Harel M

General

Full name : Harel Michal

First name : Michal

Mail : Weizmann Institute of Science Structural Biology, Rehovot 76100

Zip Code :

City :

Country : Israel

Email : michal.harel@weizmann.ac.il

Phone : 972-8-9342647

Fax : 972-8-9344159

Website :

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References (52)

Title : Proteopedia: a status report on the collaborative, 3D web-encyclopedia of proteins and other biomolecules - Prilusky_2011_J.Struct.Biol_175_244
Author(s) : Prilusky J , Hodis E , Canner D , Decatur WA , Oberholser K , Martz E , Berchanski A , Harel M , Sussman JL
Ref : J Struct Biol , 175 :244 , 2011
Abstract : Proteopedia is a collaborative, 3D web-encyclopedia of protein, nucleic acid and other biomolecule structures. Created as a means for communicating biomolecule structures to a diverse scientific audience, Proteopedia (http://www.proteopedia.org) presents structural annotation in an intuitive, interactive format and allows members of the scientific community to easily contribute their own annotations. Here, we provide a status report on Proteopedia by describing advances in the web resource since its inception three and a half years ago, focusing on features of potential direct use to the scientific community. We discuss its progress as a collaborative 3D-encyclopedia of structures as well as its use as a complement to scientific publications and PowerPoint presentations. We also describe Proteopedia's use for 3D visualization in structure-related pedagogy.
ESTHER : Prilusky_2011_J.Struct.Biol_175_244
PubMedSearch : Prilusky_2011_J.Struct.Biol_175_244
PubMedID: 21536137

Title : Acetylcholinesterase: from 3D structure to function - Dvir_2010_Chem.Biol.Interact_187_10
Author(s) : Dvir H , Silman I , Harel M , Rosenberry TL , Sussman JL
Ref : Chemico-Biological Interactions , 187 :10 , 2010
Abstract : By rapid hydrolysis of the neurotransmitter, acetylcholine, acetylcholinesterase terminates neurotransmission at cholinergic synapses. Acetylcholinesterase is a very fast enzyme, functioning at a rate approaching that of a diffusion-controlled reaction. The powerful toxicity of organophosphate poisons is attributed primarily to their potent inhibition of acetylcholinesterase. Acetylcholinesterase inhibitors are utilized in the treatment of various neurological disorders, and are the principal drugs approved thus far by the FDA for management of Alzheimer's disease. Many organophosphates and carbamates serve as potent insecticides, by selectively inhibiting insect acetylcholinesterase. The determination of the crystal structure of Torpedo californica acetylcholinesterase permitted visualization, for the first time, at atomic resolution, of a binding pocket for acetylcholine. It also allowed identification of the active site of acetylcholinesterase, which, unexpectedly, is located at the bottom of a deep gorge lined largely by aromatic residues. The crystal structure of recombinant human acetylcholinesterase in its apo-state is similar in its overall features to that of the Torpedo enzyme; however, the unique crystal packing reveals a novel peptide sequence which blocks access to the active-site gorge.
ESTHER : Dvir_2010_Chem.Biol.Interact_187_10
PubMedSearch : Dvir_2010_Chem.Biol.Interact_187_10
PubMedID: 20138030
Gene_locus related to this paper: human-ACHE

Title : Microcalorimetric study of the inhibition of butyrylcholinesterase by paraoxon - Debord_2009_Anal.Biochem_389_97
Author(s) : Debord J , Harel M , Verneuil B , Bollinger JC , Dantoine T
Ref : Analytical Biochemistry , 389 :97 , 2009
Abstract : The inhibition of horse serum butyrylcholinesterase (EC 3.1.1.8) by the organophosphorus compound paraoxon (diethyl 4-nitrophenyl phosphate) was studied by flow microcalorimetry at 37 degrees C in Tris buffer (pH 7.5) using a modification of the kinetic model described by Stojan and coworkers [J. Stojan, V. Marcel, S. Estrada-Mondaca, A. Klaebe, P. Masson, D. Fournier, A putative kinetic model for substrate metabolisation by Drosophila acetylcholinesterase, FEBS Lett. 440 (1998) 85-88]. The reversible steps of the inhibition were studied in the mixing cell of the calorimeter, whereas the irreversible step was studied in the flow-through cell. A new pseudo-first-order approximation was developed to allow the kinetic analysis of inhibition progress curves in the presence of substrate when a significant amount of substrate is transformed. This approximation also allowed one to compute an analytical expression of the calorimetric curves using a gamma distribution to describe the impulse response of the calorimeter. Fitting models to data by nonlinear regression, with simulated annealing as a stochastic optimization method, allowed the determination of all kinetic parameters. It was found that paraoxon binds to both the enzyme and acyl-enzyme, but with weak affinities (K(i) = 0.123 mM and K'(i) = 5.5 mM). A slight activation was observed at the lowest paraoxon concentrations and was attributed to the binding of the substrate to the enzyme-inhibitor complex. The bimolecular inhibition rate constant k(i) = 2.8 x 10(4) M(-1) s(-1) was in agreement with previous studies. It is hoped that the methods developed in this work will contribute to extending the application range of microcalorimetry in the field of irreversible inhibitors.
ESTHER : Debord_2009_Anal.Biochem_389_97
PubMedSearch : Debord_2009_Anal.Biochem_389_97
PubMedID: 19341699

Title : Atomic interactions of neonicotinoid agonists with AChBP: molecular recognition of the distinctive electronegative pharmacophore - Talley_2008_Proc.Natl.Acad.Sci.U.S.A_105_7606
Author(s) : Talley TT , Harel M , Hibbs RE , Radic Z , Tomizawa M , Casida JE , Taylor P
Ref : Proc Natl Acad Sci U S A , 105 :7606 , 2008
Abstract : Acetylcholine-binding proteins (AChBPs) from mollusks are suitable structural and functional surrogates of the nicotinic acetylcholine receptors when combined with transmembrane spans of the nicotinic receptor. These proteins assemble as a pentamer with identical ACh binding sites at the subunit interfaces and show ligand specificities resembling those of the nicotinic receptor for agonists and antagonists. A subset of ligands, termed the neonicotinoids, exhibit specificity for insect nicotinic receptors and selective toxicity as insecticides. AChBPs are of neither mammalian nor insect origin and exhibit a distinctive pattern of selectivity for the neonicotinoid ligands. We define here the binding orientation and determinants of differential molecular recognition for the neonicotinoids and classical nicotinoids by estimates of kinetic and equilibrium binding parameters and crystallographic analysis. Neonicotinoid complex formation is rapid and accompanied by quenching of the AChBP tryptophan fluorescence. Comparisons of the neonicotinoids imidacloprid and thiacloprid in the binding site from Aplysia californica AChBP at 2.48 and 1.94 A in resolution reveal a single conformation of the bound ligands with four of the five sites occupied in the pentameric crystal structure. The neonicotinoid electronegative pharmacophore is nestled in an inverted direction compared with the nicotinoid cationic functionality at the subunit interfacial binding pocket. Characteristic of several agonists, loop C largely envelops the ligand, positioning aromatic side chains to interact optimally with conjugated and hydrophobic regions of the neonicotinoid. This template defines the association of interacting amino acids and their energetic contributions to the distinctive interactions of neonicotinoids.
ESTHER : Talley_2008_Proc.Natl.Acad.Sci.U.S.A_105_7606
PubMedSearch : Talley_2008_Proc.Natl.Acad.Sci.U.S.A_105_7606
PubMedID: 18477694

