Israel Silman

Acetylcholinesterase, a synaptic enzyme

The Bernstein-Mason Professorial Chair of Neurochemistry

Tel: (972-8) 934-3649
Fax: (972-8) 934-4131
e-mail: israel.silman@weizmann.ac.il

I did my Ph.D. thesis under the supervision of Prof. E. Katzir at The Weizmann Institute. I returned in 1968 after postdoctoral studies in the U.S, and joined the newly founded department of Neurobiology in 1975. I am currently Head of the Nella and Leon Benoziyo Center for the Neurosciences.

Current and future research

My research concerns the enzyme acetylcholinesterase (AChE), which terminates impulse transmission at cholinergic synapses by hydrolyzing the neurotransmitter, acetylcholine (ACh). AChE is a rapid enzyme that occurs in multiple forms differing in structure and modes of attachment to the cell surface. They are generated by alternative splicing, which leads to differential posttranslational modification producing several modes of membrane-anchoring. My work deals with the structure of AChE, its mechanism, its anchoring and localization, and with regulation of its folding and assembly.

Most of our work employs a dimeric form of AChE from the electric organ of Torpedo californica. This form belongs to a family of membrane proteins, the GPI-proteins; they are anchored to the outer face of the plasma membrane by a single residue of phosphatidylinositol (PI), covalently attached, via an oligoglycan (G) extension, at the COOH-terminus. With Mike Ferguson and Steve Homans (University of Dundee), we have determined the structure of the anchoring domain.

With Joel Sussman and Michal Harel (Dept. of Structural Biology), AChE crystals suitable for X-ray crystallography were obtained. The 3D structure revealed that the active site is near the bottom of a deep and narrow cavity named the 'aromatic gorge', since it is lined by the rings of 14 conserved aromatic amino acids. The quaternary group of ACh does not interact with an array of negative charges, but with the rings of certain aromatic residues. Several conserved aromatics in the gorge are missing in the homologous enzyme, butyrylcholinesterase (BChE). Modeling and site-directed mutagenesis, the latter in collaboration with Jean Massoulie (Ecole Normale Superieure, Paris), permit assignment of functional roles to some of these residues, and to confer BChE-like characteristics on Torpedo AChE. Electrostatic calculations based on the crystal structure (with Daniel Ripoll and Carlos Faerman, Cornell University, Ithaca), indicate that it has a strong electric dipole, aligned with the active-site gorge, so that the positively charged ACh is drawn to the active site by the field. The affinity of quaternary ammoniums for aromatic rings, coupled with the electrostatic force, suggest that the gorge is an 'affinity electrophoresis' column permitting rapid and selective access of ACh to the active site.

Studies on folding and assembly of AChE take two principal lines: With Lev Weiner (Dept. of Organic Chemistry), we have shown that modifying Torpedo AChE by thiol reagents inhibits it irreversibly, even though the thiol modified is not involved in catalytic activity. Modification converts AChE to a Ômolten globule,Õ which maintains most of the secondary structure of native AChE but is devoid of significant tertiary structure. The second approach utilizes heat shock to study AChE regulation in cultured chick muscle. Heat shock proteins, rather than de novo protein synthesis, may be involved in the rapid recovery of AChE activity observed in such cultures subsequent to heat shock. With Lili Anglister (Hebrew University), we have used a novel approach to localization of GPI-anchored AChE in Torpedo tissues, combining its selective removal by GPI-specific phospholipase C with immunocytochemistry. Immunofluorescence shows this form is localized in motor and electromotor nerves and in the corresponding synaptic connections.

Selected List of Publications

Eichler, J. & Silman, I. (1995). The activity of an endoplasmic reticulum-localized pool of acetylcholinesterase is modulated by heat-shock. J. Biol. Chem. 270, 4466-4472.

Eichler, J., Tolliday, N., Toker, L. & Silman, I. (1995). Purification and characterization of HSP70 proteins from Torpedo electric organ. Comp. Biochem. Physiol. 110B, 409-415.

Kreimer, D.I., Shnyrov, V.L., Villar, E., Silman, I. & Weiner, L. (1995). Irreversible thermal denaturation of Torpedo californica acetylcholinesterase. Protein Sci. 4, 2349-2357.

