Structural basis of cell surface receptor recognition by botulinum neurotoxin B

Botulinum neurotoxins (BoNTs) are potent bacterial toxins that cause paralysis at femtomolar concentrations by blocking neurotransmitter release. A 'double receptor' model has been proposed in which BoNTs recognize nerve terminals via interactions with both gangliosides and protein receptors that mediate their entry. Of seven BoNTs (subtypes A-G), the putative receptors for BoNT/A, BoNT/B and BoNT/G have been identified, but the molecular details that govern recognition remain undefined. Here we report the crystal structure of full-length BoNT/B in complex with the synaptotagmin II (Syt-II) recognition domain at 2.6 A resolution. The structure of the complex reveals that Syt-II forms a short helix that binds to a hydrophobic groove within the binding domain of BoNT/B. In addition, mutagenesis of amino acid residues within this interface on Syt-II affects binding of BoNT/B. Structural and sequence analysis reveals that this hydrophobic groove is conserved in the BoNT/G and BoNT/B subtypes, but varies in other clostridial neurotoxins. Furthermore, molecular docking studies using the ganglioside G(T1b) indicate that its binding site is more extensive than previously proposed and might form contacts with both BoNT/B and synaptotagmin. The results provide structural insights into how BoNTs recognize protein receptors and reveal a promising target for blocking toxin-receptor recognition.     

The C(2)B Ca(2+)-binding motif of synaptotagmin is required for synaptic transmission in vivo

Synaptotagmin is a synaptic vesicle protein that is postulated to be the Ca(2+) sensor for fast, evoked neurotransmitter release. Deleting the gene for synaptotagmin (syt(null)) strongly suppresses synaptic transmission in every species examined, showing that synaptotagmin is central in the synaptic vesicle cycle. The cytoplasmic region of synaptotagmin contains two C(2) domains, C(2)A and C(2)B. Five, highly conserved, acidic residues in both the C(2)A and C(2)B domains of synaptotagmin coordinate the binding of Ca(2+) ions, and biochemical studies have characterized several in vitro Ca(2+)-dependent interactions between synaptotagmin and other nerve terminal molecules. But there has been no direct evidence that any of the Ca(2+)-binding sites within synaptotagmin are required in vivo. Here we show that mutating two of the Ca(2+)-binding aspartate residues in the C(2)B domain (D(416,418)N in Drosophila) decreased evoked transmitter release by >95%, and decreased the apparent Ca(2+) affinity of evoked transmitter release. These studies show that the Ca(2+)-binding motif of the C(2)B domain of synaptotagmin is essential for synaptic transmission.     

Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin.

Clostridial neurotoxins, including tetanus toxin and the seven serotypes of botulinum toxin (A-G), are produced as single chains and cleaved to generate toxins with two chains joined by a single disulphide bond (Fig. 1). The heavy chain (M(r) 100,000 (100K)) is responsible for specific binding to neuronal cells and cell penetration of the light chain (50K), which blocks neurotransmitter release. Several lines of evidence have recently suggested that clostridial neurotoxins could be zinc endopeptidases. Here we show that tetanus and botulinum toxins serotype B are zinc endopeptidases, the activation of which requires reduction of the interchain disulphide bond. The protease activity is localized on the light chain and is specific for synaptobrevin, an integral membrane protein of small synaptic vesicles. The rat synaptobrevin-2 isoform is cleaved by both neurotoxins at the same single site, the peptide bond Gln 76-Phe 77, but the isoform synaptobrevin-1, which has a valine at the corresponding position, is not cleaved. The blocking of neurotransmitter release of Aplysia neurons injected with tetanus toxin or botulinum toxins serotype B is substantially delayed by peptides containing the synaptobrevin-2 cleavage site. These results indicate that tetanus and botulinum B neurotoxins block neurotransmitter release by cleaving synaptobrevin-2, a protein that, on the basis of our results, seems to play a key part in neurotransmitter release.     