Title : Crystal structure of thioflavin T bound to the peripheral site of Torpedo californica acetylcholinesterase reveals how thioflavin T acts as a sensitive fluorescent reporter of ligand binding to the acylation site - Harel_2008_J.Am.Chem.Soc_130_7856
Author(s) : Harel M , Sonoda LK , Silman I , Sussman JL , Rosenberry TL
Ref : Journal of the American Chemical Society , 130 :7856 , 2008
Abstract : Acetylcholinesterase plays a key role in cholinergic synaptic transmission by hydrolyzing the neurotransmitter acetylcholine with one of the highest known catalytic rate constants. Hydrolysis occurs in a narrow and deep gorge that contains two sites of ligand binding: A peripheral site, or P-site, near the gorge entrance that contributes to catalytic efficiency both by transiently trapping substrate molecules as they enter the gorge and by allosterically accelerating the transfer of the substrate acyl group to a serine hydroxyl in an acylation site or A-site at the base of the gorge. Thioflavin T is a useful reporter of ligand interactions with the A-site. It binds specifically to the P-site with fluorescence that is enhanced approximately 1000-fold over that of unbound thioflavin T, and the enhanced fluorescence is quenched 1.5- to 4-fold when another ligand binds to the A-site in a ternary complex. To clarify the structural basis of this advantageous signal change, we here report the X-ray structure of the complex of thioflavin T with Torpedo californica acetylcholinesterase. The two aromatic rings in thioflavin T are coplanar and are packed snugly parallel to the aromatic side chains of Trp279, Tyr334, and Phe330. Overlays of this structure with the crystal structures of Torpedo californica acetylcholinesterase complexes with either edrophonium or m-( N, N, N-trimethylammonio)-2,2,2-trifluoroacetophenone, two small aromatic ligands that bind specifically to the A-site, indicate that the phenyl side chain of Phe330 must rotate to sterically accommodate both thioflavin T and the A-site ligand in the ternary complex. This rotation may allow some relaxation of the strict coplanarity of the aromatic rings in the bound thioflavin T and result in partial quenching of its fluorescence.
ESTHER : Harel_2008_J.Am.Chem.Soc_130_7856
PubMedSearch : Harel_2008_J.Am.Chem.Soc_130_7856
PubMedID: 18512913
Gene_locus related to this paper: torca-ACHE

Title : The 3D structure of the anticancer prodrug CPT-11 with Torpedo californica acetylcholinesterase rationalizes its inhibitory action on AChE and its hydrolysis by butyrylcholinesterase and carboxylesterase - Harel_2005_Chem.Biol.Interact_157-158_153
Author(s) : Harel M , Hyatt JL , Brumshtein B , Morton CL , Wadkins RM , Silman I , Sussman JL , Potter PM
Ref : Chemico-Biological Interactions , 157-158 :153 , 2005
Abstract : The anticancer prodrug CPT-11 is a highly effective camptothecin analog that has been approved for the treatment of colon cancer. The 2.6 angstroms resolution crystal structure of its complex with Torpedo californica acetylcholinesterase (TcAChE) demonstrates that CPT-11 binds to TcAChE and spans its gorge similarly to the Alzheimer drug, Aricept. The crystal structure clearly reveals the interactions, which contribute to the inhibitory action of CPT-11. Modeling of the complexes of CPT-11 with mammalian butyrylcholinesterase and carboxylesterase, both of which are known to hydrolyze the drug, shows how binding to either of the two enzymes yields a productive substrate-enzyme complex.
ESTHER : Harel_2005_Chem.Biol.Interact_157-158_153
PubMedSearch : Harel_2005_Chem.Biol.Interact_157-158_153
PubMedID: 16289500
Gene_locus related to this paper: torca-ACHE

Title : The crystal structure of the complex of the anticancer prodrug 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin (CPT-11) with Torpedo californica acetylcholinesterase provides a molecular explanation for its cholinergic action - Harel_2005_Mol.Pharmacol_67_1874
Author(s) : Harel M , Hyatt JL , Brumshtein B , Morton CL , Yoon KJ , Wadkins RM , Silman I , Sussman JL , Potter PM
Ref : Molecular Pharmacology , 67 :1874 , 2005
Abstract : The anticancer prodrug 7-ethyl-10-[4-(1-piperidino)-1-piperidino-]carbonyloxycamptothecin (CPT-11) is a highly effective camptothecin analog that has been approved for the treatment of colon cancer. It is hydrolyzed by carboxylesterases to yield 7-ethyl-10-hydroxycamptothecin (SN-38), a potent topoisomerase I poison. However, upon high-dose intravenous administration of CPT-11, a cholinergic syndrome is observed that can be ameliorated by atropine. Previous studies have indicated that CPT-11 can inhibit acetylcholinesterase (AChE), and here, we provide a detailed analysis of the inhibition of AChE by CPT-11 and by structural analogs. These studies demonstrate that the terminal dipiperidino moiety in CPT-11 plays a major role in enzyme inhibition, and this has been confirmed by X-ray crystallographic studies of a complex of the drug with Torpedo californica AChE. Our results indicate that CPT-11 binds within the active site gorge of the protein in a fashion similar to that observed with the Alzheimer drug donepezil. The 3D structure of the CPT-11/AChE complex also permits modeling of CPT-11 complexed with mammalian butyrylcholinesterase and carboxylesterase, both of which are known to hydrolyze the drug to the active metabolite. Overall, the results presented here clarify the mechanism of AChE inhibition by CPT-11 and detail the interaction of the drug with the protein. These studies may allow the design of both novel camptothecin analogs that would not inhibit AChE and new AChE inhibitors derived from the camptothecin scaffold.
ESTHER : Harel_2005_Mol.Pharmacol_67_1874
PubMedSearch : Harel_2005_Mol.Pharmacol_67_1874
PubMedID: 15772291
Gene_locus related to this paper: torca-ACHE

Title : Inhibition of acetylcholinesterase by the anticancer prodrug CPT-11 - Hyatt_2005_Chem.Biol.Interact_157-158_247
Author(s) : Hyatt JL , Tsurkan L , Morton CL , Yoon KJ , Harel M , Brumshtein B , Silman I , Sussman JL , Wadkins RM , Potter PM
Ref : Chemico-Biological Interactions , 157-158 :247 , 2005
Abstract : CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is an anticancer prodrug that has been approved for the treatment of colon cancer. It is a member of the camptothecin class of drugs and activation to the active metabolite SN-38, is mediated by carboxylesterases (CE). SN-38 is a potent topoisomerase I poison and is highly effective at killing human tumor cells, with IC50 values in the low nM range. However, upon high dose administration of CPT-11 to cancer patients, a cholinergic syndrome is observed, that can be rapidly ameliorated by atropine. This suggests a direct interaction of the drug or its metabolites with acetylcholinesterase (AChE). Kinetic studies indicated that CPT-11 was primarily responsible for AChE inhibition with the 4-piperidinopiperidine moiety, the major determinant in the loss of enzyme activity. Structural analogs of 4-piperidinopiperidine however, did not inhibit AChE, including a benzyl piperazine derivate of CPT-11. These results suggest that novel anticancer drugs could be synthesized that do not inhibit AChE, or alternatively, that novel AChE inhibitors could be designed based around the camptothecin scaffold.
ESTHER : Hyatt_2005_Chem.Biol.Interact_157-158_247
PubMedSearch : Hyatt_2005_Chem.Biol.Interact_157-158_247
PubMedID: 16257398

Title : Structure and evolution of the serum paraoxonase family of detoxifying and anti-atherosclerotic enzymes - Harel_2004_Nat.Struct.Mol.Biol_11_412
Author(s) : Harel M , Aharoni A , Gaidukov L , Brumshtein B , Khersonsky O , Meged R , Dvir H , Ravelli RB , McCarthy A , Toker L , Silman I , Sussman JL , Tawfik DS
Ref : Nat Struct Mol Biol , 11 :412 , 2004
Abstract : Members of the serum paraoxonase (PON) family have been identified in mammals and other vertebrates, and in invertebrates. PONs exhibit a wide range of physiologically important hydrolytic activities, including drug metabolism and detoxification of nerve agents. PON1 and PON3 reside on high-density lipoprotein (HDL, 'good cholesterol') and are involved in the prevention of atherosclerosis. We describe the first crystal structure of a PON family member, a variant of PON1 obtained by directed evolution, at a resolution of 2.2 A. PON1 is a six-bladed beta-propeller with a unique active site lid that is also involved in HDL binding. The three-dimensional structure and directed evolution studies permit a detailed description of PON1's active site and catalytic mechanism, which are reminiscent of secreted phospholipase A2, and of the routes by which PON family members diverged toward different substrate and reaction selectivities.
ESTHER : Harel_2004_Nat.Struct.Mol.Biol_11_412
PubMedSearch : Harel_2004_Nat.Struct.Mol.Biol_11_412
PubMedID: 15098021

