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• W2072424636 abstract "The crystal structure of mouse acetylcholinesterase at 2.9-Å resolution reveals a tetrameric assembly of subunits with an antiparallel alignment of two canonical homodimers assembled through four-helix bundles. In the tetramer, a short Ω loop, composed of a cluster of hydrophobic residues conserved in mammalian acetylcholinesterases along with flanking α-helices, associates with the peripheral anionic site of the facing subunit and sterically occludes the entrance of the gorge leading to the active center. The inverse loop-peripheral site interaction occurs within the second pair of subunits, but the peripheral sites on the two loop-donor subunits remain freely accessible to the solvent. The position and complementarity of the peripheral site-occluding loop mimic the characteristics of the central loop of the peptidic inhibitor fasciculin bound to mouse acetylcholinesterase. Tetrameric forms of cholinesterases are widely distributed in nature and predominate in mammalian brain. This structure reveals a likely mode of subunit arrangement and suggests that the peripheral site, located near the rim of the gorge, is a site for association of neighboring subunits or heterologous proteins with interactive surface loops. The crystal structure of mouse acetylcholinesterase at 2.9-Å resolution reveals a tetrameric assembly of subunits with an antiparallel alignment of two canonical homodimers assembled through four-helix bundles. In the tetramer, a short Ω loop, composed of a cluster of hydrophobic residues conserved in mammalian acetylcholinesterases along with flanking α-helices, associates with the peripheral anionic site of the facing subunit and sterically occludes the entrance of the gorge leading to the active center. The inverse loop-peripheral site interaction occurs within the second pair of subunits, but the peripheral sites on the two loop-donor subunits remain freely accessible to the solvent. The position and complementarity of the peripheral site-occluding loop mimic the characteristics of the central loop of the peptidic inhibitor fasciculin bound to mouse acetylcholinesterase. Tetrameric forms of cholinesterases are widely distributed in nature and predominate in mammalian brain. This structure reveals a likely mode of subunit arrangement and suggests that the peripheral site, located near the rim of the gorge, is a site for association of neighboring subunits or heterologous proteins with interactive surface loops. Acetylcholinesterase (AChE), 1The abbreviations used are: AChE, acetylcholinesterase; mAChE, recombinant mouse AChE; TcAChE, T. californica AChE; Fas2, fasciculin 2; DECA, decamethonium; EDR, edrophonium (ethyl-3-hydroxyphenyl dimethylammonium); MES, 2-(N-morpholino)ethanesulfonic acid; NCS, non-crystallographic symmetry; r.m.s., root mean square. a member of the family of proteins with an α/β-hydrolase fold (1Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington J.S. Silman I. Schrag J.D. Sussman J.L. Verschueren K.H.G. Goldman A. Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1851) Google Scholar, 2Cygler M. Schrag J. Sussman J.L. Harel M. Silman I. Gentry M.K. Doctor B.P. Protein Sci. 1993; 2: 366-382Crossref PubMed Scopus (539) Google Scholar), rapidly terminates cholinergic neurotransmission by hydrolysis of acetylcholine (3Massoulié J. Pezzementi L. Bon S. Krejci E. Valette F.M. Prog. Neurobiol. (Oxf.). 1993; 41: 31-91Crossref PubMed Scopus (1056) Google Scholar, 4Taylor P. Radic′ Z. Annu. Rev. Pharmacol. Toxicol. 