Substrate recognition strategy for botulinum neurotoxin serotype A

Clostridal neurotoxins (CNTs) are the causative agents of the neuroparalytic diseases botulism and tetanus. CNTs impair neuronal exocytosis through specific proteolysis of essential proteins called SNAREs. SNARE assembly into a low-energy ternary complex is believed to catalyse membrane fusion, precipitating neurotransmitter release; this process is attenuated in response to SNARE proteolysis. Site-specific SNARE hydrolysis is catalysed by the CNT light chains, a unique group of zinc-dependent endopeptidases. The means by which a CNT properly identifies and cleaves its target SNARE has been a subject of much speculation; it is thought to use one or more regions of enzyme-substrate interaction remote from the active site (exosites). Here we report the first structure of a CNT endopeptidase in complex with its target SNARE at a resolution of 2.1 A: botulinum neurotoxin serotype A (BoNT/A) protease bound to human SNAP-25. The structure, together with enzyme kinetic data, reveals an array of exosites that determine substrate specificity. Substrate orientation is similar to that of the general zinc-dependent metalloprotease thermolysin. We observe significant structural changes near the toxin's catalytic pocket upon substrate binding, probably serving to render the protease competent for catalysis. The novel structures of the substrate-recognition exosites could be used for designing inhibitors specific to BoNT/A.     

Synaptotagmins I and IV promote transmitter release independently of Ca(2+) binding in the C(2)A domain

At nerve terminals, a focal and transient increase in intracellular Ca(2+) triggers the fusion of neurotransmitter-filled vesicles with the plasma membrane. The most extensively studied candidate for the Ca(2+)-sensing trigger is synaptotagmin I, whose Ca(2+)-dependent interactions with acidic phospholipids and syntaxin have largely been ascribed to its C(2)A domain, although the C(2)B domain also binds Ca(2+) (refs 7, 8). Genetic tests of synaptotagmin I have been equivocal as to whether it is the Ca(2+)-sensing trigger of fusion. Synaptotagmin IV, a related isoform that does not bind Ca(2+) in the C(2)A domain, might be an inhibitor of release. We mutated an essential aspartate of the Ca(2+)-binding site of the synaptotagmin I C(2)A domain and expressed it in Drosophila lacking synaptotagmin I. Here we show that, despite the disruption of the binding site, the Ca(2+)-dependent properties of transmission were not altered. Similarly, we found that synaptotagmin IV could substitute for synaptotagmin I. We conclude that the C(2)A domain of synaptotagmin is not required for Ca(2+)-dependent synaptic transmission, and that synaptotagmin IV promotes rather than inhibits transmission.     

Binding of synaptotagmin to the alpha-latrotoxin receptor implicates both in synaptic vesicle exocytosis

A vertebrate neurotoxin, alpha-latrotoxin, from black widow spider venom causes synaptic vesicle exocytosis and neurotransmitter release from presynaptic nerve terminals. Although the mechanism of action of alpha-latrotoxin is not known, it does require binding of alpha-latrotoxin to a high-affinity receptor on the presynaptic plasma membrane. The alpha-latrotoxin receptor seems to be exclusively at the presynaptic plasmamembrane. Here we report that the alpha-latrotoxin receptor specifically binds to a synaptic vesicle protein, synaptotagmin, and modulates its phosphorylation. Synaptotagmin is a synaptic vesicle-specific membrane protein that binds negatively charged phospholipids and contains two copies of a putative Ca(2+)-binding domain from protein kinase C (the C2-domain), suggesting a regulatory role in synaptic vesicle fusion. Our findings suggest that a physiological role of the alpha-latrotoxin receptor may be the docking of synaptic vesicles at the active zone. The direct interaction of the alpha-latrotoxin receptor with a synaptic vesicle protein also suggests a mechanism of action for this toxin in causing neurotransmitter release.     

Synaptotagmin I functions as a calcium regulator of release probability

In all synapses, Ca2+ triggers neurotransmitter release to initiate signal transmission. Ca2+ presumably acts by activating synaptic Ca2+ sensors, but the nature of these sensors--which are the gatekeepers to neurotransmission--remains unclear. One of the candidate Ca2+ sensors in release is the synaptic Ca2+-binding protein synaptotagmin I. Here we have studied a point mutation in synaptotagmin I that causes a twofold decrease in overall Ca2+ affinity without inducing structural or conformational changes. When introduced by homologous recombination into the endogenous synaptotagmin I gene in mice, this point mutation decreases the Ca2+ sensitivity of neurotransmitter release twofold, but does not alter spontaneous release or the size of the readily releasable pool of neurotransmitters. Therefore, Ca2+ binding to synaptotagmin I participates in triggering neurotransmitter release at the synapse.     

Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C

Neurotransmitters are released at synapses by the Ca2(+)-regulated exocytosis of synaptic vesicles, which are specialized secretory organelles that store high concentrations of neurotransmitters. The rapid Ca2(+)-triggered fusion of synaptic vesicles is presumably mediated by specific proteins that must interact with Ca2+ and the phospholipid bilayer. We now report that the cytoplasmic domain of p65, a synaptic vesicle-specific protein that binds calmodulin contains an internally repeated sequence that is homologous to the regulatory C2-region of protein kinase C (PKC). The cytoplasmic domain of recombinant p65 binds acidic phospholipids with a specificity indicating an interaction of p65 with the hydrophobic core as well as the headgroups of the phospholipids. The binding specificity resembles PKC, except that p65 also binds calmodulin, placing the C2-regions in a context of potential Ca2(+)-regulation that is different from PKC. This is a novel homology between a cellular protein and the regulatory domain of protein kinase C. The structure and properties of p65 suggest that it may have a role in mediating membrane interactions during synaptic vesicle exocytosis.     

Two forms of the membrane-bound state of the first C2 domain (C2A) of synaptotagminⅠand calcium-triggered membrane insertion

The synaptic vesicle protein synaptotagmin I(syt I) is a vesicle transmembrane protein present in synaptic vesicles, which has been proposed as the Ca^2 sensor that regulates secretion. The C2A domain is the membrane proximal part of its cytoplasmic domain. The interaction between C2A and lipid bilayer has been considered to be essential for triggering neurotransmitter release. In the present work, the measurements of membrane surface tension and surface concentration showed that the C2A domain of syt I exhibited two membrane-bound states: the surface adsorption state and the membrane insertion state. The surface absorption state formed in a Ca2~-independent manner with lower affinity, while the membrane insertion state formed with high affinity was only found in the presence of Ca^2 . Both the Ca^2 -independent and Ca^2 -dependent syt I membrane interactions required anionic phospholipids, such as phosphatidylserine (PS). When expressed into rat pheo-chromocytoma (PC12) cells and human embryonic kidney (HEK-293) cells, as demonstrated by immunofluorescence staining and subcellular fractionation, most of the C2A was found at the plasma membrane, even when the cells weredepleted of Ca^2 by incubation with EGTA. These resultssuggested a new molecular mechanism of syt I as a Ca^2 sensor in membrane fusion. Ca^2 -independent surface adsorption might attach syt I to the release site during the docking or priming step. When intracellular Ca^2 increased,syt I triggered the neurotransmitter release following the Ca^2 -dependent penetration into the target membrane.     

Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization

Botulinum neurotoxin (BoNY) type A, a causative agent of botulism, is a di-chain protein (molecular weight 140,000) from Clostridium botulinum, and the most neurotoxic substance known. Some cases of sudden infant cot deaths have been attributed to such a neuroparalytic condition. BoNT inhibits irreversibly the release of acetylcholine from peripheral nerves in a highly selective manner. Hence, it is potentially an invaluable probe for studying the mechanism of transmitter release. Here we demonstrate specific labelling of murine motor nerve terminals with neurotoxic, 125I-labelled BoNT (type A) by autoradiography. We observed saturable, temperature-sensitive binding of BoNT to sites which reside solely on the nerve terminal membrane; these were distributed on all unmyelinated areas, at an average density of 150-500 per micron2 of membrane. The binding was mediated by the larger subunit of the toxin and was inhibited partially by tetanus toxin, another microbial protein. No specific binding was detectable on any other cell types examined, including noradrenergic terminals. Following binding, internalization of radioactivity was observed; this process was energy-dependent as it could be prevented totally by azide or dinitrophenol (DNP). This direct demonstration of separable steps, including highly selective binding and acceptor-mediated internalization, is reconcilable with the unique potency and the multiphasic inhibitory action of BoNT on transmitter release, as shown electrophysiologically.     

Synaptic function modulated by changes in the ratio of synaptotagmin I and IV.

Communication within the nervous system is mediated by Ca2+-triggered fusion of synaptic vesicles with the presynaptic plasma membrane. Genetic and biochemical evidence indicates that synaptotagmin I may function as a Ca2+ sensor in neuronal exocytosis because it can bind Ca2+ and penetrate into lipid bilayers. Chronic depolarization or seizure activity results in the upregulation of a distinct and unusual isoform of the synaptotagmin family, synaptotagmin IV. We have identified a Drosophila homologue of synaptotagmin IV that is enriched on synaptic vesicles and contains an evolutionarily conserved substitution of aspartate to serine that abolishes its ability to bind membranes in response to Ca2+ influx. Synaptotagmin IV forms hetero-oligomers with synaptotagmin I, resulting in synaptotagmin clusters that cannot effectively penetrate lipid bilayers and are less efficient at coupling Ca2+ to secretion in vivo: upregulation of synaptotagmin IV, but not synaptotagmin I, decreases evoked neurotransmission. These findings indicate that modulating the expression of synaptotagmins with different Ca2+-binding affinities can lead to heteromultimers that can regulate the efficiency of excitation-secretion coupling in vivo and represent a new molecular mechanism for synaptic plasticity.     