Title : Poster (48) Crystal structure of the tetramerization domain of acetylcholinesterase reveals a model of the AChE tetramer -
Author(s) : Harel M , Dvir H , Bon S , Liu WQ , Garbay C , Sussman JL , Massoulie J , Silman I
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :347 , 2004
PubMedID:

Title : The synaptic acetylcholinesterase tetramer assembles around a polyproline II helix - Dvir_2004_EMBO.J_23_4394
Author(s) : Dvir H , Harel M , Bon S , Liu WQ , Vidal M , Garbay C , Sussman JL , Massoulie J , Silman I
Ref : EMBO Journal , 23 :4394 , 2004
Abstract : Functional localization of acetylcholinesterase (AChE) in vertebrate muscle and brain depends on interaction of the tryptophan amphiphilic tetramerization (WAT) sequence, at the C-terminus of its major splice variant (T), with a proline-rich attachment domain (PRAD), of the anchoring proteins, collagenous (ColQ) and proline-rich membrane anchor. The crystal structure of the WAT/PRAD complex reveals a novel supercoil structure in which four parallel WAT chains form a left-handed superhelix around an antiparallel left-handed PRAD helix resembling polyproline II. The WAT coiled coils possess a WWW motif making repetitive hydrophobic stacking and hydrogen-bond interactions with the PRAD. The WAT chains are related by an approximately 4-fold screw axis around the PRAD. Each WAT makes similar but unique interactions, consistent with an asymmetric pattern of disulfide linkages between the AChE tetramer subunits and ColQ. The P59Q mutation in ColQ, which causes congenital endplate AChE deficiency, and is located within the PRAD, disrupts crucial WAT-WAT and WAT-PRAD interactions. A model is proposed for the synaptic AChE(T) tetramer.
ESTHER : Dvir_2004_EMBO.J_23_4394
PubMedSearch : Dvir_2004_EMBO.J_23_4394
PubMedID: 15526038
Gene_locus related to this paper: torca-ACHE

Title : The binding site for alpha-bungarotoxin in the acetylcholine receptor. -
Author(s) : Fuchs S , Kasher R , Balass M , Scherf T , Harel M , Fridkin M , Sussman JL , Katchalski-Katzir E
Ref : Cholinergic Mechanisms, CRC Press :19 , 2004
PubMedID:

Title : Poster (79) X-ray structures of TcAChE complexed with (+)-huperzine a and (-)-huperzine b: structural evidence for an active-site rearrangement -
Author(s) : Dvir H , Jiang H , Wong DM , Harel M , Chetrit M , He XC , Jin GY , Yu GL , Tang XC , Silman I , Bai DL , Sussman JL
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :362 , 2004
PubMedID:

Title : Crystal structure of the tetramerization domain of acetylcholinesterase at 2. -
Author(s) : Harel M , Dvir H , Bon S , Liu WQ , Garbay C , Sussman JL , Massoulie J , Silman I
Ref : Cholinergic Mechanisms, CRC Press :183 , 2004
PubMedID:

Title : A distinct family of acetylcholinesterases is secreted by Nippostrongylus brasiliensis - Hussein_2002_Mol.Biochem.Parasitol_123_125
Author(s) : Hussein AS , Harel M , Selkirk ME
Ref : Molecular & Biochemical Parasitology , 123 :125 , 2002
Abstract : A third variant of acetylcholinesterase (AChE A) secreted by the parasitic nematode Nippostrongylus brasiliensis has been isolated which shows 63-64% identity to AChE B and AChE C, with a truncated carboxyl terminus and a short internal insertion relative to AChEs from other species. Three of the fourteen aromatic residues which line the active site gorge in Torpedo AChE are substituted by non-aromatic residues (Y70T, W279D and F288M). All three enzymes have 8 cysteine residues in conserved positions, including 6 which have been implicated in disulphide bonds in other AChEs. Phylogenetic analysis suggests that these enzymes form a distinct group which evolved after speciation and are most closely related to ACE-2 of Caenorhabditis elegans. Recombinant AChE A secreted by Pichia pastoris was monomeric and hydrophilic, with a substrate preference for acetylthiocholine and negligible activity against butyrylthiocholine. A model structure of AChE A built from the coordinates of the Torpedo californica AChE suggests that W345 (F331 in Torpedo) limits the docking of butyrylcholine. This model is consistent with mutational analysis of the nematode enzymes. Expression of AChE A is regulated at the transcriptional level independently of the other 2 secreted variants, with maximal expression by fourth stage larvae and young adult worms. These enzymes thus appear to represent an unusual family of AChEs with conserved structural features which operate outside the normal boundaries of known functions in regulation of endogenous neurotransmitter activity.
ESTHER : Hussein_2002_Mol.Biochem.Parasitol_123_125
PubMedSearch : Hussein_2002_Mol.Biochem.Parasitol_123_125
PubMedID: 12270628
Gene_locus related to this paper: nipbr-ACHEA

Title : Kinetic and structural studies on the interaction of cholinesterases with the anti-Alzheimer drug rivastigmine - Bar-On_2002_Biochemistry_41_3555
Author(s) : Bar-On P , Millard CB , Harel M , Dvir H , Enz A , Sussman JL , Silman I
Ref : Biochemistry , 41 :3555 , 2002
Abstract : Rivastigmine, a carbamate inhibitor of acetylcholinesterase, is already in use for treatment of Alzheimer's disease under the trade name of Exelon. Rivastigmine carbamylates Torpedo californica acetylcholinesterase very slowly (k(i) = 2.0 M(-1) min(-1)), whereas the bimolecular rate constant for inhibition of human acetylcholinesterase is >1600-fold higher (k(i) = 3300 M(-1) min(-1)). For human butyrylcholinesterase and for Drosophila melanogaster acetylcholinesterase, carbamylation is even more rapid (k(i) = 9 x 10(4) and 5 x 10(5) M(-1) min(-1), respectively). Spontaneous reactivation of all four conjugates is very slow, with <10% reactivation being observed for the Torpedo enzyme after 48 h. The crystal structure of the conjugate of rivastigmine with Torpedo acetylcholinesterase was determined to 2.2 A resolution. It revealed that the carbamyl moiety is covalently linked to the active-site serine, with the leaving group, (-)-S-3-[1-(dimethylamino)ethyl]phenol, being retained in the "anionic" site. A significant movement of the active-site histidine (H440) away from its normal hydrogen-bonded partner, E327, was observed, resulting in disruption of the catalytic triad. This movement may provide an explanation for the unusually slow kinetics of reactivation.
ESTHER : Bar-On_2002_Biochemistry_41_3555
PubMedSearch : Bar-On_2002_Biochemistry_41_3555
PubMedID: 11888271
Gene_locus related to this paper: torca-ACHE