Harel, M., Kleywegt, G.J., Ravelli, R.B.G., Silman, I. & Sussman, J.L. (1995). Crystal structure of an acetylcholinesterase-fasciculin complex: interaction of a three-fingered toxin from snake venom with its target. Structure 3, 1355-1366.

Shin, I., Silman, I. & Weiner, L.M. (1996). Interaction of partially unfolded forms of Torpedo acetylcholinesterase with liposomes. Protein Sci. 5, 52-61.

Harel, M., Quinn, D.M., Nair, H.K., Silman, I. & Sussman, J.L. (1996). The structure of a transition state analog complex reveals the molecular origins of the catalytic power and substrate specificity of acetylcholinesterase. J. Am. Chem. Soc. 118, 2340-2346.

Porschke, D., Créminon, C., Cousin, X., Bon, C., Sussman, J. & Silman, I. (1996). Electrooptical measurements demonstrate a large permanent dipole moment associated with acetylcholinesterase. Biophys. J. 70, 1603-1608.

Faerman, C., Ripoll, D., Bon, S., Le Feuvre, Y., Morel, N., Massoulié, J., Sussman, J.L. & Silman, I. (1996). Site-directed mutants designed to test back-door hypotheses of acetylcholinesterase function. FEBS Lett. 386, 65-71.

Peng, L., Silman, I., Sussman, J.L. & Goeldner, M. (1996). Biochemical evaluation of photolabile precursors of choline and carbamylcholine for potential time-resolved crystallographic studies on cholinesterases. Biochemistry 35, 10854-10861.

Kreimer, D.I., Shin, I., Shnyrov, V.L., Villar, E., Silman, I. & Weiner, L. (1996).Two partially unfolded states of Torpedo acetylcholinesterase. Protein Sci. 5, 1852-1864.

Raves, M.L., Harel, M., Pang, Y.-P., Silman, I., Kozikowski, A.P. & Sussman, J.L. (1997). 3D structure of acetylcholinesterase complexed with the nootropic alkaloid, (-)-huperzine A. Nature Struct. Biol. 4, 57-63.

Shin, I., Kreimer, D.I., Silman, I. & Weiner, L. (1997). Membrane-promoted unfolding of acetylcholinesterase: A possible mechanism for insertion into the lipid bilayer. Proc. Natl. Acad. Sci. USA 94, 2848-2852.

Felder, C.E., Botti, S.A., Lifson, S., Silman, I. & Sussman, J.L. (1997). External and internal electrostatic potentials of cholinesterase models. J. Mol. Graphics & Modeling 15, 318-327.

Shin, I., Silman, I., Bon, C. & Weiner, L. (1998). Liposome-catalyzed unfolding of acetylcholinesterase from Bungarus fasciatus. Biochemistry 37, 4310-4316.

Botti, S.A., Felder, C.E., Sussman, J.L. & Silman, I. (1998). Electrotactins: A class of adhesion proteins with conserved electrostatic and structural motifs. Protein Engng. 11, 415-420.

Ravelli, R.B.G., Raves, M.L., Ren, Z., Bourgeois, D., Roth, M., Kroon, J., Silman, I. & Sussman, J.L. (1998). Static Laue diffraction studies on acetylcholinesterase. Acta Cryst. D54, 1359-1366.

Kryger, G., Silman, I. & Sussman, J.L. (1999). Structure of acetylcholinesterase complexed with E2020 (Aricept®): implications for the design of new anti-Alzheimer drugs. Structure 7, 297-307.

Morel, N., Bon, S., Greenblatt, H., Van Belle, D., Wodak, S.J., Sussman, J.L., Massoulié, J. & Silman, I. (1999). Effect of mutations within the peripheral site on the stability of acetylcholinesterase. Mol. Pharmacol. 55, 982-992.

Millard, C.B., Kryger, G., Ordentlich, A., Harel, M., Raves, M.L, Greenblatt, H.M., Segall, Y., Barak, D., Shafferman, A., Silman, I. & Sussman, J.L. (1999). Crystal structures of "aged" phosphylated Torpedo californica acetylcholinesterase: Nerve agent reaction products at the atomic level. Biochemistry 38, 7032-7039.

Weiner, L., Roth, E., Mazur, Y. & Silman, I. (1999). Photosensitized cross-linking of native and partially unfolded forms of acetylcholinesterase by hypericin. Biochemistry 38, in press.

