Synaptic vesicle exocytosis is mediated by the vesicular Ca2+-sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and with acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using both PC12 cells from Rattus norvegicus and artificial supported bilayers we now show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca2+ via PIP2. This interaction allows both Ca2+-binding sites of synaptotagmin-1 to bind to phosphatidylserine (PS) in the vesicle membrane upon Ca2+-triggering. We determined the crystal structure of the C2B-domain of synaptotagmin-1 bound to phosphoserine, allowing for developing a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca2+-influx bringing the vesicle membrane close enough for membrane fusion.

β-propellers that bind polyphosphoinositides (PROPPINs), a eukaryotic WD-40 motif-containing protein family, bind via their predicted β-propeller fold the polyphosphoinositides PtdIns3P and PtdIns(3,5) P 2 using a conserved FRRG motif. PROPPINs play a key role in macroautophagy in addition to other functions. We present the 3.0-Å crystal structure of Kluyveromyces lactis Hsv2, which shares significant sequence homologies with its three Saccharomyces cerevisiae homologs Atg18, Atg21, and Hsv2. It adopts a seven-bladed β-propeller fold with a rare nonvelcro propeller closure. Remarkably, in the crystal structure, the two arginines of the FRRG motif are part of two distinct basic pockets formed by a set of highly conserved residues. In comprehensive in vivo and in vitro studies of ScAtg18 and ScHsv2, we define within the two pockets a set of conserved residues essential for normal membrane association, phosphoinositide binding, and biological activities. Our experiments show that PROPPINs contain two individual phosphoinositide binding sites. Based on docking studies, we propose a model for phosphoinositide binding of PROPPINs.autophagy | protein-lipid interactions | X-ray crystallography | yeast

The hydrolytic endoribonuclease RNase E, which is widely distributed in bacteria and plants, plays key roles in mRNA degradation and RNA processing in Escherichia coli. The enzymatic activity of RNase E is contained within the conserved amino-terminal half of the 118 kDa protein, and the carboxy-terminal half organizes the RNA degradosome, a multi-enzyme complex that degrades mRNA co-operatively and processes ribosomal and other RNA. The study described herein demonstrates that the carboxy-terminal domain of RNase E has little structure under native conditions and is unlikely to be extensively folded within the degradosome. However, three isolated segments of 10 -40 residues, and a larger fourth segment of 80 residues, are predicted to be regions of increased structural propensity. The larger of these segments appears to be a protein -RNA interaction site while the other segments possibly correspond to sites of self-recognition and interaction with the other degradosome proteins. The carboxy-terminal domain of RNase E may thus act as a flexible tether of the degradosome components. The implications of these and other observations for the organization of the RNA degradosome are discussed.

The X-ray crystallographic analysis of redox-active systems may be complicated by photoreduction. Although radiolytic reduction by the probing X-ray beam may be exploited to generate otherwise short-lived reaction intermediates of metalloproteins, it is generally an undesired feature. Here, the X-ray-induced reduction of the three heme proteins myoglobin, cytochrome P450cam and chloroperoxidase has been followed by on-line UV-Vis absorption spectroscopy. All three systems showed a very rapid reduction of the heme iron. In chloroperoxidase the change of the ionization state from ferric to ferrous heme is associated with a movement of the heme-coordinating water molecule. The influence of the energy of the incident X-ray photons and of the presence of scavengers on the apparent reduction rate of ferric myoglobin crystals was analyzed.

Small GTPases of the Rab family not only regulate target recognition in membrane traffic but also control other cellular functions such as cytoskeletal transport and autophagy. Here we show that Rab26 is specifically associated with clusters of synaptic vesicles in neurites. Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles. Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles. Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity. We propose that Rab26 selectively directs synaptic and secretory vesicles into preautophagosomal structures, suggesting the presence of a novel pathway for degradation of synaptic vesicles.DOI: http://dx.doi.org/10.7554/eLife.05597.001