Title : 3D structure of Torpedo californica acetylcholinesterase complexed with huprine X at 2.1 A resolution: kinetic and molecular dynamic correlates - Dvir_2002_Biochemistry_41_2970
Author(s) : Dvir H , Wong DM , Harel M , Barril X , Orozco M , Luque FJ , Munoz-Torrero D , Camps P , Rosenberry TL , Silman I , Sussman JL
Ref : Biochemistry , 41 :2970 , 2002
Abstract : Huprine X is a novel acetylcholinesterase (AChE) inhibitor, with one of the highest affinities reported for a reversible inhibitor. It is a synthetic hybrid that contains the 4-aminoquinoline substructure of one anti-Alzheimer drug, tacrine, and a carbobicyclic moiety resembling that of another AChE inhibitor, (-)-huperzine A. Cocrystallization of huprine X with Torpedo californica AChE yielded crystals whose 3D structure was determined to 2.1 A resolution. The inhibitor binds to the anionic site and also hinders access to the esteratic site. Its aromatic portion occupies the same binding site as tacrine, stacking between the aromatic rings of Trp84 and Phe330, whereas the carbobicyclic unit occupies the same binding pocket as (-)-huperzine A. Its chlorine substituent was found to lie in a hydrophobic pocket interacting with rings of the aromatic residues Trp432 and Phe330 and with the methyl groups of Met436 and Ile439. Steady-state inhibition data show that huprine X binds to human AChE and Torpedo AChE 28- and 54-fold, respectively, more tightly than tacrine. This difference stems from the fact that the aminoquinoline moiety of huprine X makes interactions similar to those made by tacrine, but additional bonds to the enzyme are made by the huperzine-like substructure and the chlorine atom. Furthermore, both tacrine and huprine X bind more tightly to Torpedo than to human AChE, suggesting that their quinoline substructures interact better with Phe330 than with Tyr337, the corresponding residue in the human AChE structure. Both (-)-huperzine A and huprine X display slow binding properties, but only binding of the former causes a peptide flip of Gly117.
ESTHER : Dvir_2002_Biochemistry_41_2970
PubMedSearch : Dvir_2002_Biochemistry_41_2970
PubMedID: 11863435
Gene_locus related to this paper: torca-ACHE

Title : X-ray Structures of Torpedo californica Acetylcholinesterase Complexed with (+)-Huperzine A and (-)-Huperzine B: Structural Evidence for an Active Site Rearrangement - Dvir_2002_Biochemistry_41_10810
Author(s) : Dvir H , Jiang H , Wong DM , Harel M , Chetrit M , He XC , Jin GY , Yu GL , Tang XC , Silman I , Bai DL , Sussman JL
Ref : Biochemistry , 41 :10810 , 2002
Abstract : Kinetic and structural data are presented on the interaction with Torpedo californica acetylcholinesterase (TcAChE) of (+)-huperzine A, a synthetic enantiomer of the anti-Alzheimer drug, (-)-huperzine A, and of its natural homologue (-)-huperzine B. (+)-Huperzine A and (-)-huperzine B bind to the enzyme with dissociation constants of 4.30 and 0.33 microM, respectively, compared to 0.18 microM for (-)-huperzine A. The X-ray structures of the complexes of (+)-huperzine A and (-)-huperzine B with TcAChE were determined to 2.1 and 2.35 A resolution, respectively, and compared to the previously determined structure of the (-)-huperzine A complex. All three interact with the "anionic" subsite of the active site, primarily through pi-pi stacking and through van der Waals or C-H.pi interactions with Trp84 and Phe330. Since their alpha-pyridone moieties are responsible for their key interactions with the active site via hydrogen bonding, and possibly via C-H.pi interactions, all three maintain similar positions and orientations with respect to it. The carbonyl oxygens of all three appear to repel the carbonyl oxygen of Gly117, thus causing the peptide bond between Gly117 and Gly118 to undergo a peptide flip. As a consequence, the position of the main chain nitrogen of Gly118 in the "oxyanion" hole in the native enzyme becomes occupied by the carbonyl of Gly117. Furthermore, the flipped conformation is stabilized by hydrogen bonding of Gly117O to Gly119N and Ala201N, the other two functional elements of the three-pronged "oxyanion hole" characteristic of cholinesterases. All three inhibitors thus would be expected to abolish hydrolysis of all ester substrates, whether charged or neutral.
ESTHER : Dvir_2002_Biochemistry_41_10810
PubMedSearch : Dvir_2002_Biochemistry_41_10810
PubMedID: 12196020
Gene_locus related to this paper: torca-ACHE

Title : Structure of a complex of the potent and specific inhibitor BW284C51 with Torpedo californica acetylcholinesterase - Felder_2002_Acta.Crystallogr.D.Biol.Crystallogr_58_1765
Author(s) : Felder CE , Harel M , Silman I , Sussman JL
Ref : Acta Crystallographica D Biol Crystallogr , 58 :1765 , 2002
Abstract : The X-ray crystal structure of Torpedo californica acetylcholinesterase (TcAChE) complexed with BW284C51 [CO[-CH(2)CH(2)-pC(6)H(4)-N(CH(3))(2)(CH(2)-CH=CH(2))](2)] is described and compared with the complexes of two other active-site gorge-spanning inhibitors, decamethonium and E2020. The inhibitor was soaked into TcAChE crystals in the trigonal space group P3(1)21, yielding a complex which diffracted to 2.85 A resolution. The structure was refined to an R factor of 19.0% and an R(free) of 23.4%; the final model contains the protein, inhibitor, 132 water molecules and three carbohydrate moieties. BW284C51 binds similarly to decamethonium and E2020, with its two phenyl and quaternary amino end-groups complexed to Trp84 in the catalytic site and to Trp279 in the peripheral binding site, and its central carbonyl group hydrogen bonded very weakly to Tyr121. Possible reasons for decamethonium's weaker binding are considered. The relative strength of binding of bisquaternary inhibitors to acetylcholinesterase and the effect of several mutations of the enzyme are discussed in the context of the respective X-ray structures of their complexes with the enzyme.
ESTHER : Felder_2002_Acta.Crystallogr.D.Biol.Crystallogr_58_1765
PubMedSearch : Felder_2002_Acta.Crystallogr.D.Biol.Crystallogr_58_1765
PubMedID: 12351819
Gene_locus related to this paper: torca-ACHE

Title : Specific chemical and structural damage to proteins produced by synchrotron radiation - Weik_2000_Proc.Natl.Acad.Sci.U.S.A_97_623
Author(s) : Weik M , Ravelli RB , Kryger G , McSweeney S , Raves ML , Harel M , Gros P , Silman I , Kroon J , Sussman JL
Ref : Proceedings of the National Academy of Sciences of the United States of America , 97 :623 , 2000
Abstract : Radiation damage is an inherent problem in x-ray crystallography. It usually is presumed to be nonspecific and manifested as a gradual decay in the overall quality of data obtained for a given crystal as data collection proceeds. Based on third-generation synchrotron x-ray data, collected at cryogenic temperatures, we show for the enzymes Torpedo californica acetylcholinesterase and hen egg white lysozyme that synchrotron radiation also can cause highly specific damage. Disulfide bridges break, and carboxyl groups of acidic residues lose their definition. Highly exposed carboxyls, and those in the active site of both enzymes, appear particularly susceptible. The catalytic triad residue, His-440, in acetylcholinesterase, also appears to be much more sensitive to radiation damage than other histidine residues. Our findings have direct practical implications for routine x-ray data collection at high-energy synchrotron sources. Furthermore, they provide a direct approach for studying the radiation chemistry of proteins and nucleic acids at a detailed, structural level and also may yield information concerning putative "weak links" in a given biological macromolecule, which may be of structural and functional significance.
ESTHER : Weik_2000_Proc.Natl.Acad.Sci.U.S.A_97_623
PubMedSearch : Weik_2000_Proc.Natl.Acad.Sci.U.S.A_97_623
PubMedID: 10639129
Gene_locus related to this paper: torca-ACHE