Botti, S.A., Felder, C.E., Lifson, S., Sussman, J.L. & Silman, I. (1999). A modular treatment of molecular traffic of through the active-site of cholinesterases Biophys. J., in press.

Weik, M., Kryger, G., Schreurs, Silman, I., Sussman, J.L., Gros, P. & Kroon, J. (1999). Glass transition of water in protein crystals. Acta Cryst. D, submitted.

Tarrab-Hazdai, R., Toker, L., Silman, I. & Arnon,R. (1999).Acetylcholinesterase from Schistosoma mansoni: Interaction of globular species with heparin. Biochem. J., submitted.

Weik, M., Ravelli, R.B.G., Kryger, G., McSweeney, S., Raves, M.L., Harel, M., Gros, P., Silman, I., Kroon, J. & Sussman, J.L. (1999). Specific chemical and structural damage to proteinsby synchrotron radiation. Nature Struct. Biol., submitted.

Millard, C.B., Koellner, G., Ordentlich, A., Shafferman, A., Silman, I. & Sussman, J.L. (1999). Reaction of acetylcholinesterase with VX reveals a mobile histidine in the catalytic triad. J. Am. Chem. Soc., submitted.

Book chapters

Karlsson, E., Harvey, A.L., Cerveñansky, C., Kleywegt, G.J., Harel, M., Silman, I. & Sussman, J.L. (1998). Fasciculins, cholinesterase inhibitors from mamba venoms. In: Snake Venom Enzymes (G.S. Bailey, ed.), pp. 633-688, Alaken, Inc., Fort Collins, CO.

Silman, I. & Sussman, J.L. (1998). Structural and functional studies on acetylcholine sterase: a perspective. In: Structure and Function of Cholinesterases and Related Proteins (Doctor, B.P., Quinn, D.M., Rotundo, R.L. & Taylor, P., eds.), pp. 25-33, Plenum Press, NY.

Silman, I. (1999). Acetylcholine receptor. In: Encyclopedia of Molecular Biology (T.E. Creighton, ed.), Wiley, New York, in press.

Sussman, J.L., Harel, M., Raves, M., Quinn, D. & Silman, I. (1995). 3-D structure of acetylcholinesterase and its complexes with anticholinesterase agents. In: Modelling of Biomolecular Structures and Mechanisms (Pullman, A., Jortner, J. & Pullman, B., eds.), Kluwer Academic Publishers, Dordrecht, Holland, pp. 455-460.

Sussman, J.L., Harel, M. Raves, M., Quinn, D.M., Nair, H.K. & Silman, I. (1995). Structures of complexes of acetylcholinesterase with covalently and non-covalently bound inhibitors. In: Enzymes of the Cholinesterase Family (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P. & Taylor, P., eds.), Plenum, New York, pp. 59-65.

Ripoll, D.R., Faerman, C.H., Gillilan, R., Silman, I. & Sussman, J.L. (1995). Electrostatic properties of human acetylcholinesterase. In: Enzymes of the Cholinesterase Family (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P. & Taylor, P., eds.), Plenum, New York, pp. 67-70.

Silman, I., Kreimer, D.I., Shin, I., Dolginova, E.A., Roth, E., Goldfarb, D., Szosenfogel, R., Raves, M., Sussman, J.L., Borochov, N. & Weiner, L. (1995). Studies on partially unfolded states of Torpedo californica acetylcholinesterase. In: Enzymes of the Cholinesterase Family (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P. & Taylor, P., eds.), Plenum Press, New York, pp. 77-82.

Sussman, J.L., Harel, M., Raves, M.L., Giles, K., Ravelli, R.G.B., Peng, L., Goeldner, M., Kleywegt, G.J., Quinn, D.M., Nair, H.K. & Silman, I. (1996). Studies on the 3D structure of Torpedo acetylcholinesterase. Proc. USAMRMC 1996 Medical Defense Biosci. Rev., 13-22.

Giles, K., Raves, M., Silman, I. & Sussman, J.L. (1997). How three-fingered toxins associate with their target: acetylcholinesterase-fasciculin, a case study. In: Theoretical and Computational Methods of Genome Research (Suhai, S., ed.), Plenum Press, New York, pp. 303-315.

Sussman, J.L., Harel, M., Raves, M. & Silman, I. (1997). Crystallographic studies on complexes of acetylcholinesterase with the natural cholinesterase inhibitors huperzine A and fasciculin. Italian Biochem. Soc. Trans. 8, 62-65.

