Crystal Structure of Human Butyrylcholinesterase and of Its Complexes with Substrate and Products

Abstract

Cholinesterases are among the most efficient enzymes known. They are divided into two groups: acetylcholinesterase, involved in the hydrolysis of the neurotransmitter acetylcholine, and butyrylcholinesterase of unknown function. Several crystal structures of the former have shown that the active site is located at the bottom of a deep and narrow gorge, raising the question of how substrate and products enter and leave. Human butyrylcholinesterase (BChE) has attracted attention because it can hydrolyze toxic esters such as cocaine or scavenge organophosphorus pesticides and nerve agents. Here we report the crystal structures of several recombinant truncated human BChE complexes and conjugates and provide a description for mechanistically relevant non-productive substrate and product binding. As expected, the structure of BChE is similar to a previously published theoretical model of this enzyme and to the structure of Torpedo acetylcholinesterase. The main difference between the experimentally determined BChE structure and its model is found at the acyl binding pocket that is significantly bigger than expected. An electron density peak close to the catalytic Ser198 has been modeled as bound butyrate. Cholinesterases are among the most efficient enzymes known. They are divided into two groups: acetylcholinesterase, involved in the hydrolysis of the neurotransmitter acetylcholine, and butyrylcholinesterase of unknown function. Several crystal structures of the former have shown that the active site is located at the bottom of a deep and narrow gorge, raising the question of how substrate and products enter and leave. Human butyrylcholinesterase (BChE) has attracted attention because it can hydrolyze toxic esters such as cocaine or scavenge organophosphorus pesticides and nerve agents. Here we report the crystal structures of several recombinant truncated human BChE complexes and conjugates and provide a description for mechanistically relevant non-productive substrate and product binding. As expected, the structure of BChE is similar to a previously published theoretical model of this enzyme and to the structure of Torpedo acetylcholinesterase. The main difference between the experimentally determined BChE structure and its model is found at the acyl binding pocket that is significantly bigger than expected. An electron density peak close to the catalytic Ser198 has been modeled as bound butyrate. Cholinesterases are divided into two subfamilies according to their substrate and inhibitor specificities: acetylcholinesterase (AChE 1The abbreviations used are: AchE, acetylcholinesterase; BchE, butyrylcholinesterase; TcAChE, Torpedo californica acetylcholinesterase; DmAChE, Drosophila melanogaster acetylcholinesterase; BTC, butyrylthiocholine; Bicine, N,N-bis(2-hydroxyethyl)glycine; MES, 4-morpholineethanesulfonic acid.; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8). Acetylcholinesterase is responsible for the hydrolysis of acetylcholine released at the synaptic cleft and the neuromuscular junction in response to nerve action potential (1Massoulie J. Sussman J. Bon S. Silman I. Prog. Brain Res. 1993; 98: 139-146Crossref PubMed Scopus (125) Google Scholar). In addition, both AChE and BChE seem to be involved in roles that are independent of their catalytic activities, such as cell differentiation and development (2Meshorer E. Erb C. Gazit R. Pavlovsky L. Kaufer D. Friedman A. Glick D. Ben-Arie N. Soreq H. Science. 2002; 295: 508-512Crossref PubMed Scopus (205) Google Scholar, 3Behra M. Cousin X. Bertrand C. Vonesch J.L. Biellmann D. Chatonnet A. Strahle U. Nat. Neurosci. 2002; 5: 111-118Crossref PubMed Scopus (307) Google Scholar). The catalytic mechanism of AChE is extremely efficient approaching diffusion-controlled rates (4Quinn D. Chem. Rev. 1987; 87: 955-979Crossref Scopus (946) Google Scholar). Unexpectedly, the crystal structure of the Torpedo californica enzyme (TcAChE) showed that the active site catalytic Ser-His-Glu triad is found at the bottom of a 20-Å deep gorge lined mostly with aromatic residues (5Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2426) Google Scholar). The structure also revealed the nature and the location of the previously described peripheral and “anionic” sites; the former, located at the outer rim of the gorge, has been postulated to be the initial substrate binding site (6Szegletes T. Mallender W.D. Thomas P.J. Rosenberry T.L. Biochemistry. 