Title : Three-dimensional structures of Drosophila melanogaster acetylcholinesterase and of its complexes with two potent inhibitors - Harel_2000_Protein.Sci_9_1063
Author(s) : Harel M , Kryger G , Rosenberry TL , Mallender WD , Lewis T , Fletcher RJ , Guss JM , Silman I , Sussman JL
Ref : Protein Science , 9 :1063 , 2000
Abstract : We have crystallized Drosophila melanogaster acetylcholinesterase and solved the structure of the native enzyme and of its complexes with two potent reversible inhibitors, 1,2,3,4-tetrahydro-N-(phenylmethyl)-9-acridinamine and 1,2,3,4-tetrahydro-N-(3-iodophenyl-methyl)-9-acridinamine--all three at 2.7 A resolution. The refined structure of D. melanogaster acetylcholinesterase is similar to that of vertebrate acetylcholinesterases, for example, human, mouse, and fish, in its overall fold, charge distribution, and deep active-site gorge, but some of the surface loops deviate by up to 8 A from their position in the vertebrate structures, and the C-terminal helix is shifted substantially. The active-site gorge of the insect enzyme is significantly narrower than that of Torpedo californica AChE, and its trajectory is shifted several angstroms. The volume of the lower part of the gorge of the insect enzyme is approximately 50% of that of the vertebrate enzyme. Upon binding of either of the two inhibitors, nine aromatic side chains within the active-site gorge change their conformation so as to interact with the inhibitors. Some differences in activity and specificity between the insect and vertebrate enzymes can be explained by comparison of their three-dimensional structures.
ESTHER : Harel_2000_Protein.Sci_9_1063
PubMedSearch : Harel_2000_Protein.Sci_9_1063
PubMedID: 10892800
Gene_locus related to this paper: drome-ACHE

Title : Structures of recombinant native and E202Q mutant human acetylcholinesterase complexed with the snake-venom toxin fasciculin-II - Kryger_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_1385
Author(s) : Kryger G , Harel M , Giles K , Toker L , Velan B , Lazar A , Kronman C , Barak D , Ariel N , Shafferman A , Silman I , Sussman JL
Ref : Acta Crystallographica D Biol Crystallogr , 56 :1385 , 2000
Abstract : Structures of recombinant wild-type human acetylcholinesterase and of its E202Q mutant as complexes with fasciculin-II, a 'three-finger' polypeptide toxin purified from the venom of the eastern green mamba (Dendroaspis angusticeps), are reported. The structure of the complex of the wild-type enzyme was solved to 2.8 A resolution by molecular replacement starting from the structure of the complex of Torpedo californica acetylcholinesterase with fasciculin-II and verified by starting from a similar complex with mouse acetylcholinesterase. The overall structure is surprisingly similar to that of the T. californica enzyme with fasciculin-II and, as expected, to that of the mouse acetylcholinesterase complex. The structure of the E202Q mutant complex was refined starting from the corresponding wild-type human acetylcholinesterase structure, using the 2.7 A resolution data set collected. Comparison of the two structures shows that removal of the charged group from the protein core and its substitution by a neutral isosteric moiety does not disrupt the functional architecture of the active centre. One of the elements of this architecture is thought to be a hydrogen-bond network including residues Glu202, Glu450, Tyr133 and two bridging molecules of water, which is conserved in other vertebrate acetylcholinesterases as well as in the human enzyme. The present findings are consistent with the notion that the main role of this network is the proper positioning of the Glu202 carboxylate relative to the catalytic triad, thus defining its functional role in the interaction of acetylcholinesterase with substrates and inhibitors.
ESTHER : Kryger_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_1385
PubMedSearch : Kryger_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_1385
PubMedID: 11053835
Gene_locus related to this paper: human-ACHE

Title : Crystal structures of aged phosphonylated acetylcholinesterase: nerve agent reaction products at the atomic level - Millard_1999_Biochemistry_38_7032
Author(s) : Millard CB , Kryger G , Ordentlich A , Greenblatt HM , Harel M , Raves ML , Segall Y , Barak D , Shafferman A , Silman I , Sussman JL
Ref : Biochemistry , 38 :7032 , 1999
Abstract : Organophosphorus acid anhydride (OP) nerve agents are potent inhibitors which rapidly phosphonylate acetylcholinesterase (AChE) and then may undergo an internal dealkylation reaction (called "aging") to produce an OP-enzyme conjugate that cannot be reactivated. To understand the basis for irreversible inhibition, we solved the structures of aged conjugates obtained by reaction of Torpedo californica AChE (TcAChE) with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (sarin), or O-pinacolylmethylphosphonofluoridate (soman) by X-ray crystallography to 2.3, 2.6, or 2.2 A resolution, respectively. The highest positive difference density peak corresponded to the OP phosphorus and was located within covalent bonding distance of the active-site serine (S200) in each structure. The OP-oxygen atoms were within hydrogen-bonding distance of four potential donors from catalytic subsites of the enzyme, suggesting that electrostatic forces significantly stabilize the aged enzyme. The active sites of aged sarin- and soman-TcAChE were essentially identical and provided structural models for the negatively charged, tetrahedral intermediate that occurs during deacylation with the natural substrate, acetylcholine. Phosphorylation with DFP caused an unexpected movement in the main chain of a loop that includes residues F288 and F290 of the TcAChE acyl pocket. This is the first major conformational change reported in the active site of any AChE-ligand complex, and it offers a structural explanation for the substrate selectivity of AChE.
ESTHER : Millard_1999_Biochemistry_38_7032
PubMedSearch : Millard_1999_Biochemistry_38_7032
PubMedID: 10353814
Gene_locus related to this paper: torca-ACHE

Title : A preliminary comparison of structural models for catalytic intermediates of acetylcholinesterase - Silman_1999_Chem.Biol.Interact_119-120_43
Author(s) : Silman I , Millard CB , Ordentlich A , Greenblatt HM , Harel M , Barak D , Shafferman A , Sussman JL
Ref : Chemico-Biological Interactions , 119-120 :43 , 1999
Abstract : Determination of the three dimensional structure of Torpedo Californica acetylcholinesterase (TcAChE) provided an experimental tool for directly visualizing interaction of AChE with cholinesterase inhibitors of fundamental, pharmacological and toxicological interest. The structure revealed that the active site is located near the bottom of a deep and narrow gorge lined with 14 conserved aromatic amino acids. The structure of a complex of TcAChE with the powerful 'transition state analog' inhibitor, TMTFA, suggested that its orientation in the experimentally determined structure was very similar to that proposed for the natural substrate, acetylcholine, by manual docking. The array of enzyme-ligand interactions visualized in the TMFTA complex also are expected to envelope the unstable TI that forms with acetylcholine during acylation, and to sequester it from solvent. In our most recent studies, the crystal structures of several 'aged' conjugates of TcAChE obtained with OP nerve agents have been solved and compared with that of the native enzyme. The methylphosphonylated-enzyme obtained by reaction with soman provides a useful structural analog for the TI that forms during deacylation after the reaction of TcAChE with acetylcholine. By comparing these structures, we conclude that the same 'oxyanion hole' residues, as well as the aromatic side chains constituting the 'acyl pocket', participate in acylation (TMTFA-AChE) and deacylation (OP-AChE), and that AChE can accommodate both TIs at the bottom of the gorge without major conformational movements.
ESTHER : Silman_1999_Chem.Biol.Interact_119-120_43
PubMedSearch : Silman_1999_Chem.Biol.Interact_119-120_43
PubMedID: 10421437
Gene_locus related to this paper: torca-ACHE

Title : 3D Structure at 2.7 Resolution of Native and E202Q Mutant Human Acetylcholinesterase Complexed with Fasciculin-II -
Author(s) : Kryger G , Giles K , Harel M , Toker L , Velan B , Lazar A , Kronman C , Barak D , Ariel N , Shafferman A , Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :323 , 1998
PubMedID:

Title : 3D Structure of a Complex of Human Acetylcholinesterase with Fasciculin-II at 2.7 Resolution -
Author(s) : Kryger G , Giles K , Harel M , Toker L , Velan B , Lazar A , Kronman C , Barak D , Ariel N , Shafferman A , Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :370 , 1998
PubMedID:

Title : Crystal Structures of Complexes of E2020-Related Compounds with Torpedo Californica Acetylcholinesterase -
Author(s) : Greenblatt HM , Kryger G , Harel M , Lewis T , Taylor J , Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :371-371 , 1998
PubMedID:

