Disulfi de constrained Fabs overcome target size limitation for high-resolution single-particle cryo-EM.

High-resolution structures of proteins are critical to understanding molecular mechanisms of biological processes and in the discovery of therapeutic molecules. Cryo-EM has revolutionized structure determination of large proteins and their complexes1, but a vast majority of proteins that underlie human diseases are small (< 50 kDa) and usually beyond its reach due to low signal-to-noise images and difficulties in particle alignment2. Current strategies to overcome this problem increase the overall size of small protein targets using scaffold proteins that bind to the target, but are limited by inherent flexibility and not being bound to their targets in a rigid manner, resulting in the target being poorly resolved compared to the scaffolds3-11. Here we present an iteratively engineered molecular design for transforming Fabs (antibody fragments), into conformationally rigid scaffolds (Rigid-Fabs) that, when bound to small proteins (~20 kDa), can enable high-resolution structure determination using cryo-EM. This design introduces multiple disulfide bonds at strategic locations, generates a well-folded Fab constrained into a rigid conformation and can be applied to Fabs from various species, isotypes and chimeric Fabs. We present examples of the Rigid Fab design enabling high-resolution (2.3-2.5 Å) structures of small proteins, Ang2 (26 kDa) and KRAS (21 kDa) by cryo-EM. The strategies for designing disulfide constrained Rigid Fabs in our work thus establish a general approach to overcome the target size limitation of single particle cryo-EM.

Cryo-EM reveals an unprecedented binding site for NaV1.7 inhibitors enabling rational design of potent hybrid inhibitors.

The voltage-gated sodium (NaV) channel NaV1.7 has been identified as a potential novel analgesic target due to its involvement in human pain syndromes. However, clinically available NaV channel-blocking drugs are not selective among the nine NaV channel subtypes, NaV1.1-NaV1.9. Moreover, the two currently known classes of NaV1.7 subtype-selective inhibitors (aryl- and acylsulfonamides) have undesirable characteristics that may limit their development. To this point understanding of the structure-activity relationships of the acylsulfonamide class of NaV1.7 inhibitors, exemplified by the clinical development candidate GDC-0310, has been based solely on a single co-crystal structure of an arylsulfonamide inhibitor bound to voltage-sensing domain 4 (VSD4). To advance inhibitor design targeting the NaV1.7 channel, we pursued high-resolution ligand-bound NaV1.7-VSD4 structures using cryogenic electron microscopy (cryo-EM). Here, we report that GDC-0310 engages the NaV1.7-VSD4 through an unexpected binding mode orthogonal to the arylsulfonamide inhibitor class binding pose, which identifies a previously unknown ligand binding site in NaV channels. This finding enabled the design of a novel hybrid inhibitor series that bridges the aryl- and acylsulfonamide binding pockets and allows for the generation of molecules with substantially differentiated structures and properties. Overall, our study highlights the power of cryo-EM methods to pursue challenging drug targets using iterative and high-resolution structure-guided inhibitor design. This work also underscores an important role of the membrane bilayer in the optimization of selective NaV channel modulators targeting VSD4.

A Flexible and Scalable High-Throughput Platform for Recombinant Membrane Protein Production.

Integral membrane proteins (MP) are implicated in many disease processes and are the primary targets of numerous marketed drugs. Despite recent advances in the areas of MP solubilization, stabilization, and reconstitution, it remains a time-consuming task to identify the combination of constructs and purification conditions that will enable MP structure-function studies outside of the lipid bilayer. In this chapter, we describe a strategy for rapidly identifying and optimizing the solubilization and purification conditions for nearly any recombinant MP, based on the use of a noninvasive fluorescent probe (His-Glow) that specifically binds to the common hexahistidine affinity tag of expressed targets. This His-Glow approach permits fluorescent size-exclusion chromatography (FSEC) without the need for green fluorescent protein (GFP) fusion. A two-stage detergent screening strategy is employed at the solubilization stage, whereby appropriate detergent families are identified first, followed by optimization within these families. Screening up to 96 unique combinations of solubilization conditions and constructs can be achieved in less than 24 h. At the outset of each new project, we screen six different detergents for each construct and the subsequent implementation of a simple thermostability challenge further aids in the identification of constructs and conditions suitable for large-scale production. Our strategy streamlines the parallel optimization of appropriate production conditions for multiple MP targets to rapidly enable downstream biochemical, immunization, or structural studies.

