Function of the translocation domain belt

Abstracts Toxins 2011 / Toxicon 68 (2013) 60–123

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pain syndrome, piriformis syndrome, therapy-resistant epicondylitis, plantar fasciitis and stump pain have been treated with BoNT serotype A. However, overall, only case series and small controlled studies have provided a very low level of evidence for these indications.

Dr. Foster moved to CAMR, now the U.K. Health Protection Agency (HPA) at Porton Down, to continue the work on the neurotoxin fragments. This work established proprietary technology for the targeted delivery of clostridial neurotoxin endopeptidase activity that is the basis of Syntaxin Ltd., a spinout from HPA. Dr. Foster is an internationally recognized expert in botulinum neurotoxin biology and is currently Founder and Chief Technology Officer of Syntaxin Ltd.

Bernhard Voller, M.D., [email protected], National Institute of Neurological Disorders and Stroke, USA. Dr. Voller received his M.D. degree from the Medical University of Innsbruck, Austria, in 1994. His doctoral thesis was entitled, “Neuropsychological, MRI, and EEG findings after very Mild Traumatic Brain Injury.” He completed his residency in Neurology at the Medical University of Vienna, Austria. From 2002 to 2004, he was an intramural research fellow at the Human Motor Control Section, National Institute of Neurological Disorders and Stroke (NINDS), Bethesda, MD, under the supervision of Dr. Mark Hallett, and later, at the Human Cortical Physiology Section, under the supervision of Dr. Leonardo Cohen. He is presently Associate Professor at the Medical University of Vienna. Currently, he works as a Special Volunteer at the Human Motor Control Section, NINDS. He has more than 30 publications in the fields of botulinum toxin use, movement disorders and neurorehabilitation.

http://dx.doi.org/10.1016/j.toxicon.2012.07.031

http://dx.doi.org/10.1016/j.toxicon.2012.07.030

Re-targeting of botulinum neurotoxins K.A. Foster Syntaxin Ltd., Abingdon, UK

Syntaxin is developing a new class of biopharmaceuticals called Targeted Secretion Inhibitors (TSIs) based on engineered botulinum neurotoxins. Botulinum neurotoxins are effective in treating neuromuscular conditions, their therapeutic success resulting from specific and potent inhibition of neurotransmitter release. However, the clinical utility of botulinum neurotoxins is limited by their toxicity and restricted target cell activity. Understanding the structurefunction relationship of the neurotoxins and relating this understanding to their clinical properties has provided an opportunity to engineer novel recombinant proteins that employ specific pharmacological properties of the neurotoxins. This engineering has made available a range of proteins, the TSIs, for the treatment of diseases that are not amenable to therapy with the native neurotoxins, but for which the pharmacological properties of the neurotoxin sub-units provide clinical benefit and therapeutic advantage. A feature of TSIs is that they inhibit secretion from the target cell for a prolonged period following a single application, making them particularly suited to the treatment of chronic disease. Syntaxin's technology has broad potential for the discovery of therapeutic proteins to treat a wide range of diseases. Current programs focus on diseases with high unmet clinical needs that include pain, neurological disorders and endocrine diseases.

Blocking botulism – A journey into modules and modulators M. Montal Section of Neurobiology, Division of Biological Sciences, University of California San Diego, La Jolla, CA, USA