Title : Alternative Crystal Forms of Torpedo Californica Acetylcholinesterase -
Author(s) : Raves ML , Greenblatt HM , Kryger G , Nicolas A , Ravelli RB , Harel M , Kroon J , Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :372 , 1998
PubMedID:

Title : Kinetic and X-Ray Crystallographic Studies of the Binding of ENA-713 to Torpedo Californica Acetylcholinesterase -
Author(s) : Bar-On P , Harel M , Millard CB , Enz A , Sussman JL , Silman I
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :373 , 1998
PubMedID:

Title : Kinetic and structural studies on the interaction of the anti-Alzheimer drug, ENA-713, with Torpedo californica acetylcholinesterase -
Author(s) : Bar-On P , Harel M , Millard CB , Enz A , Sussman JL , Silman I
Ref : Journal de Physiologie (Paris) , 92 :406 , 1998
PubMedID:

Title : Crystal Structures of Aged Phosphorylated and Phosphonylated Torpedo Californica Acetylcholinesterase -
Author(s) : Millard CB , Kryger G , Ordentlich A , Harel M , Raves ML , Greenblatt HM , Segall Y , Barak D , Shafferman A , Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :425 , 1998
PubMedID:

Title : Crystal structure of Aged phosphorylated and phosphonylated Torpedo Californica Acetylcholinesterase -
Author(s) : Millard CB , Kryger G , Ordentlich A , Harel M , Raves ML , Greenblatt HM , Segall Y , Barak D , Shafferman A , Silman I , Sussman JL
Ref : In Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :425 , 1998
PubMedID:
Gene_locus related to this paper: torca-ACHE

Title : Crystal Structures of Aged Phosphorylated and Phosphonylated Torpedo Californica Acetylcholinesterase -
Author(s) : Millard CB , Kryger G , Ordentlich A , Harel M , Raves ML , Greenblatt HM , Segall Y , Barak D , Shafferman A , Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :454 , 1998
PubMedID:

Title : Structure of acetylcholinesterase complexed with the nootropic alkaloid, (-)-huperzine A - Raves_1997_Nat.Struct.Biol_4_57
Author(s) : Raves ML , Harel M , Pang YP , Silman I , Kozikowski AP , Sussman JL
Ref : Nat Struct Biol , 4 :57 , 1997
Abstract : (-)-Huperzine A (HupA) is found in an extract from a club moss that has been used for centuries in Chinese folk medicine. Its action has been attributed to its ability to strongly inhibit acetylcholinesterase (AChE). The crystal structure of the complex of AChE with optically pure HupA at 2.5 A resolution shows an unexpected orientation for the inhibitor with surprisingly few strong direct interactions with protein residues to explain its high affinity. This structure is compared to the native structure of AChE devoid of any inhibitor as determined to the same resolution. An analysis of the affinities of structural analogues of HupA, correlated with their interactions with the protein, shows the importance of individual hydrophobic interactions between HupA and aromatic residues in the active-site gorge of AChE.
ESTHER : Raves_1997_Nat.Struct.Biol_4_57
PubMedSearch : Raves_1997_Nat.Struct.Biol_4_57
PubMedID: 8989325
Gene_locus related to this paper: torca-ACHE

Title : The X-ray structure of a transition state analog complex reveals the molecular origins of the catalytic power and substrate specificity of acetylcholinesterase. -
Author(s) : Harel M , Quinn DM , Nair HK , Silman I , Sussman JL
Ref : Journal of the American Chemical Society , 118 :2340 , 1996
PubMedID:
Gene_locus related to this paper: torca-ACHE

Title : Structures of Complexes of Acetylcholinesterase with Covalently and Non-Covalently Bound Inhibitors -
Author(s) : Sussman JL , Harel M , Raves ML , Quinn DM , Nair HK , Silman I
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :59 , 1995
PubMedID:

Title : Crystal structure of an acetylcholinesterase-fasciculin complex: interaction of a three-fingered toxin from snake venom with its target - Harel_1995_Structure_3_1355
Author(s) : Harel M , Kleywegt GJ , Ravelli RB , Silman I , Sussman JL
Ref : Structure , 3 :1355 , 1995
Abstract : BACKGROUND Fasciculin (FAS), a 61-residue polypeptide purified from mamba venom, is a three-fingered toxin which is a powerful reversible inhibitor of acetylcholinesterase (AChE). Solution of the three-dimensional structure of the AChE/FAS complex would provide the first structure of a three-fingered toxin complexed with its target. RESULTS: The structure of a complex between Torpedo californica AChE and fasciculin-II (FAS-II), from the venom of the green mamba (Dendroaspis angusticeps) was solved by molecular replacement techniques, and refined at 3.0 A resolution to an R-factor of 0.231. The structure reveals a stoichiometric complex with one FAS molecule bound to each AChE subunit. The AChE and FAS conformations in the complex are very similar to those in their isolated structures. FAS is bound at the 'peripheral' anionic site of AChE, sealing the narrow gorge leading to the active site, with the dipole moments of the two molecules roughly aligned. The high affinity of FAS for AChE is due to a remarkable surface complementarity, involving a large contact area (approximately 2000 A2) and many residues either unique to FAS or rare in other three-fingered toxins. The first loop, or finger, of FAS reaches down the outer surface of the thin aspect of the gorge. The second loop inserts into the gorge, with an unusual stacking interaction between Met33 in FAS and Trp279 in AChE. The third loop points away from the gorge, but the C-terminal residue makes contact with the enzyme. CONCLUSIONS: Two conserved aromatic residues in the AChE peripheral anionic site make important contacts with FAS. The absence of these residues from chicken and insect AChEs and from butyrylcholinesterase explains the very large reduction in the affinity of these enzymes for FAS. Several basic residues in FAS make important contacts with AChE. The complementarity between FAS and AChE is unusual, inasmuch as it involves a number of charged residues, but lacks any intermolecular salt linkages.
ESTHER : Harel_1995_Structure_3_1355
PubMedSearch : Harel_1995_Structure_3_1355
PubMedID: 8747462
Gene_locus related to this paper: torca-ACHE

Title : Three-dimensional structures of acetylcholinesterase and of its complexes with anticholinesterase agents -
Author(s) : Silman I , Harel M , Axelsen PH , Raves ML , Sussman JL
Ref : Biochemical Society Transactions , 22 :745 , 1994
PubMedID: 7821677

Title : Structure and dynamics of the active site gorge of acetylcholinesterase: synergistic use of molecular dynamics simulation and X-ray crystallography - Axelsen_1994_Prot.Sci_3_188
Author(s) : Axelsen PH , Harel M , Silman I , Sussman JL
Ref : Protein Science , 3 :188 , 1994
Abstract : The active site of acetylcholinesterase (AChE) from Torpedo californica is located 20 A from the enzyme surface at the bottom of a narrow gorge. To understand the role of this gorge in the function of AChE, we have studied simulations of its molecular dynamics. When simulations were conducted with pure water filling the gorge, residues in the vicinity of the active site deviated quickly and markedly from the crystal structure. Further study of the original crystallographic data suggests that a bis-quaternary decamethonium (DECA) ion, acquired during enzyme purification, residues in the gorge. There is additional electron density within the gorge that may represent small bound cations. When DECA and 2 cations are placed within the gorge, the simulation and the crystal structure are dramatically reconciled. The small cations, more so than DECA, appear to stabilize part of the gorge wall through electrostatic interactions. This part of the gorge wall is relatively thin and may regulate substrate, product, and water movement through the active site.
ESTHER : Axelsen_1994_Prot.Sci_3_188
PubMedSearch : Axelsen_1994_Prot.Sci_3_188
PubMedID: 8003956

