Báo cáo khoa học: Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase pdf

A recombinant monomeric BChE lacking four out of nine N-glycosylation sites and the C-terminal oligomerization domain was stably expressed as a monomer in CHO cells.. three-dimensional m

Engineering of a monomeric and low-glycosylated form

of human butyrylcholinesterase

Expression, puri®cation, characterization and crystallization

Florian Nachon1, Yvain Nicolet2, Nathalie ViguieÂ1, Patrick Masson1, Juan C Fontecilla-Camps2

and Oksana Lockridge3

1 Centre de Recherches du Service de Sante des ArmeÂes, Unite d'Enzymologie, La Tronche, France; 2 Laboratoire de Cristallographie

et CristallogeÂneÁse des ProteÂines, Institut de biologie structurale ÔJ.P EbelÕ, Grenoble, France; 3 University of Nebraska Medical Center, Eppley Research Institute, Omaha, NE, USA

Human butyrylcholinesterase (BChE; EC 3.1.1.8) is of

particular interest because it hydrolyzes or scavenges a wide

range of toxic compounds including cocaine,

organophos-phorus pesticides and nerve agents The relative contribution

of each N-linked glycan for the solubility, the stability and

the secretion of the enzyme was investigated A recombinant

monomeric BChE lacking four out of nine N-glycosylation

sites and the C-terminal oligomerization domain was stably

expressed as a monomer in CHO cells The puri®ed

recom-binant BChE showed catalytic properties similar to those of

the native enzyme Tetragonal crystals suitable for X-ray

crystallography studies were obtained; they were improved

by recrystallization and found to di€ract to 2.0 AÊ resolution using synchrotron radiation The crystals belong to the tetragonal space group I422 with unit cell dimensions

a ˆ b ˆ 154.7 AÊ, c ˆ 124.9 AÊ, giving a Vmof 2.73 AÊ3per

Da (estimated 60% solvent) for a single molecule of recombinant BChE in the asymmetric unit The crystal structure of butyrylcholinesterase will help elucidate unsolved issues concerning cholinesterase mechanisms in general

Keywords: butyrylcholinesterase; crystallization; N-glycosy-lation; site-directed mutagenesis; X-ray di€raction

Acetylcholinesterase (AChE; EC 3.1.1.7) and

butyrylcho-linesterase (BChE; EC 3.1.1.8) are closely related serine

hydrolases with different substrate speci®city and inhibitor

sensitivity AChE terminates the action of the

neurotrans-mitter acetylcholine at postsynaptic membranes and

neuro-muscular junctions Although BChE is found in various

vertebrate tissues (liver, intestine, lung, heart, muscle, brain,

serum), its physiological role remains undetermined

How-ever, plasma BChE is of pharmacological and toxicological

importance because it hydrolyzes ester-containing drugs

such as succinylcholine and cocaine Consequently, puri®ed

BChE has been used for treatment of

succinylcholine-induced apnea in humans [1] and it is known to protect

rodents from the toxic effects of cocaine [2,3] To improve

the rate of hydrolysis of cocaine, a mutated enzyme has been

designed [4] However, a higher catalytic rate may be

necessary if BChE is to be used therapeutically in severe cocaine overdoses

Human BChE is also known to be a good scavenger of organophosphorus (OP) pesticides and chemical warfare nerve agents [5] For example, injections of puri®ed BChE as pretreatment against nerve agent poisoning in mice, rats and guinea pigs increased their survival with a higher ef®ciency than the classical pretreatment with pyridostigmine [6±8] Similar observations have been reported for monkeys [9,10] Mutants of human BChE (G117H) capable of hydrolyzing

OP have also been designed [11]; however, their catalytic mechanism is unclear [12] It is noteworthy that the equivalent human AChE mutant (G122H) did not acquire

OP hydrolase activity (Lockridge, O & Bartels, C.F unpublished results) Thus, BChE could be used in the near future for OP decontamination, pretreatment and treatment

of OP poisoning

Progress in engineering of BChE is currently limited by the lack of a three-dimensional structure three-dimensional models of human BChE have been built by homology to the Torpedo californica acetylcholinesterase X-ray structure [13,14] Although these models contributed to the under-standing of some aspects of the difference in speci®city between AChE and BChE, they are not satisfactory for enzyme engineering The crystal structure of human BChE

is expected to provide new insights into unsolved issues such as allosteric modulation of cholinesterase activity (BChE presents substrate activation, whereas AChE has substrate inhibition) or the traf®c of substrate, products, and water molecules in and out of the active site gorge [15,16]

Correspondence to F Nachon, Centre de Recherches du Service de

Sante des ArmeÂes, Unite d'enzymologie, 24 Avenue des Maquis du

GreÂsivaudan, BP 87±38702 La Tronche CeÂdex, France.