A Novel Inhibitor of the LolCDE ABC Transporter Essential for Lipoprotein Trafficking in Gram-Negative Bacteria.

The outer membrane is an essential structural component of Gram-negative bacteria that is composed of lipoproteins, lipopolysaccharides, phospholipids, and integral ß-barrel membrane proteins. A dedicated machinery, called the Lol system, ensures proper trafficking of lipoproteins from the inner to the outer membrane. The LolCDE ABC transporter is the inner membrane component, which is essential for bacterial viability. Here, we report a novel pyrrolopyrimidinedione compound, G0507, which was identified in a phenotypic screen for inhibitors of Escherichia coli growth followed by selection of compounds that induced the extracytoplasmic σE stress response. Mutations in lolC, lolD, and lolE conferred resistance to G0507, suggesting LolCDE as its molecular target. Treatment of E. coli cells with G0507 resulted in accumulation of fully processed Lpp, an outer membrane lipoprotein, in the inner membrane. Using purified protein complexes, we found that G0507 binds to LolCDE and stimulates its ATPase activity. G0507 still binds to LolCDE harboring a Q258K substitution in LolC (LolCQ258K), which confers high-level resistance to G0507 in vivo but no longer stimulates ATPase activity. Our work demonstrates that G0507 has significant promise as a chemical probe to dissect lipoprotein trafficking in Gram-negative bacteria.

From Constructs to Crystals - Towards Structure Determination of ß-barrel Outer Membrane Proteins.

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, α-helical and ß-barrel. While α-helical membrane proteins can be found in nearly all cellular membranes, ß-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite ß-barrel outer membrane protein (OMP), general protocols are presented for the production of target ß-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

Visual arrestin interaction with clathrin adaptor AP-2 regulates photoreceptor survival in the vertebrate retina.

Arrestins bind ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) and terminate the activation of G proteins. Additionally, nonvisual arrestin/GPCR complex can initiate G protein-independent intracellular signals through their ability to act as scaffolds that bring other signaling molecules to the internalized GPCR. Like nonvisual arrestins, vertebrate visual arrestin (ARR1) terminates G protein signaling from light-activated, phosphorylated GPCR, rhodopsin. Unlike nonvisual arrestins, its role as a transducer of signaling from internalized rhodopsin has not been reported in the vertebrate retina. Formation of signaling complexes with arrestins often requires recruitment of the endocytic adaptor protein, AP-2. We have previously shown that Lys296 → Glu (K296E), which is a naturally occurring rhodopsin mutation in certain humans diagnosed with autosomal dominant retinitis pigmentosa, causes toxicity through forming a stable complex with ARR1. Here we investigated whether recruitment of AP-2 by the K296E/ARR1 complex plays a role in generating the cell death signal in a transgenic mouse model of retinal degeneration. We measured the binding affinity of ARR1 for AP-2 and found that, although the affinity is much lower than that of the other arrestins, the unusually high concentration of ARR1 in rods would favor this interaction. We further demonstrate that p44, a splice variant of ARR1 that binds light-activated, phosphorylated rhodopsin but lacks the AP-2 binding motif, prevents retinal degeneration and rescues visual function in K296E mice. These results reveal a unique role of ARR1 in a G protein-independent signaling cascade in the vertebrate retina.

What the N-terminal domain of Atg13 looks like and what it does: a HORMA fold required for PtdIns 3-kinase recruitment.