The vasodilator-stimulated phosphoprotein (VASP) is a key regulator of actin dynamics. We have determined the 1.3-Å resolution crystal structure of the 45-residue-long tetramerization domain (TD) from human VASP. This domain forms a right-handed ␣-helical coiled-coil structure with a similar degree of supercoiling as found in the widespread left-handed coiled coils with heptad repeats. The basis for the right-handed geometry of VASP TD is a 15-residue repeat in its amino acid sequence, which reveals a characteristic pattern of hydrophobic residues. Hydrophobic interactions and a network of salt bridges render VASP TD highly thermostable with a melting point of 120°C. In an estimated 10% of all proteins (1), ␣-helical coiled coils are present, and they constitute a principal protein-folding motif. Between two and five ␣-helices are wound around each other, usually forming a left-handed superhelix (2, 3). The sequences of the left-handed coiled coils are characterized by heptad repeats, (abcdefg) n , where positions a and d are occupied mostly by apolar residues. The left-handed supercoiling is induced by a small but significant difference between 3.5 residues per turn in a heptad repeat and 3.64 residues per turn in an undistorted ␣-helix (2), by which the residues in the a and d positions form a hydrophobic core. Typically, coiled coils are very specific oligomers with high thermodynamic stability, which enables them to function as oligomerization domains in many proteins (4).In addition to the most common coiled coils with heptad repeats, structures with longer repeats are possible. It is worth noting that in 1951 Pauling et al. (5) mentioned the feasibility of other periodicities, including 11-residue repeats (three ␣-helical turns, 11͞3) and 15-residue repeats (15͞4). Although researchers have focused almost exclusively on coiled coils with heptad repeats (6), 11-residue repeats were found recently in the tetrabrachion protein from Staphylothermus marinus (7). Its crystallographic structure has indeed revealed a parallel fourstranded coiled coil with a slight right-handed supercoiling (8). Moreover, it was suggested that 15-residue repeats would induce a yet more pronounced right-handed supercoiling (6).Here, we report the crystal structure of the tetramerization domain (TD) from human vasodilator-stimulated phosphoprotein (VASP). This domain includes two consecutive 15-residue (quindecad) repeats, which result in a parallel right-handed coiled-coil structure with a similar degree of supercoiling as observed in the left-handed coiled coils with heptad repeats. Furthermore, we show that VASP TD is stabilized by a hydrophobic interface as well as a network of salt bridges, which yields a highly thermostable structure. Last, we discuss how the TD may function as a regulator of actin assembly. Materials and MethodsCrystallization and Structure Determination. The TD (residues 336-380) of human VASP was expressed and purified as described (9). Crystals were grown at 20°C by using the hanging-drop method. We mixed 5...

Chloroperoxidase (CPO) is a heme-thiolate enzyme that catalyzes hydrogen peroxide-dependent halogenation reactions. Structural data on substrate binding have not been available so far. CPO was therefore crystallized in the presence of iodide or bromide. One halide binding site was identified at the surface near a narrow channel that connects the surface with the heme. Two other halide binding sites were identified within and at the other end of this channel. Together, these sites suggest a pathway for access of halide anions to the active site. The structure of CPO complexed with its natural substrate cyclopentanedione was determined at a resolution of 1.8 Å . This is the first example of a CPO structure with a bound organic substrate. In addition, structures of CPO bound with nitrate, acetate, and formate and of a ternary complex with dimethylsulfoxide (Me 2 SO) and cyanide were determined. These structures have implications for the mechanism of compound I formation. Before binding to the heme, the incoming hydrogen peroxide first interacts with Glu-183. The deprotonated Glu-183 abstracts a proton from hydrogen peroxide. The hydroperoxo-anion then binds at the heme, yielding compound 0. Glu-183 protonates the distal oxygen of compound 0, water is released, and compound I is formed.

We have determined the crystal structure of the chloroperoxidase (CPO) hydroperoxo reaction intermediate (CPO compound 0) at 1.75-Å resolution. The intermediate was generated through controlled photoreduction of the CPO oxygen complex during x-ray data collection, which was monitored by recording of the crystal absorption spectra. Initially, the peroxo-anion species was formed and then protonated to yield compound 0. Quantum chemical calculations indicate that the peroxo-anion species is not stable and collapses instantaneously to compound 0. Compound 0 is present in the ferric low-spin doublet ground state and is characterized by a long OOO bond length of 1.5 Å and a FeOO bond distance of 1.8 Å, which is also observed in the crystal structure.crystal structure ͉ protein structure/function ͉ radiolysis ͉ reaction mechanism ͉ QM/MM calculations