Silman, I., Shin, I., Kreimer, D.I. & Weiner, L. (1997). Interaction with the lipid bilayer of native and molten globule states of aceylcholinesterase from Torpedo californica. Italian Biochem. Soc. Trans. 8, 73-76.

Silman, I., Harel, M., Raves, M. & Sussman, J.L. (1998). Crystallographic studies on complexes of acetylcholinesterase with the natural cholinesterase inhibitors fasciculin and huperzine A. In: Progress in Alzheimer's and Parkinson's Diseases (Fisher, A., Yoshida, M. & Hanin, I., eds.), Plenum Press, New York, pp. 523-530.

Botti, S.A., Felder, C., Lifson, S., Sussman, J.L. & Silman, I. (1998). An integrated model for the molecular traffic through the active site of cholinesterases. In: Structure and Function of Cholinesterases and Related Proteins (Doctor, B.P., Quinn, D.M., Rotundo, R.L. & Taylor, P., eds.), pp. 227-228, Plenum Press, NY.

Nicolas, A., Millard, C.B., Raves, M.L., Ravelli, R.B.G., Kroon, J., Silman, I. & Sussman, J.L. (1998). Activity of Torpedo californica acetylcholinesterase in the crystalline state. In: Structure and Function of Cholinesterases and Related Proteins (Doctor, B.P., Quinn, D.M., Rotundo, R.L. & Taylor, P., eds.), pp. 230-231, Plenum Press, NY.

Kryger, G., Giles, K., Harel, M., Toker, L., Velan, B., Lazar, A., Kronman, C., Barak, D., Ariel, N., Shafferman, A., Silman, I. & Sussman, J.L. (1998). 3D structure at 2.7Å resolution of native and E202Q mutant human acetylcholinesterase complexed with fasciculin-II. In: Structure and Function of Cholinesterases and Related Proteins (Doctor, B.P., Quinn, D.M., Rotundo, R.L. & Taylor, P., eds.), pp. 323-326, Plenum Press, NY.

Raves, M.L., Giles, K.,Schrag, J.D., Schmid, M.F., Phillips, G.N., Jr., Chiu, W., Howard, A.J., Silman, I. & Sussman, J.L.(1998). Quaternary structure of tetrameric acetylcholinesterase. In: Structure and Function of Cholinesterases and Related Proteins (Doctor, B.P., Quinn, D.M., Rotundo, R.L. & Taylor, P., eds.), pp. 351-356, Plenum Press, NY.

Greenblatt, H.M., Kryger, G., Harel, M., Lewis, T., Taylor, J., Silman, I. & Sussman, J.L. (1998). Crystal structures of complexes of E2020-related compounds with Torpedo californica acetylcholinesterase.In: Structure and Function of Cholinesterases and Related Proteins (Doctor, B.P., Quinn, D.M., Rotundo, R.L. & Taylor, P., eds.), p. 371, Plenum Press, NY.

Raves, M.L., Greenblatt, H.M., Kryger, G., Nicolas, A., Ravelli, R.B.G., Harel, M., Kroon, J., Silman, I. & Sussman, J.L. (1998). Alternative crystal forms of Torpedo californica acetylcholinesterase.In: Structure and Function of Cholinesterases and Related Proteins (Doctor, B.P., Quinn, D.M., Rotundo, R.L. & Taylor, P., eds.), p. 372, Plenum Press, NY.

Baron, P., Millard, C., Enz, A., Sussman, J.L. & Silman, I. (1998). Kinetic and X-ray crystallographic studies of the binding of ENA-713 to Torpedo californica acetylcholinesterase. In: Structure and Function of Cholinesterases and Related Proteins (Doctor, B.P., Quinn, D.M., Rotundo, R.L. & Taylor, P., eds.), pp. 373-374, Plenum Press, NY.

Millard, C.B., Kryger, G., Ordentlich, A., Harel, M., Raves M.L, Greenblatt, H.M., Segall, Y., Barak, D., Shafferman, A., Silman, I. & Sussman, J.L. (1998). Crystal structures of "aged" phosphorylated and phosphonylated Torpedo californica acetylcholinesterase. In: Structure and Function of Cholinesterases and Related Proteins (Doctor, B.P., Quinn, D.M., Rotundo, R.L. & Taylor, P., eds.), pp. 425-431, Plenum Press, NY.

