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.

α-Synuclein is known to play a causative role in Parkinson disease. Although its physiological functions are not fully understood, α-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 α-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 α-synuclein was built and refined by using simulated annealing molecular dynamics restrained by the immersion depths and distances. We found that α-synuclein forms an extended, curved α-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 α-synuclein, contain 11-aa repeats, but which are soluble, oligomeric proteins (rmsd = 0.82 Å). The α-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 α-synuclein in the presence of the commonly used membrane-mimetic 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.

Many of the proposed physiological functions of α-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 α-synuclein, we performed EPR analysis of 47 singly labeled α-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 α-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.

Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have comparable effects on lipid membranes, but this has not been shown directly. Here, we find that α-synuclein, β-synuclein, and apolipoprotein A-1 have the conserved functional ability to induce membrane curvature and to convert large vesicles into highly curved membrane tubules and vesicles. The resulting structures are morphologically similar to those generated by amphiphysin, a curvature-inducing protein involved in endocytosis. Unlike amphiphysin, however, synucleins and apolipoproteins do not require any scaffolding domains and curvature induction is mediated by the membrane insertion and wedging of amphipathic helices alone. Moreover, we frequently observed that α-synuclein caused membrane structures that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees well with recent findings that α-synuclein plays a role in vesicle trafficking and enhances endocytosis. Induction of membrane curvature must be under strict regulation in vivo; however, as we find it can also cause disruption of membrane integrity. Because the degree of membrane curvature induction depends on the concerted action of multiple proteins, controlling the local protein density of tubulating proteins may be important. How cellular safeguarding mechanisms prevent such potentially toxic events and whether they go awry in disease remains to be determined.

Tau Filaments are found in >20 neurodegenerative diseases. Yet, because of their enormous molecular weights and poor tendency to form highly ordered 3D crystal lattices, they have evaded high-resolution structure determination. Here, we studied 25 derivatized tau mutants by using electron paramagnetic resonance and fluorescence spectroscopy to report structural details of tau filaments. Based on strong spin exchange and pyrene excimer formation of core residues, we find that individual tau proteins form single molecule layers along the fiber axis that perfectly stack on top of each other by in-register, parallel alignment of β-strands. We suggest a model of filament growth wherein the existing filament serves as a template for the incoming, unfolded tau molecule, resulting in a new structured layer with maximized hydrogen-bonded contact surface and side-chain stacking.

Human islet amyloid polypeptide (hIAPP) misfolding is thought to play an important role in the pathogenesis of type II diabetes mellitus. It has recently been shown that membranes can catalyze the misfolding of hIAPP via an α-helical intermediate of unknown structure. To better understand the mechanism of membrane-mediated misfolding, we used site-directed spin labeling and EPR spectroscopy to generate a three-dimensional structural model of this membrane-bound form. We find that hIAPP forms a single α-helix encompassing residues 9–22. The helix is flanked by N- and C-terminal regions that do not take up a clearly detectable secondary structure and are less ordered. Residues 21 and 22 are located in a transitional region between the α-helical structure and C terminus and exhibit significant mobility. The α-helical structure presented here has important implications for membrane-mediated aggregation. Anchoring hIAPP to the membrane not only increases the local concentration but also reduces the encounter between peptides to essentially a two-dimensional process. It is significant to note that the α-helical membrane-bound form leaves much of an important amyloidogenic region of hIAPP (residues 20–29) exposed for misfolding. Misfolding of this and other regions is likely further aided by the low dielectric environment near the membrane that is known to promote secondary structure formation. Based upon these considerations, a structural model for membrane-mediated aggregation is discussed.