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• W2005353089 abstract "Hydrolysis of acetylcholine catalyzed by acetylcholinesterase (AChE), one of the most efficient enzymes in nature, occurs at the base of a deep and narrow active center gorge. At the entrance of the gorge, the peripheral anionic site provides a binding locus for allosteric ligands, including substrates. To date, no structural information on substrate entry to the active center from the peripheral site of AChE or its subsequent egress has been reported. Complementary crystal structures of mouse AChE and an inactive mouse AChE mutant with a substituted catalytic serine (S203A), in various complexes with four substrates (acetylcholine, acetylthiocholine, succinyldicholine, and butyrylthiocholine), two non-hydrolyzable substrate analogues (m-(N,N,N-trimethylammonio)-trifluoroacetophenone and 4-ketoamyltrimethylammonium), and one reaction product (choline) were solved in the 2.05-2.65-Å resolution range. These structures, supported by binding and inhibition data obtained on the same complexes, reveal the successive positions and orientations of the substrates bound to the peripheral site and proceeding within the gorge toward the active site, the conformations of the presumed transition state for acylation and the acyl-enzyme intermediate, and the positions and orientations of the dissociating and egressing products. Moreover, the structures of the AChE mutant in complexes with acetylthiocholine and succinyldicholine reveal additional substrate binding sites on the enzyme surface, distal to the gorge entry. Hence, we provide a comprehensive set of structural snapshots of the steps leading to the intermediates of catalysis and the potential regulation by substrate binding to various allosteric sites at the enzyme surface. Hydrolysis of acetylcholine catalyzed by acetylcholinesterase (AChE), one of the most efficient enzymes in nature, occurs at the base of a deep and narrow active center gorge. At the entrance of the gorge, the peripheral anionic site provides a binding locus for allosteric ligands, including substrates. To date, no structural information on substrate entry to the active center from the peripheral site of AChE or its subsequent egress has been reported. Complementary crystal structures of mouse AChE and an inactive mouse AChE mutant with a substituted catalytic serine (S203A), in various complexes with four substrates (acetylcholine, acetylthiocholine, succinyldicholine, and butyrylthiocholine), two non-hydrolyzable substrate analogues (m-(N,N,N-trimethylammonio)-trifluoroacetophenone and 4-ketoamyltrimethylammonium), and one reaction product (choline) were solved in the 2.05-2.65-Å resolution range. These structures, supported by binding and inhibition data obtained on the same complexes, reveal the successive positions and orientations of the substrates bound to the peripheral site and proceeding within the gorge toward the active site, the conformations of the presumed transition state for acylation and the acyl-enzyme intermediate, and the positions and orientations of the dissociating and egressing products. Moreover, the structures of the AChE mutant in complexes with acetylthiocholine and succinyldicholine reveal additional substrate binding sites on the enzyme surface, distal to the gorge entry. Hence, we provide a comprehensive set of structural snapshots of the steps leading to the intermediates of catalysis and the potential regulation by substrate binding to various allosteric sites at the enzyme surface. The principal role of acetylcholinesterase (AChE) 3The abbreviations used are: AChE, acetylcholinesterase; (mAChE, recombinant from mouse; S203A, mutant from mouse; TcAChE, from T. californica); BChE, butyrylcholinesterase; ACh, acetylcholine; ATCh, acetylthiocholine; BTCh, butyrylthiocholine; SCh, succinyldicholine; 4K-TMA, 4-ketoamyltrimethylammonium; PAS, peripheral anionic site; r.m.s., root mean