Coiled-coil fibrous domains mediate ligand binding by macrophage scavenger receptor type II

The macrophage scavenger receptor, which has been implicated in the pathogenesis of atherosclerosis, has an unusually broad binding specificity. Ligands include modified low-density lipoprotein and some polyanions (for example, poly(I) but not poly(C]. The scavenger receptor type I (ref. 3) has three principal extracellular domains that could participate in ligand binding: two fibrous coiled-coil domains (alpha-helical coiled-coil domain IV and collagen-like domain V), and the 110-amino-acid cysteine-rich C-terminal domain VI. We have cloned complementary DNAs encoding a second scavenger receptor which we have termed type II. This receptor is identical to the type I receptor, except that the cysteine-rich domain is replaced by a six-residue C terminus. Despite this truncation, the type II receptor mediates endocytosis of chemically modified low-density lipoprotein with high affinity and specificity, similar to that of the type I receptor. Therefore one or both of the extracellular fibrous domains are responsible for the unusual ligand-binding specificity of the receptor.     

Different domains of synaptotagmin control the choice between kiss-and-run and full fusion

Exocytosis-the release of the contents of a vesicle--proceeds by two mechanisms. Full fusion occurs when the vesicle and plasma membranes merge. Alternatively, in what is termed kiss-and-run, vesicles can release transmitter during transient contacts with the plasma membrane. Little is known at the molecular level about how the choice between these two pathways is regulated. Here we report amperometric recordings of catecholamine efflux through individual fusion pores. Transfection with synaptotagmin (Syt) IV increased the frequency and duration of kiss-and-run events, but left their amplitude unchanged. Endogenous Syt IV, induced by forskolin treatment, had a similar effect. Full fusion was inhibited by mutation of a Ca2+ ligand in the C2A domain of Syt I; kiss-and-run was inhibited by mutation of a homologous Ca2+ ligand in the C2B domain of Syt IV. The Ca2+ sensitivity for full fusion was 5-fold higher with Syt I than Syt IV, but for kiss-and-run the Ca2+ sensitivities differed by a factor of only two. Syt thus regulates the choice between full fusion and kiss-and-run, with Ca2+ binding to the C2A and C2B domains playing an important role in this choice.     

Synaptic vesicle-associated Ca2+/calmodulin-dependent protein kinase II is a binding protein for synapsin I.

Synapsin I is a synaptic vesicle-associated phosphoprotein that is involved in the modulation of neurotransmitter release. Ca2+/calmodulin-dependent protein kinase II, which phosphorylates two sites in the carboxy-terminal region of synapsin I, causes synapsin I to dissociate from synaptic vesicles and increases neurotransmitter release. Conversely, the dephosphorylated form of synapsin I, but not the form phosphorylated by Ca2+/calmodulin-dependent protein kinase II, inhibits neurotransmitter release. The amino-terminal region of synapsin I interacts with membrane phospholipids, whereas the C-terminal region binds to a protein component of synaptic vesicles. Here we demonstrate that the binding of the C-terminal region of synapsin I involves the regulatory domain of a synaptic vesicle-associated form of Ca2+/calmodulin-dependent protein kinase II. Our results indicate that this form of the kinase functions both as a binding protein for synapsin I, and as an enzyme that phosphorylates synapsin I and promotes its dissociation from the vesicles.     