1999; 38: 122-133Crossref PubMed Scopus (152) Google Scholar). The binding of ligand to this site has been proposed to slow down the traffic of substrate and product at the acylation site (6Szegletes T. Mallender W.D. Thomas P.J. Rosenberry T.L. Biochemistry. 1999; 38: 122-133Crossref PubMed Scopus (152) Google Scholar, 7De Ferrari G.V. Mallender W.D. Inestrosa N.C. Rosenberry T.L. J. Biol. Chem. 2001; 276: 23282-23287Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Although a similar peripheral site has been described for human BChE, site-directed mutagenesis and photo-affinity labeling studies showed that its location and the response upon ligand binding differ significantly from those of AChE (8Nachon F. Ehret-Sabatier L. Loew D. Colas C. van Dorsselaer A. Goeldner M. Biochemistry. 1998; 37: 10507-10513Crossref PubMed Scopus (52) Google Scholar, 9Masson P. Legrand P. Bartels C.F. Froment M.T. Schopfer L.M. Lockridge O. Biochemistry. 1997; 36: 2266-2277Crossref PubMed Scopus (133) Google Scholar). The site to which the positively charged quaternary ammonium of choline moiety productively binds is found half-way down the gorge, in between the peripheral and acylation sites. Originally, there was a great deal of controversy concerning the nature of the residues involved in this site. Both the crystal structure and labeling experiments showed that positively charged ligands form π-cation interactions with Phe 330 and Trp 84 (numbering in italics corresponds to that of torpedo AChE) (10Harel 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 (845) Google Scholar). The physiological role of BChE remains unclair (11Chatonnet A. Lockridge O. Biochem. J. 1989; 260: 625-634Crossref PubMed Scopus (472) Google Scholar, 12Mack A. Robitzki A. Prog. Neurobiol. 2000; 60: 607-628Crossref PubMed Scopus (122) Google Scholar). Although it is capable of hydrolyzing ACh and other acylcholines, so far no endogenous natural substrate has been described for this enzyme. Because BChE is relatively abundant in plasma (about 3 mg/liter), and can degrade a large number of ester-containing compounds, it plays important pharmacological and toxicological roles (13Lockridge O. Masson P. Neurotoxicology. 2000; 21: 113-126PubMed Google Scholar). For instance, BChE is a potential detoxifying enzyme to be used as a prophylactic scavenger against neurotoxic organophosphates such as the nerve gas soman (14Allon N. Raveh L. Gilat E. Cohen E. Grunwald J. Ashani Y. Toxicol. Sci. 1998; 43: 121-128PubMed Google Scholar, 15Broomfield C.A. Maxwell D.M. Solana R.P. Castro C.A. Finger A.V. Lenz D.E. J. Pharmacol. Exp. Ther. 1991; 259: 633-638PubMed Google Scholar, 16Raveh L. Grunwald J. Marcus D. Papier Y. Cohen E. Ashani Y. Biochem. Pharmacol. 1993; 45: 2465-2474Crossref PubMed Scopus (173) Google Scholar). We have recently published the engineering and crystallization of a monomeric and partially glycosylated recombinant human BchE (17Nachon F. Nicolet Y. Viguie N. Masson P. Fontecilla-Camps J.C. Lockridge O. Eur. J. Biochem. 2002; 269: 630-637Crossref PubMed Scopus (121) Google Scholar). Here we report several crystal structures of BChE complexed with a substrate, products, and conjugated to soman after aging. From these structures we propose alternative substrate and product binding that may be related to the high catalytic efficiency of the choline esterases. Crystallization of Recombinant BChE and Its Complexes—Recombinant human BChE suitable for crystallization was obtained, purified, and crystallized as described previously (17Nachon F. Nicolet Y. Viguie N. Masson P. Fontecilla-Camps J.C. Lockridge O. Eur. J. Biochem. 2002; 269: 630-637Crossref PubMed Scopus (121) Google Scholar). No butyrate was present in the culture medium and none was added during any step of the purification procedure. The 3-bromopropionate-BChE complex was obtained by soaking crystals for a few minutes in the mother liquor containing 100 mm bromopropionate (Sigma). The BChE-choline complex was obtained by soaking BChE crystals grown from a 2.1 m (NH4)2SO4 100 mm Bicine (Fluka), pH 9.0, crystallization solution, in the mother liquor containing 100 mm choline chloride. Crystallization of soman-aged BChE: racemic soman (pinacolyl methylphosphonofluoridate) was obtained from CEB Le Bouchet (Vert-le-Petit, France). The purified enzyme (6.6 mg/ml) was inhibited in the presence of 0.5 mm soman (∼5.5-fold molar excess) in 10 mm MES buffer, pH 6.5. The reaction mixture was further incubated for 3 days at 4 °C, allowing enough time for completion of the aging reaction and the disappearance of the remaining unreacted soman. The inhibited enzyme was crystallized under the same conditions as the uninhibited BChE except that the mother liquor was buffered at pH 8.0 using a 0.1 m Tris/HCl buffer solution. Soman-aged BChE and butyrylthiocholine (BTC) were cocrystallized at pH 6.5 (0.1 m MES buffer, 2.1 m (NH4)2SO4) with 10 mm BTC (Sigma). X-ray data were collected from a 4-day-old crystal to limit the loss of substrate by spontaneous hydrolysis. X-ray Data Collection and Structure Solution—All crystals were flash-cooled at 100 K in a nitrogen stream using 15–20% glycerol in the mother liquor as cryoprotectant. Data sets were collected at the following beamlines of the European Synchrotron Radiation Facility (Grenoble, France): ID14-eh1 for the soman-aged BChE, ID14-eh2 for the native BChE, BM30 for the choline-BChE complex and the somanaged BTC complex, and ID14-eh4 for the 3-bromopropionate-BChE complex (see Table I). Data sets for the native, the soman-aged BchE, and soman-aged BChE-BTC complex crystals were integrated, scaled, and reduced using MOSFLM, SCALA, and TRUNCATE from the CCP4 suite (18Collaborative Computational Project No. 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar). Data sets from the choline-BChE complex and the 3-bromopropionate-BChE species were processed using the program XDS (19Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3233) Google Scholar). Data collection from the 3-bromopropionate-BChE crystal was performed at a wavelength of 0.915 Å to measure the Br anomalous scattering signal. Subsequent data processing was performed without merging the Friedel mates to better evaluate the anomalous signal of the bromine atom. Molecular replacement was carried out with the native data set between 15- and 3.5-Å resolution using the program AMoRe (20Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5029) Google Scholar) and the TcAChE structure (Protein Data Bank ID code: 2ACE) as a search model. A well constrasted solution with R-factor = 42.4% and correlation coefficient = 46.7% was obtained for one monomer per asymmetric unit. This solution was used as a starting model for manual rebuilding and refinement of BChE against all data to 2.0-Å resolution with the programs TURBO (21Roussel A. Cambillaud C. TURBO computer program. Silicon Graphics, Mountain View, CA1989Google Scholar) and CNS (22Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gross P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. 1998; D54: 905-921Crossref Scopus (16967) Google Scholar), respectively. Observed structure factors were scaled anisotropically and a bulk solvent correction was applied. Several cycles of refinement, manual rebuilding, and solvent addition led to a model with good statistics (Table I). Residues 1–3, 378–379, and 455 did not have matching electron density and, consequently, were not included in the model. Carbohydrate chains corresponding to five of the six expected glycosylation sites (17Nachon F. Nicolet Y. Viguie N. Masson P. Fontecilla-Camps J.C. Lockridge O. Eur. J. Biochem. 2002; 269: 630-637Crossref PubMed Scopus (121) Google Scholar) have been included in the crystallographic model. They correspond to those connected to Asn57, Asn106, Asn241, Asn341, and Asn485. Although Asn256 was expected to be glycosylated, no electron density was observed for carbohydrate. Further examination of the electron density maps led to the inclusion of three molecules of glycerol, used as cryoprotectant, two sulfate ions, the precipitanting agent, one molecule of MES buffer, and two chloride ions. During refinement, an unexpected residual electron density was observed the of the catalytic the of refinement and after several at this density as corresponding to added during purification or a good was obtained butyrate was used as a model (see and refinement choline-BChE complex was to cell a = = of data processing was carried out without merging the Friedel mates to evaluate the anomalous signal from the bromine from The choline-BChE complex was to data processing was carried out without merging the Friedel mates to evaluate the anomalous signal from the bromine atom. in a The soman-aged BChE structure was using the refinement from the program The model statistics are shown in Table I. Residues 