square; TCh, thiocholine; TMTFA, m-(N,N,N-trimethylammonio)trifluoroacetophenone; Mes, 2-(N-morpholino)ethanesulfonic acid. 3The abbreviations used are: AChE, acetylcholinesterase; (mAChE, recombinant from mouse; S203A, mutant from mouse; TcAChE, from T. californica); BChE, butyrylcholinesterase; ACh, acetylcholine; ATCh, acetylthiocholine; BTCh, butyrylthiocholine; SCh, succinyldicholine; 4K-TMA, 4-ketoamyltrimethylammonium; PAS, peripheral anionic site; r.m.s., root mean square; TCh, thiocholine; TMTFA, m-(N,N,N-trimethylammonio)trifluoroacetophenone; Mes, 2-(N-morpholino)ethanesulfonic acid. at cholinergic synapses is to terminate neurotransmission by fast hydrolysis of the substrate, acetylcholine (ACh) (1Massoulie J. Sussman J. Bon S. Silman I. Prog. Brain Res. 1993; 98: 139-146Crossref PubMed Scopus (125) Google Scholar, 2Taylor P. Radić Z. Annu. Rev. Pharmacol. Toxicol. 1994; 34: 281-320Crossref PubMed Scopus (600) Google Scholar). The AChE active center, containing the catalytic triad, Glu334-His447-Ser203 in mammals (3Rachinsky T.L. Camp S. Li Y. Ekstrom T.J. Newton M. Taylor P. Neuron. 1990; 5: 317-327Abstract Full Text PDF PubMed Scopus (136) Google Scholar), is located centrosymmetric to the subunit and at the base of a deep and narrow gorge (4Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2406) Google Scholar, 5Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar). AChE-catalyzed hydrolysis of ACh and other carboxyl esters proceeds via formation of an initial noncovalent enzyme-substrate complex. Nucleophilic attack of the substrate carbonyl carbon by the Ser203 hydroxyl generates a transient tetrahedral oxyanion intermediate, which collapses into a short-lived (t½ ∼ 50 μs) acyl-enzyme (ester) intermediate and a released choline molecule (6Froede H.C. Wilson I.B. J. Biol. Chem. 1984; 259: 11010-11013Abstract Full Text PDF PubMed Google Scholar). Deacylation through hydrolytic attack on the ester carbonyl by a water molecule leads to a second tetrahedral intermediate, which then collapses into a regenerated enzyme and an acetate molecule. Rapid rates of substrate association and choline product dissociation contribute to the AChE high specific activity and catalytic throughput. Ser203 is rendered more nucleophilic by catalytic triad residues Glu334 and His447. Residue Trp86, located at the very base of the active center gorge, orients the ACh trimethylammonium group prior to hydrolysis, whereas the oxyanion hole amide hydrogens from Gly121, Gly122, and Ala204, presumably stabilize the carbonyl oxygen of ACh in the transition states for acylation and deacylation. Inhibitors of AChE bind to the active site or to the peripheral anionic site (PAS), an allosteric site located at the active center gorge entrance, or they span the two sites thereby occupying much of the active center gorge (7Changeux J.P. Mol. Pharmacol. 1966; 2: 369-392PubMed Google Scholar, 8Taylor P. Lappi S. Biochemistry. 1975; 14: 1989-1997Crossref PubMed Scopus (329) Google Scholar, 9Mooser G. Sigman D.S. Biochemistry. 1974; 13: 2299-2307Crossref PubMed Scopus (100) Google Scholar). PAS inhibitors limit the catalytic rate by steric and electrostatic blockade of ligand trafficking through the gorge and by altering the active center conformation (10Radić Z. Reiner E. Taylor P. Mol. Pharmacol. 1991; 39: 98-104PubMed Google Scholar, 11Szegletes T. Mallender W.D. Thomas P.J. Rosenberry T.L. Biochemistry. 1999; 38: 122-133Crossref PubMed Scopus (150) Google Scholar, 12Mallender W.D. Szegletes T. Rosenberry T.L. Biochemistry. 2000; 39: 7753-7763Crossref PubMed Scopus (124) Google Scholar, 13Johnson J.L. Cusack B. Hughes T.F. McCullough E.H. Fauq A. Romanovskis P. Spatola A.F. Rosenberry T.L. J. Biol. Chem. 2003; 278: 38948-38955Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 14Johnson J.L. Cusack B. Davies M.P. Fauq A. Rosenberry T.L. Biochemistry. 