Botulinum neurotoxin (BoNT), the causative agent of botulism, is acknowledged to be one of the most feared biological weapons of the 21st century (CDC Category A). The imminent threat of a terrorist attack empowered by BoNT remains an issue of major concern nationally and globally. For BoNT, a modular nanomachine, functional complexity emerges from its modular design and the tight interplay between its component modules–a partnership with consequences that surpass the simple sum of the individual component's action. This presentation focuses on the dynamic interplay between modules, which is inextricably linked not only to the activity of the toxin, but also to its sensitivity to small-molecule translocation inhibitors that possess antibotulinal activity. Targeting the protein-conducting channel of BoNT is anticipated to open a new path for developing countermeasures, an endeavor of paramount significance to health science and biodefense. Mauricio Montal, M.D., Ph.D., [email protected], University of California San Diego, USA. Mauricio Montal received his M.D. from the National University of Mexico (1971) and his Ph.D. from the University of Pennsylvania (1970). In 1976, he joined the faculty at the University of California San Diego, where he is a Distinguished Professor of Biological Sciences in the Section of Neurobiology. His research expertise areas include the botulinum neurotoxins and mechanisms of synaptic exocytosis; the structure, function and dysfunction of channel proteins; mechanisms of neuronal excitability and synaptic transmission; and neuronal survival and the connection between excitability and apoptotic death. He is the recipient of many honors that include earning his M.D. summa cum laude, serving as the Founding Chair of the Neurosciences Department at the Roche Institute of Molecular Biology, and receiving a Guggenheim Fellowship. He has also been recognized by the Cole Award of the Biophysical Society and by a Research Scientist Award from the National Institute of Mental Health. Dr. Montal is a Fellow of the Biophysical Society and a Member of the National Academy of Sciences of Mexico. He was selected to be the 17th Friedrich Merz Guest Professor at Goethe-University and Merz Pharma, Frankfurt, Germany. Dr. Montal serves as Editor of FEBS LETTERS (Federation of European Biochemical Societies Letters) and as a consultant to pharmaceutical and biotechnological companies. http://dx.doi.org/10.1016/j.toxicon.2012.07.032

Keith Foster, Ph.D., [email protected], Syntaxin Ltd., UK. Dr. Foster completed his doctorate in the biochemistry of permeability changes to eukaryotic cell membranes caused by enveloped viruses at St. George's Hospital Medical School, University of London, in 1980. Following postdoctoral research into inositol phosphate metabolism in neuronal tissue with Professor Tim Hawthorne at the University of Nottingham, he moved to Beecham Pharmaceuticals and subsequently to SmithKline Beecham (SB), where his research focused on arachidonic acid metabolism and signal transduction in inflammatory cells. He left SB in 1993 to join the newly established Speywood Laboratory Ltd. Here he was responsible for establishing, de novo, a research facility and team to undertake studies into the therapeutic potential of botulinum neurotoxin fragments. In 1995,

Function of the translocation domain belt M. Galloux a, A. Araye-Guet a, H. Vitrac b, C. Montagner b, S. Raffestin a, M.R. Popoff c, A. Chenal d, V. Forge b, D. Gillet a a

SIMOPRO-IBITECS, CEA, Gif sur Yvette, France IRTSV, CEA, Grenoble, France Anaerobic Bacteria and Toxins Unit, Pasteur Institute, Paris, France d Biochemistry of Macromolecular Interactions Unit, Pasteur Institute, Paris, France b c

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Abstracts Toxins 2011 / Toxicon 68 (2013) 60–123

We describe the successive steps by which the botulinum neurotoxin A translocation domain binds and penetrates a phospholipid membrane as a function of pH. A focus was made on the belt region sticking out of the translocation (T) domain and embracing the catalytic domain of the toxin. Four recombinant versions of the translocation domain were produced with an intact, partially or fully truncated loop. Interaction with membranes was studied from pH 7.5 to pH 3.5 by tryptophane fluorescence, FRET with dansyl groups in the polar head groups of the phospholipids, and tryptophane fluorescence quenching by phospholipids brominated on their acyl chains. Large unilamelar vesicle permeabilization by the T domains was also monitored. The interaction with membranes does not involve major secondary or tertiary structure changes, as reported for other toxins such as diphtheria toxin. The T domain becomes insoluble around its pI value and then penetrates into the membrane. At that stage the translocation domain becomes able to permeabilize lipid vesicles. This event occurs for pH values lower than 5.5, as in the endosomes. Electrostatic interactions mediated by the belt region, which contains numerous negatively-charged residues, limits the protein-membrane interaction. Indeed, interaction with the membrane of the protein deleted of this extremity takes place for higher pH values than for the entire T domain. The N-terminal belt of the translocation domain of botulinum neurotoxin A, which surrounds the catalytic domain, plays an important role in membrane penetration of the toxin, enabling the interaction with the membrane at a pH lower than in the absence of the belt. Daniel Gillet, Ph.D., [email protected], CEA, France. Daniel Gillet obtained a Ph.D. in molecular biology from University Paris 7-Denis-Diderot in 1989. He joined the team of André Ménez at the CEA in Saclay in 1990, where he worked on the production of recombinant tracers for diagnostics. He spent a year in John R. Murphy's laboratory at Boston University in 1993 studying the possibility of using diphtheria toxin as a translocator for DNA and proteins. Following his return to CEA, he worked on fundamental aspects of diphtheria toxin translocation and biotechnological applications of bacterial and animal toxins. Since 2005, he has been involved in research for inhibitors of toxins that include ricin and botulinum toxins. Daniel Gillet is a Coordinator for Biology of the French CBRN Research and Development program for civil biodefense conducted at the CEA. http://dx.doi.org/10.1016/j.toxicon.2012.07.033