1994; 34: 281-320Crossref PubMed Scopus (608) Google Scholar). In mammals, AChE is encoded by a single gene, yet a multiplicity of molecular forms arise through alternative mRNA processing and association of the catalytic subunits with structural subunits. Alternative mRNA processing gives rise to three splicing options: (a) a soluble monomer without the capacity for disulfide linkage; (b) a glycophospholipid-linked form that is membrane-associated and exists as monomers and dimers; and (c) an amphiphilic form found as monomers, dimers and tetramers (in the tetramer, amphiphilic character may be lost presumably through occlusion of the hydrophobic surfaces). Further structural complexity is achieved in the tetrameric assemblies through disulfide association of the catalytic subunits with structural subunits that are either amphipathic or collagen-like, giving rise to predominant forms in brain and skeletal muscle. The tetrameric forms are dimers of dimers where one set of disulfides links with a polyproline-containing structural subunit (5Bon S. Coussen F. Massoulié J. J. Biol. Chem. 1997; 272: 3016-3021Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) and the other set is formed between monomers (3Massoulié J. Pezzementi L. Bon S. Krejci E. Valette F.M. Prog. Neurobiol. (Oxf.). 1993; 41: 31-91Crossref PubMed Scopus (1056) Google Scholar, 4Taylor P. Radic′ Z. Annu. Rev. Pharmacol. Toxicol. 1994; 34: 281-320Crossref PubMed Scopus (608) Google Scholar). However, there is currently little structural information about the subunit orientation in the tetramer and the association of tetramers with structural subunits. Differences in the molecular forms of the cholinesterases are the primary determinants of their cellular disposition (3Massoulié J. Pezzementi L. Bon S. Krejci E. Valette F.M. Prog. Neurobiol. (Oxf.). 1993; 41: 31-91Crossref PubMed Scopus (1056) Google Scholar). Abnormal associations of AChE are apparent in Alzheimer's dementia, where a selective loss of the amphiphilic tetramers is observed (6Atack J.R. Perry E.K. Bonham J.R. Perry R.H. Tomlinson B.E. Blessed G. Fairbairn A. Neurosci. Lett. 1983; 40: 199-204Crossref PubMed Scopus (178) Google Scholar, 7Schegg K.M. Harrington L.S. Nielsen S. Zweig R.M. Peacock J.H. Neurobiol. Aging. 1992; 13: 697-704Crossref PubMed Scopus (32) Google Scholar). AChE was suggested to associate with or to nucleate the neuritic plaques found in the disease (8Geula C. Mesulam M.M. Terry R.D. Katzman R. Bick K.L. Alzheimer Disease. Raven Press, New York1994: 263-291Google Scholar, 9Carson K.A. Geula C. Mesulam M.M. Brain Res. 1991; 540: 2204-2208Crossref Scopus (81) Google Scholar, 10Moran M.A. Mufson E.J. Gomez-Ramos P. Acta Neuropathol. 1993; 85: 362-369Crossref PubMed Scopus (168) Google Scholar, 11Ulrich J. Meier-Ruge W. Probst A. Meier E. Ipsen S. Acta Neuropathol. 1990; 80: 624-628Crossref PubMed Scopus (106) Google Scholar, 12Alvarez A. Alarcon R. Opazo C. Campos E.O. Munoz F.J. Calderon F.H. Dajas F. Gentry M.K. Doctor B.P. De Mello F.G. Inestrosa N. J. Neurosci. 1998; 18: 3213-3223Crossref PubMed Google Scholar); however, the mode of this association is unknown. The active center of AChE, which consists of the triad Ser203-Glu334-His447 in mammals (13Rachinsky T.L. Camp S. Li Y. Ekström J. Newton M. Taylor P. Neuron. 1990; 5: 317-327Abstract Full Text PDF PubMed Scopus (135) Google Scholar), is nearly centrosymmetric to the subunit and is located at the bottom of a narrow gorge (14Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2437) Google Scholar). Inhibitors may bind at the active center or at a distant allosteric site, the peripheral anionic site, located at the gorge rim. The structure of recombinant monomeric mouse AChE (mAChE) in a complex with the peptidic inhibitor fasciculin 2 (Fas2), bound to the peripheral anionic site, provided the first mammalian cholinesterase template (15Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar). Only slight differences were observed in the conformation of Fas2-associated mAChE compared with uncomplexedTorpedo californica AChE (TcAChE) (14Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2437) Google Scholar). However, it was not possible to distinguish species-related differences from changes in conformation accompanying Fas2 binding. Indeed, little difference in TcAChE conformation was observed between structures of the apoenzyme (14Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2437) Google Scholar) and the Fas2 complex (16Harel M. Kleywegt G.J. Ravelli R.B.G. Silman I. Sussman J.L. Structure. 