liposomebound ␣-synuclein (18-20). The latter studies revealed an ordering of the N-terminal repeat regions induced upon membrane binding whereas the highly charged C terminus remained unstructured and, therefore, was not involved in membrane interaction. Beyond these data, however, direct structural information, such as the precise location, length, orientation, and number of...

High resolution (1.43-1.8 A) crystal structures and the corresponding electron paramagnetic resonance (EPR) spectra were determined for T4 lysozyme derivatives with a disulfide-linked nitroxide side chain [-CH(2)-S-S-CH(2)-(3-[2,2,5,5-tetramethyl pyrroline-1-oxyl]) identical with R1] substituted at solvent-exposed helix surface sites (Lys65, Arg80, Arg119) or a tertiary contact site (Val75). In each case, electron density is clearly resolved for the disulfide group, revealing distinct rotamers of the side chain, defined by the dihedral angles X(1) and X(2). The electron density associated with the nitroxide ring in the different mutants is inversely correlated with its mobility determined from the EPR spectrum. Residue 80R1 assumes a single g(+)()g(+)() conformation (Chi(1) = 286, X(2) = 294). Residue 119R1 has two EPR spectral components, apparently corresponding to two rotamers, one similar to that for 80R1 and the other in a tg(-)() conformation (Chi(1) = 175, X(2) = 54). The latter state is apparently stabilized by interaction of the disulfide with a Gln at i + 4, a situation also observed at 65R1. R1 residues at helix surface site 65 and tertiary contact site 75 make intra- as well as intermolecular contacts in the crystal and serve to identify the kind of molecular interactions possible for the R1 side chain. A single conformation of the entire 75R1 side chain is stabilized by a variety of interactions with the nitroxide ring, including hydrophobic contacts and two unconventional C-H.O hydrogen bonds, one in which the nitroxide acts as a donor (with tyrosine) and the other in which it acts as an acceptor (with phenylalanine). The interactions revealed in these structures provide an important link between the dynamics of the R1 side chain, reflected in the EPR spectrum, and local protein structure. A library of such interactions will provide a basis for the quantitative interpretation of EPR spectra in terms of protein structure and dynamics.

The 37-residue islet amyloid polypeptide (IAPP) is thought to play an important role in the pathogenesis of type II diabetes. Despite a growing body of evidence implicating membrane interaction in IAPP toxicity, the membrane-bound form has not yet been well characterized. Here we used circular dichroism (CD) and fluorescence spectroscopy to investigate the molecular details of the interaction of IAPP with lipid membranes of varying composition. In the presence of membranes containing negatively charged phosphatidylserine (PS), we observed significant acceleration in the formation of IAPP aggregates. This acceleration is strongly modulated by the PS concentration and ionic strength, and is also observed at physiologically relevant PS concentrations. CD spectra of IAPP obtained immediately after the addition of membranes containing PS revealed features characteristic of an alpha-helical conformation approximately approximately 15-19 residues in length. After a longer incubation with membranes, IAPP gave rise to CD spectra characteristic of a beta-sheet conformation. Taken together, our CD and fluorescence data indicate that conditions that promote weakly stable alpha-helical conformations may promote IAPP aggregation. The potential roles of IAPP-membrane interaction and the novel membrane-bound alpha-helical conformation in IAPP aggregation are discussed.

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Electron coupling through a beta strand has been investigated by measurement of the intramolecular electron-transfer (ET) rates in ruthenium-modified derivatives of the beta barrel blue copper protein Pseudomonas aeruginosa azurin. Surface histidines, introduced on the methionine-121 beta strand by mutagenesis, were modified with a Ru(2,2'-bipyridine)2(imidazole)2+ complex. The Cu+ to Ru3+ rate constants yielded a distance-decay constant of 1.1 per angstrom, a value close to the distance-decay constant of 1.0 per angstrom predicted for electron tunneling through an idealized beta strand. Activationless ET rate constants in combination with a tunneling-pathway analysis of the structures of azurin and cytochrome c confirm that there is a generally efficient network for coupling the internal (native) redox center to the surface of both proteins.