2003; 42: 5438-5452Crossref PubMed Scopus (66) Google Scholar). Mutagenesis and structural studies have revealed the functional role of residues Tyr72, Asp74, Tyr124, Trp286, and Tyr341 at the PAS (5Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar, 15Radić Z. Pickering N.A. Vellom D.C. Camp S. Taylor P. Biochemistry. 1993; 32: 12074-12084Crossref PubMed Scopus (419) Google Scholar, 16Shafferman A. Kronman C. Flashner Y. Leitner M. Grosfeld H. Ordentlich A. Gozes Y. Cohen S. Ariel N. Barak D. J. Biol. Chem. 1992; 267: 17640-17648Abstract Full Text PDF PubMed Google Scholar, 17Barak D. Kronman C. Ordentlich A. Ariel N. Bromberg A. Marcus D. Lazar A. Velan B. Shafferman A. J. Biol. Chem. 1994; 269: 6296-6305Abstract Full Text PDF PubMed Google Scholar, 18Harel M. Kleywegt G.J. Ravelli R.B. Silman I. Sussman J.L. Structure. 1995; 3: 1355-1366Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 19Bourne Y. Taylor P. Radić Z. Marchot P. EMBO J. 2003; 22: 1-12Crossref PubMed Scopus (324) Google Scholar, 20Bourne Y. Kolb H.C. Radić Z. Sharpless K.B. Taylor P. Marchot P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1449-1454Crossref PubMed Scopus (305) Google Scholar). Kinetics of cationic substrate hydrolysis catalyzed by AChE deviate from Michaelis-Menten kinetics (21Nachmansohn D. Wilson I.B. Adv. Enzymol. Relat. Subj. Biochem. 1951; 12: 259-339PubMed Google Scholar, 22Aldridge W.N. Reiner E. Biochem. J. 1969; 115: 147-162Crossref PubMed Scopus (75) Google Scholar). Cationic substrates, including ACh, inhibit their own catalysis at concentrations exceeding the Km (≥1mm, i.e. ≥20 × Km for ATCh and mouse AChE (mAChE) (15Radić Z. Pickering N.A. Vellom D.C. Camp S. Taylor P. Biochemistry. 1993; 32: 12074-12084Crossref PubMed Scopus (419) Google Scholar)). AChE from Drosophila, but not from vertebrates, also shows substrate activation at low concentrations (23Marcel V. Palacios L.G. Pertuy C. Masson P. Fournier D. Biochem. J. 1998; 329: 329-334Crossref PubMed Scopus (63) Google Scholar, 24Stojan J. Marcel V. Estrada-Mondaca S. Klaebe A. Masson P. Fournier D. FEBS Lett. 1998; 440: 85-88Crossref PubMed Scopus (38) Google Scholar). Competitive displacement, by ACh, of the specific PAS ligands, propidium and the peptidic toxin fasciculin, provides evidence that ACh binds the PAS in addition to the active site (10Radić Z. Reiner E. Taylor P. Mol. Pharmacol. 1991; 39: 98-104PubMed Google Scholar, 25Karlsson E. Mbugua P.M. Rodriguez-Ithurralde D. J. Physiol. 1984; 79: 232-240Google Scholar, 26Marchot P. Khelif A. Ji Y.H. Mansuelle P. Bougis P.E. J. Biol. Chem. 1993; 268: 12458-12467Abstract Full Text PDF PubMed Google Scholar). Electrostatic calculations based on the TcAChE structure and subsequent molecular dynamics simulation suggested that AChE may also have a “back door,” distinct from the gorge entrance and whose transient opening would contribute to the high rate of traffic of substrates, products, and water into and out of the active center gorge (27Axelsen P.H. Harel M. Silman I. Sussman J.L. Protein Sci. 1994; 3: 188-197Crossref PubMed Scopus (152) Google Scholar, 28Gilson M.K. Straatsma T.P. McCammon J.A. Ripoll D.R. Faerman C.H. Axelsen P.H. Silman I. Sussman J.L. Science. 1994; 263: 1276-1278Crossref PubMed Scopus (240) Google Scholar). Opening of this putative door would involve a displacement of the Trp86 side chain, which constitutes the thin wall separating the choline binding site in the active site from the outside solvent. Existence of residual catalytic activity of AChE complexes with the large fasciculin molecule, which binds the PAS to seal the gorge entrance (5Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar, 18Harel M. Kleywegt G.J. Ravelli R.B. Silman I. Sussman J.L. Structure. 