1995; 3: 1355-1366Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). In addition, currently available crystal structures of AChE reveal the entrance of the active site gorge to be either occluded by a symmetry-related molecule (14Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2437) Google Scholar, 17Harel 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 (851) Google Scholar, 18Axelsen P.H. Harel M. Silman I. Sussman J.L. Protein Sci. 1994; 3: 188-197Crossref PubMed Scopus (154) Google Scholar) or sealed with high surface complementarity by Fas2 (15Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar, 16Harel M. Kleywegt G.J. Ravelli R.B.G. Silman I. Sussman J.L. Structure. 1995; 3: 1355-1366Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar), therefore precluding structural analysis of an unprotected peripheral anionic site region. The crystal structure of mAChE refined to 2.9-Å resolution has good stereochemistry, with an R-factor of 21.5% for data in the 15 to 2.9-Å resolution range, and contains four mAChE monomers assembled as a tetramer, three GlcNAc moieties and one GlcNAc-β1,4-GlcNAc-α1,6-Fuc trisaccharide moiety linked to Asn residues, four decamethonium (DECA) molecules, four phosphate groups, four glycerol molecules, and 205 water molecules in the asymmetric unit. The structure of mAChE provides significantly improved accuracy in the positions of the main and side chains of the molecule over the Fas2·mAChE structure, reveals distinctive features of the mouse enzyme, and permits direct comparison of solvent-exposed and -occluded peripheral anionic sites within the same crystal unit. More importantly, this structure highlights surface determinants that could participate in formation of oligomers upon assembly in normal and pathological states, in allosteric modulation of catalysis, and in forming heterologous cell contacts. Soluble mAChE expressed in HEK-293 cells was purified by affinity chromatography with desorption using either 100 mm DECA (structures A and C) or 10 mm edrophonium (EDR) (structure B); extensively dialyzed against 1 mm MES, pH 6.5, 50 mm NaCl, and 0.01% (w/v) NaN3; and prepared as described previously (19Marchot P. Ravelli R.B.G. 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). Crystallization was achieved at 4 °C by vapor diffusion using hanging drops (4 μl) and a protein/well solution ratio of 1:1. Well solutions were made of 1.7 mNaKPO4, pH 7.0, and 10 mm CaCl2(structure A) and 1.9 m NaKPO4, pH 7.0, and 10 mm MgCl2 (structure B), which were ice-cooled and centrifuged (10,000 × g, 4 °C) to remove precipitated material, or 0.95 m sodium citrate, pH 7.0 (structure C). Single crystals grew within 2–4 weeks to an average size of 0.2 × 0.05 × 0.05 mm whether NaKPO4 or sodium citrate was the precipitating salt. The crystals were flash-cooled at 100 K using 5–20% glycerol in the well solution as cryoprotectant. Oscillation images were integrated with DENZO (20.Otwinowski, Z., Proceedings of the CCP4 Study Weekend on Data Collection & Processing, Sawyer, L., Issacs, N., Burley, S., 1993, 56, 62, Science and Engineering Research Council/Daresbury Laboratory, Warrington, England.Google Scholar) and scaled and merged with SCALA (21.Evans, P. R., Proceedings of the CCP4 Study Weekend on Data Collection & Processing, Sawyer, L., Issacs, N., Burley, S., 1993, 114, 122, Science and Engineering Research Council/Daresbury Laboratory, Warrington, England.Google Scholar) (TableI). Amplitude factors were generated with TRUNCATE (22CCP4 Collaborative Computational Project No. 4 Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19793) Google Scholar). All three crystals belonged to the orthorhombic space group P212121 with unit cell dimensions a = 136.5 Å, b = 173.1 Å, and c = 224.2 Å, giving V m values of 5.1 Å3/Da (76% solvent) and 2.55 Å3/Da (38%) for four and eight mAChE molecules, respectively, in the asymmetric unit (23Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7925) Google Scholar).Table IData collection and refinement statisticsStructure AStructure BStructure CData collectionSynchrotron sourceDESYDESYLUREBeam lineX11BW7BDW32Resolution