Atg13 is a subunit of the Atg1 complex that is involved in autophagy. The middle and C-terminal regions of Atg13 are intrinsically disordered and rich in regulatory phosphorylation sites. Thus far, there have been no structural data for any part of Atg13, and no function assigned to its N-terminal domain. We crystallized this domain, and found that it has a HORMA (Hop1, Rev7, Mad2) fold. We showed that the Atg13 HORMA domain is required for autophagy and for recruitment of the phosphatidylinositol (PtdIns) 3-kinase subunit Atg14, but is not required for Atg1 interaction or Atg13 recruitment to the PAS. The HORMA domain of Atg13 is similar to the closed conformation of the spindle checkpoint protein Mad2. A pair of conserved arginines was identified in the structure, and tested functionally in yeast. These residues are important for autophagy, as mutations abrogate autophagy and block Atg14 recruitment. The location of these Arg residues in the structure suggests that the Atg13 HORMA domain could act as a phosphorylation-dependent conformational switch.

A HORMA domain in Atg13 mediates PI 3-kinase recruitment in autophagy.

Autophagy-related 13 (Atg13) is a key early-acting factor in autophagy and the major locus for nutrient-dependent regulation of autophagy by Tor. The 2.3-Å resolution crystal structure of the N-terminal domain of Atg13 reveals a previously unidentified HORMA (Hop1p, Rev1p and Mad2) domain similar to that of the spindle checkpoint protein Mad2. Mad2 has two different stable conformations, O-Mad2 and C-Mad2, and the Atg13 HORMA structure corresponds to the C-Mad2 state. The Atg13 HORMA domain is required for autophagy and for recruitment of the phosphatidylinositol (PI) 3-kinase subunit Atg14 but is not required for Atg1 interaction or Atg13 recruitment to the preautophagosomal structure. The Atg13 HORMA structure reveals a pair of conserved Arg residues that constitute a putative phosphate sensor. One of the Arg residues is in the region corresponding to the "safety belt" conformational switch of Mad2, suggesting conformational regulation of phosphate binding. These two Arg residues are essential for autophagy, suggesting that the Atg13 HORMA domain could function as a phosphoregulated conformational switch.

Membrane binding and self-association of the epsin N-terminal homology domain.

Epsin possesses a conserved epsin N-terminal homology (ENTH) domain that acts as a phosphatidylinositol 4,5-bisphosphate-lipid-targeting and membrane-curvature-generating element. Upon binding phosphatidylinositol 4,5-bisphosphate, the N-terminal helix (H(0)) of the ENTH domain becomes structured and aids in the aggregation of ENTH domains, which results in extensive membrane remodeling. In this article, atomistic and coarse-grained (CG) molecular dynamics (MD) simulations are used to investigate the structure and the stability of ENTH domain aggregates on lipid bilayers. EPR experiments are also reported for systems composed of different ENTH-bound membrane morphologies, including membrane vesicles as well as preformed membrane tubules. The EPR data are used to help develop a molecular model of ENTH domain aggregates on preformed lipid tubules that are then studied by CG MD simulation. The combined computational and experimental approach suggests that ENTH domains exist predominantly as monomers on vesiculated structures, while ENTH domains self-associate into dimeric structures and even higher-order oligomers on the membrane tubes. The results emphasize that the arrangement of ENTH domain aggregates depends strongly on whether the local membrane curvature is isotropic or anisotropic. The molecular mechanism of ENTH-domain-induced membrane vesiculation and tubulation and the implications of the epsin's role in clathrin-mediated endocytosis resulting from the interplay between ENTH domain membrane binding and ENTH domain self-association are also discussed.

Computer modeling of nitroxide spin labels on proteins.

Electron paramagnetic resonance using site-directed spin labeling can be used as an approach for determination of protein structures that are difficult to solve by other methods. One important aspect of this approach is the measurement of interlabel distances using the double electron-electron resonance (DEER) method. Interpretation of experimental data could be facilitated by a computational approach to calculation of interlabel distances. We describe an algorithm, PRONOX, for rapid computation of interlabel distances based on calculation of spin label conformer distributions at any site of a protein. The program incorporates features of the label distribution established experimentally, including weighting of favorable conformers of the label. Distances calculated by PRONOX were compared with new DEER distances for amphiphysin and annexin B12 and with published data for FCHo2 (F-BAR), endophilin, and α-synuclein, a total of 44 interlabel distances. The program reproduced these distances accurately (r(2) = 0.94, slope = 0.98). For 9 of the 11 distances for amphiphysin, PRONOX reproduced the experimental data to within 2.5 Å. The speed and accuracy of PRONOX suggest that the algorithm can be used for fitting to DEER data for determination of protein tertiary structure.