Morel, M., Bon, S., Sussman, J., Massoulié, J. & Silman, I. (1998). Surface residues near the peripheral site affect the stability of Torpedo californica acetylcholinesterase. In: Structure and Function of Cholinesterases and Related Proteins (Doctor, B.P., Quinn, D.M., Rotundo, R.L. & Taylor, P., eds.), p.435, Plenum Press, NY.

Shin, I., Silman, I., Bon, C. & Weiner, L. (1998). Membrane-promoted unfolding of Torpedo californica and Bungarus fasciatus acetylcholinesterase. In: Structure and Function of Cholinesterases and Related Proteins (Doctor, B.P., Quinn, D.M., Rotundo, R.L. & Taylor, P., eds.), pp. 438-439, Plenum Press, NY.

Giles, K., Ben-Yohanan, R., Velan, B., Shafferman, A., Sussman, J.L. & Silman, I. (1998). Assembly of acetylcholinesterase subunits in vitro. In: Structure and Function of Cholinesterases and Related Proteins (Doctor, B.P., Quinn, D.M., Rotundo, R.L. & Taylor, P., eds.), pp. 442-443, Plenum Press, NY.

Botti, S.A., Felder, C., Sussman, J.L. & Silman, I. (1998). The conjunction of a conserved electrostatic motif and a common cholinesterase fold defines a class of adhesion proteins. In: Structure and Function of Cholinesterases and Related Proteins (Doctor, B.P.,Quinn, D.M., Rotundo, R.L. & Taylor, P., eds.), pp. 448-449, Plenum Press, NY.

Giles, K., Silman, I. & Sussman, J.L. (1998). Expression and tissue distribution of cholinesterases via EST analysis. In: Structure and Function of Cholinesterases and Related Proteins (Doctor, B.P., Quinn, D.M., Rotundo, R.L. & Taylor, P., eds.), p. 450, Plenum Press, NY.

Kryger, G., Silman, I. & Sussman, J.L. (1998). 3D structure of a complex of the anti-Alzheimer drug, E2020, with acetylcholinesterase from at 2.5 Å resolution. In: Structure and Function of Cholinesterases and Related Proteins (Doctor, B.P., Quinn, D.M., Rotundo, R.L. & Taylor, P., eds.), pp. 469-475, Plenum Press, NY.

Kryger, G., Silman, I. & Sussman, J.L. (1998). Three-dimensional structure of a complex of E2020 with acetylcholinesterase from Torpedo californica. J. Physiol. (Paris) 92, 53-57.

Bar-On, P., Harel, M., Millard, C.B., Enz, A., Sussman, J.L. & Silman, I. (1998). Kinetic and structural studies on the interaction of the anti-Alzheimer drug, ENA-713, with Torpedo californica acetylcholinesterase. J. Physiol. (Paris) 92, 406-407.

Botti, S., Felder, C., Sussman, J.L. & Silman, I. (1998). Electrostatic homology modelling of a set of ChE-like neural adhesion proteins identifies a shared `annular' motif with ChEs. Structural implications for a cell-recognition role of ChEs. J. Physiol. (Paris) 92, 414-416.

Tarrab-Hazdai, R., Silman, I., Toker, L., Schechtman, D. & Arnon, R. (1998). Acetylcholinesterase of S. mansoni (Trematode). Interaction of globular species with glycosaminoglycans. J. Physiol. (Paris) 92, 501-502.

Silman, I., Millard, C.B., Ordentlich, A., Greenblatt, H.M., Harel, M., Barak, D., Shafferman, A. & Sussman, J.L. (1998). A preliminary comparison of structural models for catalytic intermediates of acetylcholinesterase. Proc. 3rd International Meeting on Esterases Reacting with Organophosphorus Compounds, Dubrovnik, April 1998, Chem.-Biol. Interactions, in press.

Millard, C.B., Kryger, G., Ordentlich, A., Harel, M., Raves, M.L, Greenblatt, H.M., Segall, Y., Barak, D., Shafferman, A., Silman, I. & Sussman, J.L. (1998). Structural models for the acylation and deacylation transition states of acetylcholinesterase: a preliminary comparison. Proc. US Army Medical Defence Bioscience Review, Hunt Valley, MD, June 1998, in press.

A molecular model of the enzyme acetylcholinesterase.