range (Å)15 to 2.915 to 3.115 to 3.1Total observations604,052727,622636,975Unique reflections107,37999,32692,186Multiplicity2.82.63.3Completeness (%)Overall (final shell)92 (90)90 (78)96 (92)R merge (final shell)1-aR merge = Σ ∣ I− 〈I〉 ∣ / Σ I, where I is an individual reflection measurement and 〈I〉 is the mean intensity for symmetry-related reflections.10.5 (37)12 (39)13 (42)RefinementR-factor (%)1-bR-factor = Σ ∣ ∣ Fo ∣ − ∣F c ∣ ∣ / Σ ∣ F o ∣, where F o and F c are observed and calculated structure factors, respectively.21.523.023.0R free(%)1-cR free is calculated for 2% of randomly selected reflections excluded from refinement.24.526.026.0r.m.s. deviation1-dr.m.s. deviation from ideal values.Bonds (Å)0.0110.0110.011Angles (°)1.61.61.6Average B-factor (Å)1-bR-factor = Σ ∣ ∣ Fo ∣ − ∣F c ∣ ∣ / Σ ∣ F o ∣, where F o and F c are observed and calculated structure factors, respectively.Main chains40.642.343.2Side chains45.346.248.1Solvent42ND1-eND, not determined.NDDecamethonium/edrophonium6827NDOligosaccharide757874Ramachandran plotResidues in most favored regions (%)848484Residues in additionally allowed regions (%)15.515.515.51-a R merge = Σ ∣ I− 〈I〉 ∣ / Σ I, where I is an individual reflection measurement and 〈I〉 is the mean intensity for symmetry-related reflections.1-b R-factor = Σ ∣ ∣ Fo ∣ − ∣F c ∣ ∣ / Σ ∣ F o ∣, where F o and F c are observed and calculated structure factors, respectively.1-c R free is calculated for 2% of randomly selected reflections excluded from refinement.1-d r.m.s. deviation from ideal values.1-e ND, not determined. Open table in a new tab Initial phases for an in-house 4.5-Å resolution data set were obtained by molecular replacement using the mAChE molecule present in the structure of the Fas2·mAChE complex (Protein Data Bank 1MAH) (15Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar) as a search model with the AMoRe program package (24Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar). Four mAChE subunits were positioned within the asymmetric unit (correlation = 75.2%,R-factor = 29.9% in the 15 to 4.5-Å resolution range) and found to assemble as a dimer of the same dimers as seen in the Fas2·mAChE structure. Rigid-body refinement applied to the whole tetramer, each of the two dimers, and the individual subunits was then performed with X-PLOR (25Brünger A.T. Kuriyan J. Karplus M. Science. 1987; 235: 1118-1135Google Scholar) using synchrotron data between 8 and 3 Å and gave an R-factor of 33%. For 2% of the reflections against which the model was not refined, R freewas 33%. Several cycles of Powell conjugate-gradient minimization were then performed, and electron density maps calculated with the subsequent model were inspected with TURBO-FRODO (26Roussel A. Cambillau C. Silicon Graphics Committee Silicon Graphics Geometry Partners Directory. Silicon Graphics, Mountain View, CA1989: 77-78Google Scholar). The positions of misplaced amino and carboxyl termini and of a few side chains were adjusted, and the Pro258–Gly264portion, which was missing in the search model, was built into 2F o − F c andF o − F c electron density maps. In each of the subunits in the tetramer, a DECA molecule was fitted into a residual density observed within the active site (see “Results”). A glycerol molecule arising from the cryoprotection solution was positioned into a density found in the vicinity of Glu81, with the glycerol oxygen atoms hydrogen-bonded to the Glu452, Glu81, and Thr436 oxygen atoms and the carbon atoms in van der Waals interactions with the side chain of Met85. A phosphate group arising from the crystallization liquor was positioned between the Lys332 and Arg395 side chains, with its oxygen atoms bound to the Trp442 carbonyl oxygen and Lys332 nitrogen atoms. Since the non-crystallographic symmetry (NCS) was not restrained throughout these early stages of model building and refinement, subunits were then superimposed within each dimer, and different NCS constraints were applied along the molecule and used during all subsequent refinement steps. This model was refined with REFMAC (27Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13908) Google Scholar), including all low resolution data. A conservative number of solvent molecules were manually added into the model; most of them were found to be located at nearly identical positions in the 2.5-Å resolution structure of TcAChE (Protein Data Bank 2ACE) (28Raves M.L. Harel M. Pang Y.