1995; 3: 1355-1366Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar), may also argue for the need of an alternative entry portal(s) for the substrate (26Marchot P. Khelif A. Ji Y.H. Mansuelle P. Bougis P.E. J. Biol. Chem. 1993; 268: 12458-12467Abstract Full Text PDF PubMed Google Scholar, 29Eastman J. Wilson E.J. Cervenansky C. Rosenberry T.L. J. Biol. Chem. 1995; 270: 19694-19701Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 30Radić Z. Duran R. Vellom D.C. Li Y. Cervenansky C. Taylor P. J. Biol. Chem. 1994; 269: 11233-11239Abstract Full Text PDF PubMed Google Scholar, 31Radić Z. Quinn D.M. Vellom D.C. Camp S. Taylor P. J. Biol. Chem. 1995; 270: 20391-20399Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Yet, crystal structures of fasciculin-AChE complexes did not reveal an open back door. To date, the functioning of the back door remains hypothetical and, apart from some speculation (32Bartolucci C. Perola E. Cellai L. Brufani M. Lamba D. Biochemistry. 1999; 38: 5714-5719Crossref PubMed Scopus (92) Google Scholar, 33Bencharit S. Morton C.L. Howard-Williams E.L. Danks M.K. Potter P.M. Redinbo M.R. Nat. Struct. Biol. 2002; 9: 337-342Crossref PubMed Scopus (150) Google Scholar), evidence for its existence has not been reported. Although a structural perspective on the AChE catalytic mechanism should include the acylation and deacylation steps and related intermediates, the rapid substrate turnover precludes their entrapment and visualization in a crystalline state. Initial manual docking of an ACh molecule into the Torpedo californica (TcAChE) structure (4Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2406) Google Scholar) was followed by structures of AChE complexes with competitive, reversible inhibitors (34Harel M. Schalk I. Ehret-Sabatier L. Bouet F. Goeldner M. Hirth C. Axelsen P.H. Silman I. Sussman J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9031-9035Crossref PubMed Scopus (839) Google Scholar, 35Raves M.L. Harel M. Pang Y.P. Silman I. Kozikowski A.P. Sussman J.L. Nat. Struct. Biol. 1997; 4: 57-63Crossref PubMed Scopus (390) Google Scholar, 36Ravelli R.B. Raves M.L. Ren Z. Bourgeois D. Roth M. Kroon J. Silman I. Sussman J.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 1359-1366Crossref PubMed Google Scholar, 37Bartolucci C. Perola E. Pilger C. Fels G. Lamba D. Proteins. 2001; 42: 182-191Crossref PubMed Scopus (126) Google Scholar, 38Bar-On P. Millard C.B. Harel M. Dvir H. Enz A. Sussman J.L. Silman I. Biochemistry. 2002; 41: 3555-3564Crossref PubMed Scopus (287) Google Scholar), covalent organophosphate or carbamate inhibitors (39Millard C.B. Kryger G. Ordentlich A. Greenblatt H.M. Harel M. Raves M.L. Segall Y. Barak D. Shafferman A. Silman I. Sussman J.L. Biochemistry. 1999; 38: 7032-7039Crossref PubMed Scopus (256) Google Scholar), bifunctional inhibitors (20Bourne Y. Kolb H.C. Radić Z. Sharpless K.B. Taylor P. Marchot P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1449-1454Crossref PubMed Scopus (305) Google Scholar, 32Bartolucci C. Perola E. Cellai L. Brufani M. Lamba D. Biochemistry. 1999; 38: 5714-5719Crossref PubMed Scopus (92) Google Scholar, 34Harel M. Schalk I. Ehret-Sabatier L. Bouet F. Goeldner M. Hirth C. Axelsen P.H. Silman I. Sussman J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9031-9035Crossref PubMed Scopus (839) Google Scholar, 40Kryger G. Silman I. Sussman J.L. J. Physiol. 1998; 92: 191-194Google Scholar, 41Felder C.E. Harel M. Silman I. Sussman J.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1765-1771Crossref PubMed Scopus (49) Google Scholar), PAS inhibitors (5Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar, 18Harel M. Kleywegt G.J. Ravelli R.B. Silman I. Sussman J.L. Structure. 