Title : Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins - Cygler_1993_Protein.Sci_2_366
Author(s) : Cygler M , Schrag JD , Sussman JL , Harel M , Silman I , Gentry MK , Doctor BP
Ref : Protein Science , 2 :366 , 1993
Abstract : Based on the recently determined X-ray structures of Torpedo californica acetylcholinesterase and Geotrichum candidum lipase and on their three-dimensional superposition, an improved alignment of a collection of 32 related amino acid sequences of other esterases, lipases, and related proteins was obtained. On the basis of this alignment, 24 residues are found to be invariant in 29 sequences of hydrolytic enzymes, and an additional 49 are well conserved. The conservation in the three remaining sequences is somewhat lower. The conserved residues include the active site, disulfide bridges, salt bridges, and residues in the core of the proteins. Most invariant residues are located at the edges of secondary structural elements. A clear structural basis for the preservation of many of these residues can be determined from comparison of the two X-ray structures.
ESTHER : Cygler_1993_Protein.Sci_2_366
PubMedSearch : Cygler_1993_Protein.Sci_2_366
PubMedID: 8453375

Title : Quaternary ligand binding to aromatic residues in the active-site gorge of acetylcholinesterase - Harel_1993_Proc.Natl.Acad.Sci.U.S.A_90_9031
Author(s) : Harel M , Schalk I , Ehret-Sabatier L , Bouet F , Goeldner M , Hirth C , Axelsen PH , Silman I , Sussman JL
Ref : Proceedings of the National Academy of Sciences of the United States of America , 90 :9031 , 1993
Abstract : Binding sites of Torpedo acetylcholinesterase (EC 3.1.1.7) for quaternary ligands were investigated by x-ray crystallography and photoaffinity labeling. Crystal structures of complexes with ligands were determined at 2.8-A resolution. In a complex with edrophonium, and quaternary nitrogen of the ligand interacts with the indole of Trp-84, and its m-hydroxyl displays bifurcated hydrogen bonding to two members of the catalytic triad, Ser-200 and His-440. In a complex with tacrine, the acridine is stacked against the indole of Trp-84. The bisquaternary ligand decamethonium is oriented along the narrow gorge leading to the active site; one quaternary group is apposed to the indole of Trp-84 and the other to that of Trp-279, near the top of the gorge. The only major conformational difference between the three complexes is in the orientation of the phenyl ring of Phe-330. In the decamethonium complex it lies parallel to the surface of the gorge; in the other two complexes it is positioned to make contact with the bound ligand. This close interaction was confirmed by photoaffinity labelling by the photosensitive probe 3H-labeled p-(N,N-dimethylamino)benzenediazonium fluoroborate, which labeled, predominantly, Phe-330 within the active site. Labeling of Trp-279 was also observed. One mole of label is incorporated per mole of AcChoEase inactivated, indicating that labeling of Trp-279 and that of Phe-330 are mutually exclusive. The structural and chemical data, together, show the important role of aromatic groups as binding sites for quaternary ligands, and they provide complementary evidence assigning Trp-84 and Phe-330 to the "anionic" subsite of the active site and Trp-279 to the "peripheral" anionic site.
ESTHER : Harel_1993_Proc.Natl.Acad.Sci.U.S.A_90_9031
PubMedSearch : Harel_1993_Proc.Natl.Acad.Sci.U.S.A_90_9031
PubMedID: 8415649
Gene_locus related to this paper: torca-ACHE

Title : Three-dimensional structure of acetylcholinesterase and of its complexes with anticholinesterase drugs - Sussman_1993_Chem.Biol.Interact_87_187
Author(s) : Sussman JL , Harel M , Silman I
Ref : Chemico-Biological Interactions , 87 :187 , 1993
Abstract : Based on our recent X-ray crystallographic determination of the structure of acetylcholinesterase (AChE) from Torpedo californica, we can see for the first time, at atomic resolution, a protein binding pocket for the neurotransmitter, acetylcholine. It was found that the active site consists of a catalytic triad (S200-H440-E327) which lies close to the bottom of a deep and narrow gorge, which is lined with the rings of 14 aromatic amino acid residues. Despite the complexity of this array of aromatic rings, we suggested, on the basis of modelling which involved docking of the acetylcholine (ACh) molecule in an all-trans configuration, that the quaternary group of the choline moiety makes close contact with the indole ring of W84. In order to study the interaction of AChE with anticholinesterase drugs at the structural level, we have incorporated into the acetylcholinesterase crystals several different inhibitors, and have recently determined the 3-D structure of AChE:edrophonium and AChE:tacrine complexes. The crystal structures of both of these complexes are in good agreement with our model building of the ACh bound in the active site of AChE and indicate the interactions of these two drugs with the enzyme.
ESTHER : Sussman_1993_Chem.Biol.Interact_87_187
PubMedSearch : Sussman_1993_Chem.Biol.Interact_87_187
PubMedID: 8343975

Title : Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding - Shafferman_1992_J.Biol.Chem_267_17640
Author(s) : Shafferman A , Kronman C , Flashner Y , Leitner M , Grosfeld H , Ordentlich A , Gozes Y , Cohen S , Ariel N , Barak D , Harel M , Silman I , Sussman JL , Velan B
Ref : Journal of Biological Chemistry , 267 :17640 , 1992
Abstract : Evidence for the involvement of Ser-203, His-447, and Glu-334 in the catalytic triad of human acetylcholinesterase was provided by substitution of these amino acids by alanine residues. Of 20 amino acid positions mutated so far in human acetylcholinesterase (AChE), these three were unique in abolishing detectable enzymatic activity (less than 0.0003 of wild type), yet allowing proper production, folding, and secretion. This is the first biochemical evidence for the involvement of a glutamate in a hydrolase triad (Schrag, J.D., Li, Y., Wu, M., and Cygler, M. (1991) Nature 351, 761-764), supporting the x-ray crystal structure data of the Torpedo californica acetylcholinesterase (Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. and Silman, I. (1991) Science 253, 872-879). Attempts to convert the AChE triad into a Cys-His-Glu or Ser-His-Asp configuration by site-directed mutagenesis did not yield effective AChE activity. Another type of substitution, that of Asp-74 by Gly or Asn, generated an active enzyme with increased resistance to succinylcholine and dibucaine; thus mimicking in an AChE molecule the phenotype of the atypical butyrylcholinesterase natural variant (D70G mutation). Mutations of other carboxylic residues Glu-84, Asp-95, Asp-333, and Asp-349, all conserved among cholinesterases, did not result in detectable alteration in the recombinant AChE, although polypeptide productivity of the D95N mutant was considerably lower. In contrast, complete absence of secreted human AChE polypeptide was observed when Asp-175 or Asp-404 were substituted by Asn. These two aspartates are conserved in the entire cholinesterase/thyroglobulin family and appear to play a role in generating and/or maintaining the folded state of the polypeptide. The x-ray structure of the Torpedo acetylcholinesterase supports this assumption by revealing the participation of these residues in salt bridges between neighboring secondary structure elements.
ESTHER : Shafferman_1992_J.Biol.Chem_267_17640
PubMedSearch : Shafferman_1992_J.Biol.Chem_267_17640
PubMedID: 1517212

Title : The alpha\/beta hydrolase fold - Ollis_1992_Prot.Engin_5_197
Author(s) : Ollis DL , Cheah E , Cygler M , Dijkstra B , Frolow F , Franken SM , Harel M , Remington SJ , Silman I , Schrag JD , Sussman JL , Verschueren KHG , Goldman A
Ref : Protein Engineering , 5 :197 , 1992
Abstract : We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.
ESTHER : Ollis_1992_Prot.Engin_5_197
PubMedSearch : Ollis_1992_Prot.Engin_5_197
PubMedID: 1409539