-P. Silman I. Kozikowski A.P. Sussman J.L. Nat. Struct. Biol. 1997; 4: 57-63Crossref PubMed Scopus (393) Google Scholar). The conformations of some side chains were corrected using mAChE coordinates from a 2.7-Å resolution structure of the Fas2·mAChE complex. 2Y. Bourne, unpublished data. The final structure A comprises mAChE residues Glu4–Thr543 (subunits A and D) and residues Glu1–Cys257 and Asn265–Ala547 (subunits B and C) (see Figs. 1and 2). High temperature factors and weak electron density include mAChE residues Glu4–Gln7 and Arg493–Pro498 in all four subunits; Pro258–Gly264 is not visible in electron density maps of subunits B and C. The r.m.s. deviation between the two mAChE subunits of a canonical dimer is 0.06 Å for tight-constraint NCS atoms, 0.16 Å for medium-constraint NCS atoms, and 0.64 Å for loose-constraint NCS atoms. Structures B and C were solved using the refined structure A (inhibitor and solvent molecules removed) as an initial model and refined to 3.1 Å. The r.m.s. deviation values for the backbone atoms and all atoms are 0.21 and 0.3 Å between structures A and B, 0.13 and 0.2 Å between structures A and C, and 0.19 and 0.26 Å between structures B and C, respectively. The stereochemistry of all three structures was analyzed with PROCHECK (29Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and WHATIF (30Hooft R.W.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Crossref PubMed Scopus (1815) Google Scholar) (TableI). Figures were generated with the programs RIBBON (31Carson M. J. Appl. Crystallogr. 1991; 24: 958-961Crossref Scopus (784) Google Scholar), GRASP (32Nicholls A. GRAPS: Graphical Representation and Analysis of Surface Properties. Columbia University, New York1992Google Scholar), and TURBO-FRODO (26Roussel A. Cambillau C. Silicon Graphics Committee Silicon Graphics Geometry Partners Directory. Silicon Graphics, Mountain View, CA1989: 77-78Google Scholar).Figure 2Overall view of the mAChE molecule and localization of the short Ω loop relative to other secondary structure elements. The molecule (subunit D) is viewed down to the gorge entrance. Residues belonging to the peripheral anionic site (Tyr72, Asp74, Tyr124, Trp286, and Tyr341) and residues within the active center (Trp86, Ser203, Glu334, and His447) are displayed asgreen and gray bonds with colored spheres, respectively. The short Ω loop Cys257–Cys272 and helices α16,7 and α26,7 are displayed in red and green with ayellow disulfide bridge to the left the gorge entrance. The large Ω loop Cys69–Cys96 is displayed inorange above the gorge entrance. Helices α37,8 and α10, which contribute to the dimer four-helix bundle, are displayed in magenta at the bottom of the molecule. The GlcNAc moiety and the GlcNAc-β1,4-GlcNAc-α1,6-Fuc trisaccharide moiety linked to Asn265 on the left of the molecule and to Asn350 on the right, respectively, are displayed asgray bonds with colored spheres. The labels N and C indicate the amino and carboxyl termini of the molecule, respectively. The carboxyl-terminal segment Ala544–Ala547 is not resolved in this subunit.View Large Image Figure ViewerDownload (PPT) The mAChE molecule, which consists of a 12-stranded central-mixed β-sheet surrounded by 14 α-helices, has the same overall conformation as found for the Fas2·mAChE complex (15Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar): the r.m.s. deviations between the free and complexed mAChEs are 0.5 Å for the backbone atoms and 0.6 Å for all atoms, with the largest deviations occurring in surface loops (Ala24–Gly26, Pro108–Ser110, Gln322–Leu324, Asp372–Ala374, Leu386–Pro388, and Asp491–Ser497), in the position of domain Tyr341–Glu399, and in the orientation of Tyr337. mAChE, which is devoid of the carboxyl-terminal amphipathic helix and intersubunit disulfide-linking Cys, is monomeric in dilute solution (19Marchot P. Ravelli R.B.G. 