1995; 3: 1355-1366Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 19Bourne Y. Taylor P. Radić Z. Marchot P. EMBO J. 2003; 22: 1-12Crossref PubMed Scopus (324) Google Scholar), and the substrate analogue, tri-methylammoniotrifluoroacetophenone (TMTFA) (42Harel M. Quinn D.M. Nair H.K. Silman I. Sussman J.L. J. Am. Chem. Soc. 1996; 118: 2340-2346Crossref Scopus (340) Google Scholar). The latter provides a close structural mimic of the substrate-AChE tetrahedral transition state. To enhance the opportunity of observing intact substrate molecules bound with high occupancies at the various sites, we expressed a catalytically inactive mAChE mutant, S203A, and crystallized it together with the wild type mAChE in a form that keeps potential ligand binding sites at the enzyme surface free of packing contacts (19Bourne Y. Taylor P. Radić Z. Marchot P. EMBO J. 2003; 22: 1-12Crossref PubMed Scopus (324) Google Scholar, 20Bourne Y. Kolb H.C. Radić Z. Sharpless K.B. Taylor P. Marchot P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1449-1454Crossref PubMed Scopus (305) Google Scholar). We then soaked the crystals in concentrated solutions of the substrates ACh and acetylthiocholine (ATCh), the slowly hydrolyzable substrates succinyldicholine (SCh) and butyrylthiocholine (BTCh), the non-hydrolyzable substrate analogues TMTFA and 4-ketoamyltrimethylammonium (4K-TMA), and the reaction product choline (Scheme 1). The eight resulting crystalline complexes and conjugates, supported by binding and inhibition data, show complementary views of the transition state, the acyl-enzyme and the deacetylated enzyme, and substrate binding to the PAS. In addition, substrate binding sites remote from the active site and the PAS were identified. Hence these structures provide a comprehensive set of snapshots of the reaction intermediates along the gorge pathway and the likely modes of regulation by substrate binding to low affinity sites at the enzyme surface. Chemicals—4K-TMA iodide was from ICN and ambenonium dichloride from Tocris Cookson. ACh, ATCh, SCh, BTCh, and choline, as chloride or iodine salts, were from Sigma. TMTFA iodide and decidium diiodide were respective gifts from Daniel M. Quinn, University of Iowa, Iowa City, UT, and Harvey A. Berman, State University of New York, Buffalo, NY. Polyethylene glycols were from Hampton Research, Fluka, or Sigma. Enzymes—Soluble mAChE expressed in human embryonic kidney-293 cells (15Radić Z. Pickering N.A. Vellom D.C. Camp S. Taylor P. Biochemistry. 1993; 32: 12074-12084Crossref PubMed Scopus (419) Google Scholar, 43Marchot P. Ravelli R.B. Raves M.L. Bourne Y. Vellom D.C. Kanter J. Camp S. Sussman J.L. Taylor P. Protein Sci. 1996; 5: 672-679Crossref PubMed Scopus (59) Google Scholar) was purified by affinity chromatography with elution using either 5 mm propidium diiodide (19Bourne Y. Taylor P. Radić Z. Marchot P. EMBO J. 2003; 22: 1-12Crossref PubMed Scopus (324) Google Scholar) or 100 mm SCh dichloride; in the latter case, the eluted enzyme was immediately dialyzed to avoid precipitation/inactivation due to medium acidification through slow SCh hydrolysis. The mAChE mutant S203A was generated by site-directed mutagenesis, expressed in human embryonic kidney-293 cells, and purified using 100 mm decamethonium bromide (15Radić Z. Pickering N.A. Vellom D.C. Camp S. Taylor P. Biochemistry. 1993; 32: 12074-12084Crossref PubMed Scopus (419) Google Scholar, 43Marchot P. Ravelli R.B. Raves M.L. Bourne Y. Vellom D.C. Kanter J. Camp S. Sussman J.L. Taylor P. Protein Sci. 1996; 5: 672-679Crossref PubMed Scopus (59) Google Scholar). The enzyme and mutant were extensively dialyzed against 1 mm Mes, pH 6.5, 1 m NaCl, 40 mm MgCl2, 0.01% NaN3 (w/v), and desalted by gel-filtration FPLC on Superdex-200 (Amersham Biosciences) in 1 mm Mes, pH 6.5, 50 mm NaCl, 0.01% NaN3 (w/v) (crystallization buffer), or by extensive dialysis against this buffer. They were concentrated to about 10 mg/ml by ultrafiltration. Crystallization, Complex Formation, and Data Collection—Crystallization was achieved at 4 °C by vapor diffusion using hanging drops (1-2 μl) and a protein-to-well solution ratio of 1:1, with P550MME or P600 (25-32%) (v/v) in either Hepes or sodium acetate (60-100 mm), pH 6.5-8.0, as the well solution (19Bourne Y. Taylor P. Radić Z. Marchot P. EMBO J. 2003; 22: 1-12Crossref PubMed Scopus (324) Google Scholar). The TMTFA-mAChE complex was formed in solution using apo-mAChE at 5.0 mg/ml (∼80 μm; 1.5 1010 × Ki (31Radić Z. Quinn D.M. Vellom D.C. Camp S. Taylor