1–3, 378–379, and 455 and to Asn256 and no matching electron density and were not included in the model. The soman-aged BChE structure also the three glycerol the two chloride ions, and one of the two sulfate previously observed in the native The same refinement was to this and the choline-BChE structure (Table I). The structure of the 3-bromopropionate-BChE complex was using the native BChE structure as a starting set in which the modeled butyrate been difference and difference maps were with the program The starting Data Bank and for were obtained using R. M. R.W. J. PubMed Scopus Google Scholar). it was to the electron density for bromine and the other were obtained the to that the moiety was partially during the soaking of BChE crystal structure was by the replacement and to 2.0-Å resolution using CNS (see and Table I). As expected M. Sussman J.L. E. Bon S. P. J. Silman I. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, D. A. D. N. C. A. Biochemistry. 2001; PubMed Scopus Google Scholar, S. P. Biochemistry. 1993; PubMed Scopus Google Scholar, A. Jiang Lockridge O. Biochemistry. 1997; 36: PubMed Scopus Google Scholar, Y. S. P. Biochemistry. 1993; PubMed Scopus Google Scholar), the structure of BChE is similar to that of BChE not form the observed in structures of TcAChE (5Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2426) Google Scholar, M. Silman I. Sussman J.L. Structure Full Text Full Text PDF PubMed Scopus Google Scholar), AChE Y. P. P. Full Text PDF PubMed Scopus Google Scholar, Y. P. P. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and human AChE Harel M. Toker L. A. C. D. N. A. Silman I. Sussman J.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; PubMed Scopus Google Scholar). In the involved in the are and the active site are located of the Although are found at the BChE and are not form a and the active site are located the same of the from the crystal be responsible for these between BChE and TcAChE are to the residues the gorge, the former enzyme has several of the aromatic of the by the with the of Phe and Phe of TcAChE by and these it for the binding of the butyrate substrate moiety in BChE and the of the acyl In addition, the catalytic is connected to a large electron density peak is in the We in that a previously modeled structure of BChE M. Sussman J.L. E. Bon S. P. J. Silman I. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) from the structure at these a the AChE starting As an the acyl pocket of the model is significantly than the one observed in the crystal A to the During the crystallographic refinement it that the catalytic was bound to an moiety that was modeled as butyrate with well matching electron density and this we native crystals with the butyrate in which the is by a bromine atom. we collected data at wavelength bromine to a anomalous signal. of the moiety by its with no in the of the corresponding electron In the of the enzyme the of the the acyl to the catalytic to with no residual in the difference electron density the other maps after crystallographic refinement of butyrate or the that one is a good model for the We are the of the using density Although the of the moiety is it to be for crystallization because were extremely to In the few crystals grown from these the butyrate bound to crystals were obtained or BTC, was added to the crystallization This may be related to the of the acyl pocket in BChE by butyrate. A we the native TcAChE, and the has the same (see residues to the acyl have in the three in BChE the acyl the at the catalytic by butyrate or soman The reaction of soman and other related esters with the catalytic of can be by such as in J. Appl. Toxicol. 1994; PubMed Scopus Google with of an the a J. Science. PubMed Scopus Google Scholar). for its the is a of both the we have in the BChE crystals and of the postulated The a with one of the a was also observed in the crystal structures of and TcAChE A. Harel M. Y. D. A. Silman I. Sussman J.L. Biochemistry. 