Membrane curvature sensing by amphipathic helices: a single liposome study using α-synuclein and annexin B12.

Preferential binding of proteins on curved membranes (membrane curvature sensing) is increasingly emerging as a general mechanism whereby cells may effect protein localization and trafficking. Here we use a novel single liposome fluorescence microscopy assay to examine a common sensing motif, the amphipathic helix (AH), and provide quantitative measures describing and distinguishing membrane binding and sensing behavior. By studying two AH-containing proteins, α-synuclein and annexin B12, as well as a range of AH peptide mutants, we reveal that both the hydrophobic and hydrophilic faces of the helix greatly influence binding and sensing. Although increased hydrophobic and electrostatic interactions with the membrane both lead to greater densities of bound protein, the former yields membrane curvature-sensitive binding, whereas the latter is not curvature-dependent. However, the relative contributions of both components determine the sensing of AHs. In contrast, charge density in the lipid membrane seems important primarily in attracting AHs to the membrane but does not significantly influence sensing. These observations were made possible by the ability of our assay to distinguish within our samples liposomes with and without bound protein as well as the density of bound protein. Our findings suggest that the description of membrane curvature-sensing requires consideration of several factors such as short and long range electrostatic interactions, hydrogen bonding, and the volume and structure of inserted hydrophobic residues.

Membrane curvature induction and tubulation are common features of synucleins and apolipoproteins.

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, ß-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.

A combinatorial NMR and EPR approach for evaluating the structural ensemble of partially folded proteins.

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 alpha-synuclein (alphaS), 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 alphaS, 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 antiparallel alphaS 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 alphaS correlate with the degree of protein-induced departures from free micelle dimensions.

Multiple modes of endophilin-mediated conversion of lipid vesicles into coated tubes: implications for synaptic endocytosis.

Endophilin A1 is a BAR (Bin/amphiphysin/Rvs) protein abundant in neural synapses that senses and induces membrane curvature, contributing to neck formation in presynaptic endocytic vesicles. To investigate its role in membrane remodeling, we used cryoelectron microscopy to characterize structural changes induced in lipid vesicles by exposure to endophilin. The vesicles convert rapidly to coated tubules whose morphology reflects the local concentration of endophilin. Their diameters and curvature resemble those of synaptic vesicles in situ. Three-dimensional reconstructions of quasicylindrical tubes revealed arrays of BAR dimers, flanked by densities that we equate with amphipathic helices whose folding and membrane insertion were attested by EPR. We also observed the compression of bulbous coated tubes into 70-A-wide cylindrical micelles, which appear to mimic the penultimate (hemi-fission) stage of endocytosis. Our findings suggest that the adaptability of endophilin-lipid interactions underlies dynamic changes of endocytic membranes.

Roles of amphipathic helices and the bin/amphiphysin/rvs (BAR) domain of endophilin in membrane curvature generation.

Control of membrane curvature is required in many important cellular processes, including endocytosis and vesicular trafficking. Endophilin is a bin/amphiphysin/rvs (BAR) domain protein that induces vesicle formation by promotion of membrane curvature through membrane binding as a dimer. Using site-directed spin labeling and EPR spectroscopy, we show that the overall BAR domain structure of the rat endophilin A1 dimer determined crystallographically is maintained under predominantly vesiculating conditions. Spin-labeled side chains on the concave surface of the BAR domain do not penetrate into the acyl chain interior, indicating that the BAR domain interacts only peripherally with the surface of a curved bilayer. Using a combination of EPR data and computational refinement, we determined the structure of residues 63-86, a region that is disordered in the crystal structure of rat endophilin A1. Upon membrane binding, residues 63-75 in each subunit of the endophilin dimer form a slightly tilted, amphipathic alpha-helix that directly interacts with the membrane. In their predominant conformation, these helices are located orthogonal to the long axis of the BAR domain. In this conformation, the amphipathic helices are positioned to act as molecular wedges that induce membrane curvature along the concave surface of the BAR domain.