P. J. Biol. Chem. 1995; 270: 20391-20399Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar)) and a 3-fold molar excess of the ligand over the enzyme, and concentrated prior to crystallization. The other seven complexes were generated by crystal soaking, carried out at 4 °C in sitting drops (20 μl) made of the well solution supplemented with the ligand (250 mm) and polyethylene glycol up to 35% (∼0.6 m) to ensure cryoprotection; the soaking drops were renewed twice at 12-h intervals and again just before direct crystal flash-cooling in the nitrogen gas stream. Crystals belong to the orthorhombic space group P212121 with unit cell dimensions a = 79 Å, b = 112 Å, c = 227 Å. Oscillation images were integrated with DENZO (44Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38446) Google Scholar) and data were scaled and merged with SCALA (45P4 CC Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19733) Google Scholar) (Table 1).TABLE 1Data collection and refinement statisticsmAChES203A mutantTMTFA4K-TMASChCholineAChATChSChBTChBinding site(s)ASaAS, active site; PAS, peripheral anionic site; BDR, back door region.AS/PASAS/PASAS/PASAS/PASAS/PAS/BDRAS/PAS/BDRPASPanel in Fig. 2ABFHCDEGData collectionbValues in parentheses are those for the last shell.Beamline (ESRF)ID14-EH1ID14-EH1ID14-EH4ID14-EH4ID14-EH2ID14-EH4ID14-EH4ID14-EH2Wavelength (Å)0.9330.9330.9750.9750.9330.9750.9750.933Resolution range (Å)20-2.430-2.230-2.0530-2.2530-2.5530-2.1530-2.2520-2.65Observations276,837351,519327,048302,093259,660363,316337,398231,897Unique reflections78,296102,641112,41592,59065,68698,80289,06757,849Multiplicity3.5 (3.6)3.4 (3.3)2.9 (1.7)3.3 (2.5)3.9 (3.9)3.7 (3.1)3.8 (3.1)4.0 (4.0)Completeness (%)99.4 (99.5)100 (98.9)90.1 (55.1)96.2 (96.2)99.9 (99.9)96.9 (96.9)95.4 (69.4)99.9 (99.9)I/σ(I)11.3 (3.3)11.9 (2.8)10.6 (1.4)11.9 (3.0)15.2 (4.3)13.0 (3.4)14.3 (3.1)16.0 (4.0)RmergecRmerge = ∑hkl ∑i|Ii(hkl) - 〈Ihkl 〉|/∑hkl ∑i Ii(hkl), where I is an individual reflection measurement and 〈I 〉 is the mean intensity for symmetry-related reflections.8.2 (44.7)6.5 (45.1)6.1 (38.5)7.3 (33.8)6.8 (42.4)6.2 (38.3)5.6 (48.7)6.8 (46.7)Wilson plot B-factor (Å2)52.641.135.341.742.335.151.349.2RefinementRcrystdRcryst = ∑hkl || Fo| - |Fc||/∑hkl|Fo|, where Fo and Fc are observed and calculated structure factors, respectively. Rfree is calculated for 2% of randomly selected reflections excluded from refinement/Rfree (%)18.6/21.418.3/21.719.2/22.117.5/19.818.9/22.417.7/20.917.8/19.919.9/24.2R.m.s. deviationeRoot mean square deviations from ideal values.Bonds (Å)/Angles (°)0.01/1.290.01/1.280.01/1.410.01/1.260.01/1.300.01/1.230.01/1.370.01/1.24Chiral volume (Å3)0.0950.0970.1120.0750.0910.0740.0810.086Average B-factor (Å2)Main/side chains65.4 (66.4)57.6/58.958.2/59.452.9/54.138.8/39.656.7/57.849.8/51.346.2/47.5Solvent/carbohydrate-PEG63.0/87.359.6/64.562.5/72.752.9/82.537.2/57.364.2/83.650.5/61.239.5/42.8Ligand (AS/PAS/BDR)*57.6/-/-49.6/74.2/-83.8/-/-81.3/87.3/-52.6/55.3/-76.4/87.2/80.477.7/-/77.366.8/85.3/-Main/side chain ΔB for bonded atoms (Å2)0.93/1.340.94/1.480.98/1.520.72/1.230.86/1.430.82/1.330.85/1.441.07/1.72PDB accession code2H9Y2HA02HA22HA32HA42HA52HA62HA7a AS, active site; PAS, peripheral anionic site; BDR, back door region.b Values in parentheses are those for the last shell.c Rmerge = ∑hkl ∑i|Ii(hkl) - 〈Ihkl 〉|/∑hkl ∑i Ii(hkl), where I is an individual reflection measurement and 〈I 〉 is the mean intensity for symmetry-related reflections.d Rcryst = ∑hkl || Fo| - |Fc||/∑hkl|Fo|, where Fo and Fc are observed and calculated structure factors, respectively. Rfree is calculated for 2% of randomly selected reflections excluded from refinemente Root mean square deviations from ideal values. Open table in a new tab Structure Determination and Refinement—The apo-mAChE structure (Protein Data Bank entry 1J06 (19Bourne Y. Taylor P. Radić Z. Marchot P. EMBO J. 2003; 22: 1-12Crossref PubMed Scopus (324) Google Scholar)) without solvent was used as a starting