A型肉毒杆菌毒素的基因测序与分析

对国内A型肉毒杆菌毒素基因测序,分析了解毒素基因结构并推测其氨基酸序列。提取肉毒杆菌总DNA,利用自行设计的引物对目的基因进行PCR扩增,把目的基因导入E.Coli JM109,筛选含目的基因的阳性克隆菌进行测序和分析,从而了解毒素基因结构并推测其氮基酸序列。对A型肉毒杆菌毒素基因的核苷酸序列成功测序并与GenBank中的相应序列比较,其序列同源性为96％。推测其氨基酸序列同源性为100％。成功地对A型肉毒杆菌毒素的基因进行了测序,这为肉毒杆菌毒素中毒的快速诊断,以及进一步探求以基因工程方法生产此毒素奠定基础。     

Structure of the dimerized hormone-binding domain of a guanylyl-cyclase-coupled receptor

The atrial natriuretic peptide (ANP) hormone is secreted by the heart in response to an increase in blood pressure. ANP exhibits several potent anti-hypertensive actions in the kidney, adrenal gland and vascular system. These actions are induced by hormone binding extracellularly to the ANP receptor, thereby activating its intracellular guanylyl cyclase domain for the production of cyclic GMP. Here we present the crystal structure of the glycosylated dimerized hormone-binding domain of the ANP receptor at 2.0-A resolution. The monomer comprises two interconnected subdomains, each encompassing a central beta-sheet flanked by alpha-helices, and exhibits the type I periplasmic binding protein fold. Dimerization is mediated by the juxtaposition of four parallel helices, arranged two by two, which brings the two protruding carboxy termini into close relative proximity. From affinity labelling and mutagenesis studies, the ANP-binding site maps to the side of the dimer crevice and extends to near the dimer interface. A conserved chloride-binding site is located in the membrane distal domain, and we found that hormone binding is chloride dependent. These studies suggest mechanisms for hormone activation and the allostery of the ANP receptor.     

Control of transmembrane lipid asymmetry in chromaffin granules by an ATP-dependent protein

The Ca2+-dependent binding of annexin proteins to secretory granule membranes seems to be involved in the early stage of exocytosis. Binding studies have shown that these proteins have a specificity for phosphatidylserine (PtdS) interfaces. Furthermore, aminolipids are necessary for contact and fusion between lipid vesicles or between liposomes and chromaffin granules. Thus, PtdS must be present on the granule outer (cytoplasmic) monolayer. We report here that chromaffin granules possess a mechanism to maintain PtdS orientation, comparable to the ATP-dependent aminophospholipid translocase from human erythrocytes. The translocase, in granules, selectively transports PtdS from the luminal to the cytoplasmic monolayer, provided the incubation medium contains ATP. As this protein shares several properties with the granule vanadate-sensitive ATPase II, we infer that this ATPase, of relative molecular mass 115,000, is the protein responsible for aminophospholipid translocation. This is the first evidence for an ATP-dependent specific phospholipid 'flippase' in intracellular organelles.     

Salsolinol合成酶的克隆和表达

 Salsolinol 合成酶是一种催化多巴胺和乙醛生成Salsolinol 的酶,与帕金森病发病机制密切相关。研究发现Salsolinol 合成酶与泛素的氨基酸序列高度相似,只有4 个氨基酸位点有差异。本研究以泛素基因为模板,采用聚合酶链式反应技术对4 个位点进行定点突变,将突变基因片段克隆到载体pET30a-GST 上,构建pET30a-GST-Sal synthase 重组载体,转化BL21 后,IPTG 诱导重组菌表达融合蛋白,经亲和层析柱纯化。结果表明,实现目的位点的定点突变,获得Sal 合成酶基因,成功构建了GST-Sal synthase 原核表达质粒,在大肠杆菌中表达纯化后得到较高纯度的GST-Sal synthase 融合蛋白。     

Crystal structure of the anthrax lethal factor.

Lethal factor (LF) is a protein (relative molecular mass 90,000) that is critical in the pathogenesis of anthrax. It is a highly specific protease that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family near to their amino termini, leading to the inhibition of one or more signalling pathways. Here we describe the crystal structure of LF and its complex with the N terminus of MAPKK-2. LF comprises four domains: domain I binds the membrane-translocating component of anthrax toxin, the protective antigen (PA); domains II, III and IV together create a long deep groove that holds the 16-residue N-terminal tail of MAPKK-2 before cleavage. Domain II resembles the ADP-ribosylating toxin from Bacillus cereus, but the active site has been mutated and recruited to augment substrate recognition. Domain III is inserted into domain II, and seems to have arisen from a repeated duplication of a structural element of domain II. Domain IV is distantly related to the zinc metalloprotease family, and contains the catalytic centre; it also resembles domain I. The structure thus reveals a protein that has evolved through a process of gene duplication, mutation and fusion, into an enzyme with high and unusual specificity.