Mode of substrate binding and cleavage S. Swaminathan, R. Agarwal, D. Kumaran

three-dimensional structures of their catalytic domains and their almost identical active site architectures, clostridial neurotoxins have the ability to recognize specific SNARE proteins and precisely position a unique peptide bond at the active site for cleavage. This precision is possibly due to the presence of multiple recognition sites that define the specificity of peptide cleavage. BoNT/A and /C cleave adjacent peptide bonds of the same substrate SNAP-25, while BoNT/F and /D do the same with VAMP. This versatile mechanism of this class of enzymes is still not understood completely. Although extensive biochemical and modeling studies have been carried out to understand the mechanism of recognition, only two substrate-enzyme complex structures are available. An overview of the mode of recognition of substrate binding sites and peptide bond cleavage will be presented. Subramanyam Swaminathan, Ph.D., [email protected], Brookhaven National Laboratory, USA. Dr. Swaminathan received his Ph.D. in Crystallography from the University of Madras, India. As a post-doctoral fellow at the University of Pittsburgh, he worked on the charge density analysis of small molecules using both X-ray and neutron diffraction and then worked on the structure of superantigens (the staphylococcal enterotoxins) at the Veterans Administration Medical Center in Pittsburgh, PA. He currently holds a Senior Scientist position at Brookhaven National Laboratory, New York. His major research interest is in understanding the structure– function relationships in proteins, especially the Clostridium botulinum neurotoxins, via macromolecular crystallography. He is also involved in discovery of drugs for treating botulism. He is a member of the Institute of Chemical Biology and Drug Discovery at Stony Brook University, New York. http://dx.doi.org/10.1016/j.toxicon.2012.07.034

Molecular mechanism of calcium-triggered vesicle fusion M. Kyoung a, b, c, d, e, f, A. Srivastava a, b, c, d, e, Y. Zhang f, J. Diao a, b, c, d, e, M. Vrljic a, b, c, d, e, P. Grob g, E. Nogales g, h, S. Chu h, i,1, A.T. Brunger a, b, c, d, e a Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA b Department of Neurology and Neurological Science, Stanford University, Stanford, CA, USA c Department of Structural Biology, Stanford University, Stanford, CA, USA d Department of Photon Science, Stanford University, Stanford, CA, USA e Howard Hughes Medical Institute, Stanford, CA, USA f California Institute for Quantitative Biosciences, University of California Berkeley, Berkeley CA, USA g Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA h Lawrence Berkeley National Laboratory, Berkeley, CA, USA i Departments of Physics and Molecular and Cell Biology, University of California, Berkeley, CA, USA

Biology Department, Brookhaven National Laboratory, Upton, New York, USA

Botulinum neurotoxins (BoNTs) and tetanus neurotoxins (TeNT) inhibit neurotransmitter release by cleaving a single peptide bond in one of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNARE) that causes flaccid and spastic paralysis, respectively. The seven serotypes (A–G) of BoNTs and TeNT cleave a unique peptide bond in a specific SNARE protein, synaptosomal-associated protein 25 kDa (SNAP-25), vesicle-associated membrane protein (VAMP), or syntaxin. Only BoNT/B and TeNT cleave the same scissile bond in VAMP, and BoNT/C cleaves both SNAP-25 and syntaxin. Clostridial neurotoxins are unique in that their substrates are large polypeptides. In spite of the similar

The highly conserved SNARE protein family mediates membrane fusion in eukaryotic cells. Over the last decade, an in vitro ensemble lipid-mixing assay has been commonly used for studying SNARE-mediated membrane fusion. There are inherent limitations with this lipid mixing assay since it cannot distinguish different stages of fusion, such as docking, hemifusion, and full fusion (i.e., pore opening). Since lipid mixing was often misinterpreted as complete fusion, it may have contributed to sometimes conflicting views of the mechanism of certain accessory proteins such as complexin 1

Present address: Department of Energy, Washington, DC, USA.