model to refine the reported structures with REFMAC (46Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13818) Google Scholar) using the maximum likelihood approach and incorporating bulk solvent corrections, anisotropic Fobs versus Fcalc scaling and TLS refinement with each subunit defining a TLS group (Table 1). Typically, rigid-body refinement was first performed on each of the two subunits forming the crystalline mAChE dimer (19Bourne Y. Taylor P. Radić Z. Marchot P. EMBO J. 2003; 22: 1-12Crossref PubMed Scopus (324) Google Scholar) using all data followed by cycles of restrained refinements. Random sets of reflections were set aside for cross-validation purposes. For each structure, the resulting σA-weighted 2Fo - Fc and Fo - Fc electron density maps were used to position the ligand. Automated solvent building was performed with ARP/wARP (47Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2563) Google Scholar) and manual adjustment with the graphics program TURBO-FRODO (66Roussel A. Cambillau C. Silicon Graphics Committee TURBO-FRODO; Silicon Graphics Geometry Partners Directory. Mountain View, CA1989Google Scholar) and COOT (48Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23012) Google Scholar). The final structures, one apo-S203A and eight S203A or mAChE complexes, comprise residues Glu1-Thr543 and Glu4-Thr543 for the two mAChE/S203A molecules in the asymmetric unit, respectively, and GlcNAc moieties linked to Asn350 and Asn464. High temperature factors and weak electron densities are observed for residues within the surface loop region Asp491-Pro498. The average root mean square (r.m.s.) deviation value between the nine structures and the apo-mAChE structure is 0.24 Å for 535 Cα atoms (from 0.19 Å for the SCh-mAChE complex to 0.294 Å for the 4K-TMA-mAChE complex). The stereochemistries of the structures were analyzed with PROCHECK (49Laskowski R. MacArthur M. Moss D. Thornton J. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar); with the exception of residue Ser/Ala203, no residues were found in the disallowed regions of the Ramachandran plot. The atomic coordinates and structure factors of the four S203A (ACh, ATCh, SCh, and BTCh) and four mAChE complexes (TMTFA, 4K-TMA, SCh, and choline) have been deposited with the RCSB Protein Data Bank (Table 1). Figures were generated with PyMOL (50DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar). Inhibition and Binding Studies—Inhibition by SCh, 4K-TMA, and choline of mAChE-catalyzed ATCh hydrolysis was measured spectrophotometrically (51Ellman G.L. Courtney K.D. Andres Jr., V. Feather-Stone R.M. Biochem. Pharmacol. 1961; 7: 88-95Crossref PubMed Scopus (21126) Google Scholar) in 0.1 m phosphate buffer, pH 7.0, at 22 °C. The inhibition constants, Ki and αKi, were determined from the dependence of slopes and y intercepts of Lineweaver-Burk plots on inhibitor concentration (15Radić Z. Pickering N.A. Vellom D.C. Camp S. Taylor P. Biochemistry. 1993; 32: 12074-12084Crossref PubMed Scopus (419) Google Scholar). Binding of ligands ACh, ATCh, SCh, BTCh, 4K-TMA, and choline to mAChE (200 nm) and the S203A mutant (100 nm) was measured from the dependence of the pseudo-first order association rate, kobs, of the reversible bisquaternary inhibitors ambenonium (2 or 5 μm) (52Hodge A.S. Humphrey D.R. Rosenberry T.L. Mol. Pharmacol. 1992; 41: 937-942PubMed Google Scholar) or decidium (1 μm) (53Berman H.A. Decker M.M. Biochim. Biophys. Acta. 1986; 872: 125-133Crossref PubMed Scopus (20) Google Scholar) on ligand concentration (from 1 μm to 30 mm for 4K-TMA and to 300 mm for the other ligands). Rates were monitored in a millisecond time frame by stopped-flow measurements of intrinsic Trp fluorescence quenching of mAChE or its mutant as described (54Radić Z. Taylor P. J. Biol. Chem. 2001; 276: 4622-4633Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Dissociation constants, Kd, were determined from semi-logarithmic plots of kobs versus ligand concentration ([L]) and calculated by nonlinear regression using Equation 1, kobs=kobs0/(1+[L]/Kd)(Eq. 1) where kobs0 is the pseudo-first order rate constant for ambenonium or decidium association in the absence of ligand. When kobs versus [L] plots were biphasic, two dissociation constants, Kd and KdL, characterizing ligand binding to the enzyme and the enzyme-ligand complex, respectively, were calculated using Equation 2, kobs=(kobs0-kobsL,0)/(1+[L]/Kd)+kobsL,0/(1+[L]/KdL)(Eq. 2) where kobsL,0 is the limiting first-order rate constant for competitor association with the enzyme-ligand complex. The 2.65-Å resolution structure of the S203A mutant, compared with apo-mAChE (r.m.s. deviation value: 0.26 Å for 535 Cα atoms), shows an undisrupted catalytic site architecture despite the absence of the Ser hydroxyl, as well as unaltered topography for the PAS region (Fig. 1). Soaking a S203A crystal with 50 mm ATCh (∼103 × Km and 4 × Kss for mAChE; ∼50 × Kd for the mutant; Table 2) led to a 2.25-Å resolution structure in which only the reaction products thiocholine (TCh) and acetate were found, well ordered and with high occupancy, in the active center gorge (not shown). Despite the small size of choline, the side chain of Tyr337, located in the constricted region of the gorge, was found rotated by 24° and" @default.
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• W2005353089 title "Substrate and Product Trafficking through the Active Center Gorge of Acetylcholinesterase Analyzed by Crystallography and Equilibrium Binding" @default.
• W2005353089 cites W125799310 @default.
• W2005353089 cites W1480316552 @default.
• W2005353089 cites W1520730837 @default.
• W2005353089 cites W1536459857 @default.
• W2005353089 cites W1539796472 @default.
• W2005353089 cites W1577601626 @default.
• W2005353089 cites W1578889779 @default.
• W2005353089 cites W1587234113 @default.
• W2005353089 cites W1968516288 @default.
• W2005353089 cites W1971048984 @default.
• W2005353089 cites W1972719099 @default.
• W2005353089 cites W1979854220 @default.
• W2005353089 cites W1986191025 @default.
• W2005353089 cites W1988940766 @default.
• W2005353089 cites W1990280069 @default.
• W2005353089 cites W1993672505 @default.
• W2005353089 cites W1993676814 @default.
• W2005353089 cites W1996861305 @default.
• W2005353089 cites W2001641653 @default.
• W2005353089 cites W2009008858 @default.
• W2005353089 cites W2011000826 @default.
• W2005353089 cites W2021931130 @default.
• W2005353089 cites W2022964379 @default.
• W2005353089 cites W2027955282 @default.
• W2005353089 cites W2028067831 @default.
• W2005353089 cites W2028884143 @default.
• W2005353089 cites W2030128867 @default.
• W2005353089 cites W2035043090 @default.
• W2005353089 cites W2038840577 @default.
• W2005353089 cites W2039867374 @default.
• W2005353089 cites W2041706446 @default.
• W2005353089 cites W2044555017 @default.
• W2005353089 cites W2045542952 @default.
• W2005353089 cites W2046006267 @default.
• W2005353089 cites W2049873111 @default.
• W2005353089 cites W2051679721 @default.
• W2005353089 cites W2058185609 @default.
• W2005353089 cites W2065159016 @default.
• W2005353089 cites W2069294211 @default.
• W2005353089 cites W2072519718 @default.
• W2005353089 cites W2077207000 @default.
• W2005353089 cites W2078301842 @default.
• W2005353089 cites W2085958374 @default.
• W2005353089 cites W2086758152 @default.
• W2005353089 cites W2089015588 @default.
• W2005353089 cites W2098437204 @default.
• W2005353089 cites W2109490221 @default.
• W2005353089 cites W2121140853 @default.
• W2005353089 cites W2125296772 @default.
• W2005353089 cites W2136220160 @default.
• W2005353089 cites W2140642212 @default.
• W2005353089 cites W2140788274 @default.
• W2005353089 cites W2144081223 @default.
• W2005353089 cites W2149196013 @default.
• W2005353089 cites W2150578242 @default.
• W2005353089 cites W2152147380 @default.
• W2005353089 cites W981498444 @default.
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