Structure of membrane-bound alpha-synuclein from site-directed spin labeling and computational refinement.

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

Mechanism of endophilin N-BAR domain-mediated membrane curvature.

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.

Calcium- and membrane-induced changes in the structure and dynamics of three helical hairpins in annexin B12.

Annexins are a family of soluble proteins that can undergo reversible Ca(2+)-dependent interaction with the interfacial region of phospholipid membranes. The helical hairpins on the convex face of the crystal structure of soluble annexins are proposed to mediate binding to membranes, but the mechanism is not defined. For this study, we used a site-directed spin labeling (SDSL) experimental approach to investigate Ca(2+) and membrane-induced structural and dynamic changes that occurred in the helical hairpins encompassing three of the four D and E helices of annexin B12. Electron paramagnetic resonance (EPR) parameters were analyzed for the soluble and Ca(2+)-dependent membrane-bound states of the following nitroxide scans of annexin B12: a continuous 24-residue scan of the D and E helices in the third repeat (residues 219-242) and short scans encompassing the D-E loop regions of the first repeat (residues 68-74) and the fourth repeat (300-305). EPR mobility and accessibility parameters of most sites were similar when the protein was in solution or in the membrane-bound state, and both sets of data were consistent with the crystal structure of the protein. However, membrane-induced changes in mobility and accessibility were observed in all three loop regions, with the most dramatic changes noted at sites corresponding to the highly conserved serine and glycine residues in the loops. EPR accessibility parameters clearly established that nitroxide side chains placed at these sites made direct contact with the bilayer. EPR mobility parameters showed that these sites were very mobile in solution, but immobilized on the EPR time scale in the membrane-bound state. Since the headgroup regions of bilayer phospholipids are relatively mobile in the absence of annexins, Ca(2+)-dependent binding of annexin B12 appears to form a complex in which the mobility of the D-E loop region of the protein and the headgroup region of the phospholipid are highly constrained. Possible biological consequences of annexin-induced restriction of membrane mobility are discussed.

The conserved core domains of annexins A1, A2, A5, and B12 can be divided into two groups with different Ca2+-dependent membrane-binding properties.

The hallmark of the annexin super family of proteins is Ca(2+)-dependent binding to phospholipid bilayers, a property that resides in the conserved core domain of these proteins. Despite the structural similarity between the core domains, studies reported herein showed that annexins A1, A2, A5, and B12 could be divided into two groups with distinctively different Ca(2+)-dependent membrane-binding properties. The division correlates with the ability of the annexins to form Ca(2+)-dependent membrane-bound trimers. Site-directed spin-labeling and Forster resonance energy transfer experimental approaches confirmed the well-known ability of annexins A5 and B12 to form trimers, but neither method detected self-association of annexin A1 or A2 on bilayers. Studies of chimeras in which the N-terminal and core domains of annexins A2 and A5 were swapped showed that trimer formation was mediated by the core domain. The trimer-forming annexin A5 and B12 group had the following Ca(2+)-dependent membrane-binding properties: (1) high Ca(2+) stoichiometry for membrane binding ( approximately 12 mol of Ca(2+)/mol of protein); (2) binding to membranes was very exothermic (> -60 kcal/ mol of protein); and (3) binding to bilayers that were in the liquid-crystal phase but not to bilayers in the gel phase. In contrast, the nontrimer-forming annexin A1 and A2 group had the following Ca(2+)-dependent membrane-binding properties: (1) lower Ca(2+) stoichiometry for membrane binding (

Structure of membrane-bound alpha-synuclein studied by site-directed spin labeling.

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