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), but dimeric in the Fas2·mAChE crystals, where a homodimer assembles through a tightly packed four-helix bundle (15Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar). In the mAChE crystal, two identical homodimers assemble as a tetramer in which the two four-helix bundles are aligned antiparallel (Fig.1). The main axes of the two dimers are tilted by ∼35° from each other to form a compact, pseudo-square planar tetramer with overall dimensions of 90 × 90 × 100 Å. In the tetramer, a short surface loop and flanking α-helices from subunit A of the first dimer tightly associate with the peripheral site region at the gorge entrance of the facing subunit, subunit C of the second dimer (Fig. 1). The inverse loop-peripheral site interaction involves the loop and α-helices of subunit D in the second dimer and the peripheral site region of subunit B in the first dimer. As a result, peripheral sites of the two loop-acceptor subunits, B and C, are occluded, whereas peripheral sites of the two loop-donor subunits, A and D, are accessible to the solvent, a feature unique to this structure. At the tetramer center, the carboxyl-terminal α-helices in the four-helix bundles converge at the interface. The dimer-dimer interface, which extends over 90 Å in a direction roughly perpendicular to the four-helix bundle axis, buries to a 1.6-Å probe radius, a 2500-Å2 surface area for the two pairwise dimer-dimer interfaces, an area falling in the highest range for functionally relevant crystal packing interfaces (33Janin J. Nat. Struct. Biol. 1997; 4: 973-974Crossref PubMed Scopus (202) Google Scholar). Including the area buried at the four-helix bundle interface within each dimer (15Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar), the total mAChE surface area buried within the tetramer encompasses 6000 Å2. At the dimer-dimer interface, mAChE loop 257–272, a short Ω loop (34Fetrow J.S. FASEB J. 1995; 9: 708-717Crossref PubMed Scopus (169) Google Scholar) that is bridged by two Cys residues and contains the Pro-Pro-Gly-Gly-Ala-Gly-Gly sequence conserved in mammalian AChEs along with flanking α-helices α16,7 and α26,7 (Fig. 2), is associated with the peripheral anionic site at the gorge entrance of the facing mAChE subunit. The helix-loop-helix domain sterically occludes access to the active center gorge (Figs. Figure 1, Figure 2, Figure 3). This part of the interface involves hydrophobic and polar contacts of 12 residues from each subunit (Fig. 3). The Gly260–Gly264 cluster of hydrophobic residues at the loop tip packs against peripheral site residues Tyr72, Trp286, and Tyr341, with predominant interactions between dipeptide Gly260-Gly261 and Trp286 and between dipeptide Gly261-Ala262 and Tyr341. Two discrete patches of polar interactions involve the loop extremities and flanking helices with the boundary of the peripheral anionic site and appear to form three hydrogen bonds. The guanidinyl moieties of Arg245 and Arg253, located near the amino and carboxyl termini of helix α16,7, respectively, form key hydrogen bonds with the side chain carboxylate and the amide backbone nitrogen atom of Glu292, and the Gly263 nitrogen forms a hydrogen bond with the Tyr341 carbonyl oxygen. The loop shows high complementarity to the peripheral anionic site, a feature arising from the flexibility imparted by the vicinal Gly residues; limited internal stabilization of conformation is achieved from the adjacent Pro residues, with Pro258 in the cis-conformation and in van der Waals contacts with Ala262. Flexibility of the loop is evident since it cannot be seen in the absence of external stabilization, as found for subunits B and C in the tetramer (Fig. 1) and in the Fas2·mAChE complex at 3.2-Å resolution (15Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar); only the Pro doublet is seen in the Fas2·mAChE complex at 2.7-Å resolution.2 The second region of the dimer-dimer interface is located centrosymmetric to the tetramer where the two