Title : Conversion of acetylcholinesterase to butyrylcholinesterase: modeling and mutagenesis - Harel_1992_Proc.Natl.Acad.Sci.U.S.A_89_10827
Author(s) : Harel M , Sussman JL , Krejci E , Bon S , Chanal P , Massoulie J , Silman I
Ref : Proceedings of the National Academy of Sciences of the United States of America , 89 :10827 , 1992
Abstract : Torpedo acetylcholinesterase (AcChoEase, EC 3.1.1.7) and human butyrylcholinesterase (BtChoEase, EC 3.1.1.8), while clearly differing in substrate specificity and sensitivity to inhibitors, possess 53% sequence homology; this permitted modeling human BtChoEase on the basis of the three-dimensional structure of Torpedo AcChoEase. The modeled BtChoEase structure closely resembled that of AcChoEase in overall features. However, six conserved aromatic residues that line the active-site gorge, which is a prominent feature of the AcChoEase structure, are absent in BtChoEase. Modeling showed that two such residues, Phe-288 and Phe-290, replaced by leucine and valine, respectively, in BtChoEase, may prevent entrance of butyrylcholine into the acyl-binding pocket. Their mutation to leucine and valine in AcChoEase, by site-directed mutagenesis, produced a double mutant that hydrolyzed butyrylthiocholine almost as well as acetylthiocholine. The mutated enzyme was also inhibited well by the bulky, BtChoEase-selective organophosphate inhibitor (tetraisopropylpyrophosphoramide, iso-OMPA). Trp-279, at the entrance of the active-site gorge in AcChoEase, is absent in BtChoEase. Modeling designated it as part of the "peripheral" anionic site, which is lacking in BtChoEase. The mutant W279A displayed strongly reduced inhibition by the peripheral site-specific ligand propidium relative to wild-type Torpedo AcChoEase, whereas inhibition by the catalytic-site inhibitor edrophonium was unaffected.
ESTHER : Harel_1992_Proc.Natl.Acad.Sci.U.S.A_89_10827
PubMedSearch : Harel_1992_Proc.Natl.Acad.Sci.U.S.A_89_10827
PubMedID: 1438284

Title : Three Dimensional Structure of Acetylcholinesterase -
Author(s) : Sussman JL , Harel M , Silman I
Ref : In Multidisciplinary approaches to cholinesterase functions - Proceedings of Fourth International Meeting on Cholinesterases , (Shafferman, A. and Velan, B., Eds) Plenum Press, New York :95 , 1992
PubMedID:

Title : A Model of Butyrylcholinesterase Based on the X-Ray Structure of Acetylcholinesterase Indicates Differences in Specificity -
Author(s) : Harel M , Silman I , Sussman JL
Ref : In Multidisciplinary approaches to cholinesterase functions - Proceedings of Fourth International Meeting on Cholinesterases , (Shafferman, A. and Velan, B., Eds) Plenum Press, New York :189 , 1992
PubMedID:

Title : Refined crystal structures of aged and non-aged organophosphoryl conjugates of gamma-chymotrypsin - Harel_1991_J.Mol.Biol_221_909
Author(s) : Harel M , Su CT , Frolow F , Ashani Y , Silman I , Sussman JL
Ref : Journal of Molecular Biology , 221 :909 , 1991
Abstract : "Aged" organophosphoryl conjugates of serine hydrolases differ from the corresponding "non-aged" conjugates in their striking resistance to nucleophilic reactivation. The refined X-ray structures of "aged" and "non-aged" organophosphoryl conjugates of gamma-chymotrypsin were compared in order to understand the molecular basis for this resistance of "aged" conjugates. "Aged" and "non-aged" crystalline organophosphoryl-gamma-chymotrypsin conjugates were obtained by prolonged soaking of native gamma-chymotrypsin crystals with appropriate organophosphates. Thus, a representative "non-aged" conjugate, diethylphosphoryl-gamma-chymotrypsin, was obtained by soaking native crystals with paraoxon (diethyl-p-nitrophenyl phosphate), and a closely related "aged" conjugate, monoisopropyl-gamma-chymotrypsin, was obtained by soaking with diisopropylphosphorofluoridate. In both crystalline conjugates, the refined structures clearly reveal a high occupancy of the active site by the appropriate organophosphoryl moiety within covalent bonding distance of Ser195 O gamma. Whereas in the "non-aged" conjugate both ethyl groups can be visualized clearly, in the putative "aged" conjugate, as expected, only one isopropyl group is present. There is virtually no difference between the "aged" and "non-aged" conjugates either with respect to the conformation of the polypeptide backbone as a whole or with respect to the positioning of the side-chains within the active site. In the "aged" conjugate, however, close proximity (2.6 A) of the negatively charged phosphate oxygen atom of the dealkylated organophosphoryl group to His57 N epsilon 2 indicates the presence of a salt bridge between these two moieties. In contrast, in the "non-aged" conjugate the DEP moiety retains its two alkyl groups; thus, lacking a negative oxygen atom, it does not enter into such a charge-charge interaction and its nearest oxygen atom is 3.6 A away from His57 N epsilon 2. It is suggested that steric constraints imposed by the salt bridge in the "aged" conjugate lie at the basis of its resistance to reactivation.
ESTHER : Harel_1991_J.Mol.Biol_221_909
PubMedSearch : Harel_1991_J.Mol.Biol_221_909
PubMedID: 1942036

Title : Poster: Physicochemical and crystallographic studies on the stability and structure of aged and nonaged organophosphoryl conjugates of chymotrypsin -
Author(s) : Su CT , Steinberg N , Silman I , Harel M , Sussman JL , Grunwald J , Ashani Y
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :274 , 1991
PubMedID:

Title : Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein - Sussman_1991_Science_253_872
Author(s) : Sussman JL , Harel M , Frolow F , Oefner C , Goldman A , Toker L , Silman I
Ref : Science , 253 :872 , 1991
Abstract : The three-dimensional structure of acetylcholinesterase from Torpedo californica electric organ has been determined by x-ray analysis to 2.8 angstrom resolution. The form crystallized is the glycolipid-anchored homodimer that was purified subsequent to solubilization with a bacterial phosphatidylinositol-specific phospholipase C. The enzyme monomer is an alpha/beta protein that contains 537 amino acids. It consists of a 12-stranded mixed beta sheet surrounded by 14 alpha helices and bears a striking resemblance to several hydrolase structures including dienelactone hydrolase, serine carboxypeptidase-II, three neutral lipases, and haloalkane dehalogenase. The active site is unusual because it contains Glu, not Asp, in the Ser-His-acid catalytic triad and because the relation of the triad to the rest of the protein approximates a mirror image of that seen in the serine proteases. Furthermore, the active site lies near the bottom of a deep and narrow gorge that reaches halfway into the protein. Modeling of acetylcholine binding to the enzyme suggests that the quaternary ammonium ion is bound not to a negatively charged "anionic" site, but rather to some of the 14 aromatic residues that line the gorge.
ESTHER : Sussman_1991_Science_253_872
PubMedSearch : Sussman_1991_Science_253_872
PubMedID: 1678899
Gene_locus related to this paper: torca-ACHE

Title : Structural Studies on Acetylcholinesterase from Torpedo californica -
Author(s) : Sussman JL , Harel M , Frolow F , Oefner C , Toker L , Silman I
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :7 , 1991
PubMedID:

Title : Purification and crystallization of a dimeric form of acetylcholinesterase from Torpedo californica subsequent to solubilization with phosphatidylinositol-specific phospholipase C - Sussman_1988_J.Mol.Biol_203_821
Author(s) : Sussman JL , Harel M , Frolow F , Varon L , Toker L , Futerman AH , Silman I
Ref : Journal of Molecular Biology , 203 :821 , 1988
Abstract : A dimeric form of acetylcholinesterase from Torpedo californica was purified to homogeneity by affinity chromatography subsequent to solubilization with a phosphatidylinositol-specific phospholipase C of bacterial origin. Bipyramidal crystals of the enzyme were obtained from solutions in polyethylene glycol 200. The crystals diffract to 2.0 A (1 A = 0.1 nm) resolution. They were found to be orthorhombic, space group P2221, with a = 163.4(+/- 0.2) A, b = 112.1(+/- 0.2) A, c = 81.3(+/- 0.1) A.
ESTHER : Sussman_1988_J.Mol.Biol_203_821
PubMedSearch : Sussman_1988_J.Mol.Biol_203_821
PubMedID: 2850366