1999; 38: PubMed Scopus Google Scholar). A of the acyl has also been for the crystal structure of these in that the acyl is and are with that the AChE active site the gorge, this or an alternative located it J. Chem. 1998; Scopus Google Scholar). As of the acyl by butyrate to be for at the an of the Ser198 in BChE, we crystals in a 100 mm choline chloride solution, pH maps with data collected from a crystal no electron density corresponding to the In this structure the from Ser198 two a one it a with the catalytic and a expected it has from the and a with the main from at the a and is that this is to the of at pH 9.0, because crystals grown at pH 6.5 similar Ser198 with or and a native structure at pH has the butyrate bound to the active site in this structure is the of the bound as expected, its quaternary ammonium binds close to a π-cation the the gorge and a with a molecule in with the main from Although choline binding may not be relevant the high choline in the soaking solution and the it that this reaction product may at two at the active the as of bound to the active site and an alternative as observed a and The of an alternative choline binding to an crystals obtained from previously inhibited with soman were in a solution containing the substrate In the complex between soman-aged BChE and BTC the binds with its ammonium close to and its to a molecule to the one described for the BChE-choline complex This the that substrate binding π-cation interactions is and that a bound to the catalytic is with site substrate binding as by the soman-aged BChE-BTC data for all the structures are in Table I. the in with data from the concerning the catalytic mechanism of enzymes to an step in the reaction is by the and extremely efficient is of the substrate binding sites. sites as for substrate at the outer rim of the gorge by a not From one or several of these substrate can into an to the one observed in the soman-aged BChE-BTC as by Masson P. Legrand P. Bartels C.F. Froment M.T. Schopfer L.M. Lockridge O. Biochemistry. 1997; 36: 2266-2277Crossref PubMed Scopus (133) Google Scholar), a of the molecule its quaternary ammonium bound at the π-cation can the substrate to binding. of the of BTC to a than to may this has been proposed that the of the catalytic triad in at the bottom of a gorge the of the in a M. D.M. Silman I. Sussman J.L. J. Chem. Scopus Google Scholar). The provide an for the location of the catalytic triad as the presence of peripheral and sites at the gorge for a of substrate it to the active site in a most efficient The presence of a butyrate bound to Ser198 be related to the of the catalytic This is by the by Y. P. P. J. PubMed Scopus Google Scholar) described a or molecule found close to the catalytic of AChE in complex with a peripheral site other structures of AChE also electron close to the catalytic M. Rosenberry T.L. Mallender W.D. T. R.J. Silman I. Sussman J.L. Sci. 2000; PubMed Scopus Google Scholar, M. S. Harel M. P. Silman I. J. Sussman J.L. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). Although the between of Ser198 and of of Å is with the between and is a that is for that of In this were to the of the choline moiety be have been For the A structure at resolution T. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. PubMed Scopus Google Scholar) a of Å and the is far from that the is or to the of of the is to propose that the of to to Ser198 this is not this to be well and be in all the structures the other we have observed that in several BChE complexes the of Ser198 is and no to to for a report by Masson P. F. Bartels C.F. Froment M.T. F. C. Lockridge O. Eur. J. Biochem. PubMed Scopus Google Scholar) that in the presence of substrate and BChE is under these previously data T. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. PubMed Scopus Google Scholar, P. F. Bartels C.F. Froment M.T. F. C. Lockridge O. Eur. J. Biochem. PubMed Scopus Google Scholar), the of the catalytic by during alternative to of the catalytic by during be a product binding at the catalytic The presence of a butyrate moiety in the purified BChE at this As upon substrate binding at the active site the its with the a the and an the the of the of Ser198 the partially atom. This the step of the reaction by the the of the A similar mechanism a molecule the X-ray structures of both and soman-aged BChE have a glycerol molecule bound to the π-cation site. As glycerol was used as (see the its presence in the active site gorge the of the π-cation site and that the substrate is to residues and the acyl The presence of a moiety to the catalytic is Several of that in it is most butyrate as it can be by it is by and the presence of butyrate is for experiments be to its and structure factors have been with the Data Bank with with and with soman and and be released upon We are to the of and beamlines from the European Synchrotron Radiation is for with data collection from the 3-bromopropionate-BChE