four-helix bundles converge (Fig. 1). The carboxyl-terminal region, not seen in the Fas2·mAChE structure, is ordered sufficiently to be seen in subunits B and C, with only the carboxyl-terminal Pro548 being disordered. The two Thr543 residues at and near the carboxyl-terminal ends of helices α10 of subunits A and C, respectively, face each other and are separated by only 7.5 Å. The segment from Ala544 to Ala547 is not part of helix α10; instead, it deviates from the helix axis and exits the plane of the tetramer, perhaps because of charge repulsion between Glu546 residues of neighboring subunits in the tetramer. Accordingly, the carboxyl-terminal helix α10ends at Thr543, the last residue encoded by exon 4 before the region of alternative splicing (13Rachinsky T.L. Camp S. Li Y. Ekström J. Newton M. Taylor P. Neuron. 1990; 5: 317-327Abstract Full Text PDF PubMed Scopus (135) Google Scholar, 19Marchot P. Ravelli R.B.G. 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). GlcNAc moieties are observed in two of the three consensus sequences for N-linked glycosylation, Asn350 in subunit A, also seen in the Fas2·mAChE complex (15Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar), and Asn265 in subunits A and D, although the density is weaker. In addition, a GlcNAc-β1,4-GlcNAc-α1,6-Fuc trisaccharide moiety is linked to Asn350 in subunit D (Figs. 1 and 2) (35Saxena A. Ashani Y. Raveh L. Stevenson D. Patel T. Doctor B.P. Mol. Pharmacol. 1998; 53: 112-122Crossref PubMed Scopus (69) Google Scholar, 36Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3778) Google Scholar). The structure of mAChE at 2.9-Å resolution provides greater delineation of the positions of the α-carbon and side chains than the Fas2·mAChE complex (3.2 Å) (15Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar) and establishes several distinctive features of mAChE compared with TcAChE (Protein Data Bank 2ACE) (28Raves M.L. Harel M. Pang Y.-P. Silman I. Kozikowski A.P. Sussman J.L. Nat. Struct. Biol. 1997; 4: 57-63Crossref PubMed Scopus (393) Google Scholar). Thus, the conformations of the amino- and carboxyl-terminal segments were resolved, and the mAChE-specific positioning of the Met85 side chain in its stacking interaction with the indole of Trp86 was confirmed. The Tyr341–Ser399 rigid-body motion that was found when comparing the Fas2·mAChE and TcAChE structures is not apparent in mAChE; hence, movement of this domain is induced by Fas2 association (15Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar). The most prominent feature arising upon superimposition of the structures is the close resemblance of the two peripheral site-occluding loops, the short Ω loop of mAChE subunit A and the central loop, loop II, of Fas2 bound to mAChE, in their positions and surface complementarity (Fig. 4). The mAChE loop and the tip of Fas2 loop II, although positioned roughly perpendicular with their respective Pro doublets not superimposable, overlap at the gorge entrance. Several side chains and backbone carbons of the mAChE loop mimic the side chains of Fas2 loop II in their interactions with residues in the peripheral anionic site. In particular, interaction of Gly261 in mAChE subunit A with Trp286 at the gorge entry of mAChE subunit C mimics the key interaction of Met33 in Fas2 with Trp286 in Fas2-associated mAChE; backbone atoms of Gly263-Gly264 in mAChE align with the side chain of Lys32 in Fas2; and Gly260 in mAChE establishes the same van der Waals contacts with Tyr72 as does Fas2 Leu35. Several residues in helix α16,7 of mAChE subunit A also adopt positions similar to residues in bound Fas2: the side chain of Arg253in" @default.
• W2072424636 created "2016-06-24" @default.
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• W2072424636 date "1999-01-01" @default.
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• W2072424636 title "Crystal Structure of Mouse Acetylcholinesterase" @default.
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• W2072424636 doi "https://doi.org/10.1074/jbc.274.5.2963" @default.
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