Fax: + 33 4 76 63 69 61, Tel.: + 33 4 76 63 69 88,

E-mail: ¯orian@nachon.net

Abbreviations: AChE, acetylcholinesterase; BChE,

butyrylcholinest-erase, CCD, charge coupled device; ChE, cholinesterase; CHO,

Chi-nese hamster ovary; DMEM, Dulbecco's modi®ed Eagle's medium;

Nbs 2 , 5,5¢-dithiobis-2-nitrobenzoic acid; HEK, human embryonic

kidney cells; OP, organophosphorus ester.

(Received 6 August 2001, revised 19 November 2001, accepted 20

November 2001)

During the past decade, the crystallization of puri®ed

plasma BChE has not been successful, despite an

exhaus-tive screening program in one of our laboratories Human

BChE is a heavily glycosylated homotetramer of 340 kDa

with nine N-glycosylation sites per catalytic subunit

representing almost 25% of its mass [17,18] It is known

that the glycan moieties often perturb crystallization

[19,20] Human BChE oligosaccharides, which are of the

complex biantennary type [21,22], could shield the protein

surface and prevent or reduce favorable crystal contacts

Therefore, several attempts to deglycosylate the native

enzyme were made Chemical deglycosylation with

tri¯u-oroacetic acid, which was successfully used on horseradish

peroxidase [23], as well as enzymatic partial

deglycosyla-tion using neuraminidase and galactosidase (Masson, P

unpublished results) led to aggregation Due to the

presence of fucose residues, enzymatic deglycosylation

using large amounts of recombinant

GST±N-glycosi-dase F fusion protein [24] was not ef®cient except under

mild denaturing conditions Thus, we decided to

investi-gate the effects of the suppression of N-glycosylation sites

to produce a low-glycosylated recombinant BChE suitable

for crystallization

M A T E R I A L S A N D M E T H O D S

Mutagenesis

4sugOff17/455/481/486BChEDwas obtained by PCR using Pfu

polymerase Carbohydrate attachment sites at N17, N455,

N481, and N486 were deleted by mutating Asn residues to

Gln residues The tetramerization domain at the C-terminus

of BChE was deleted by placing a stop codon at position

530 [25,26] The stop codon deleted 45 amino acids from the

C-terminus to yield a protein containing 529 amino acids

and six carbohydrate chains PCR fragments were cloned

into the expression plasmid pGS and resequenced to

con®rm that only the desired mutations were present

Plasmid pGS has the CMV promoter and rat glutamine

synthetase for selection

Other mutants from which carbohydrate attachment sites

were deleted were also constucted by PCR In each case, a

codon for Asn was replaced by a codon for Gln The

expression plasmid pGS was suitable for both transient and

stable expression

Transient expression

BChE mutants were transiently expressed in human

embryonic kidney cell line 293T/17, used with permission

from D Baltimore (Rockefeller University of New York;

ATCC No CRL 11268) Cells were grown to 80±90%

con¯uence in 100 mm dishes and then transfected by

calcium phosphate co-precipitation of 20 lg plasmid DNA

per dish Four days after transfection, the culture medium

[5% fetal bovine serum in Dulbecco's modi®ed Eagle's

medium (DMEM)] was harvested for a BChE activity

assay Each mutant BChE was transfected into ®ve dishes

Large scale production of recombinant human BChE

4sugOff17/455/481/486BChEDin pGS was expressed in CHO

cells and stably transfected as previously described [11]

Selective pressure to retain the plasmid was provided by

25 lMmethionine sulfoximine Secreted BChE was collected into serum-free and glutamine-free culture medium, Ultra-culture (BioWhittaker, Walkersville, MD, USA; catalogue

no 12±725B), thus avoiding contamination by AChE pre-sent in fetal bovine serum No antibiotics were added to the culture medium The cells were grown in 1-L roller bottles The culture medium (150 mL per bottle) in the roller bottles was changed every 2±4 days A roller bottle yielded enzyme continuously for as long as 6 months Each L of culture medium contained 3±5 mg of 4sugOff17/455/481/486BChED Puri®cation of 4sugOff17/455/481/486BChED

Units of activity are expressed as lmoles of substrate hydrolyzed per minute Protein concentration was estimated from absorbance at 280 nm (E1%ˆ 18) A speci®c activity

of 720 Uámg)1, measured at 25 °C with 1 mM butyrylthi-ocholine in 0.1M potassium phosphate pH 7.0, was the standard for 100% pure native BChE All puri®cation steps were conducted at 4 °C

Serum-free culture medium was collected from roller bottles over a period of 6 months Twenty-six liters of culture medium containing 100 mg of 4sugOff17/455/481/486

BChED were loaded onto 400 mL of procainamide± Sepharose packed in a XK50/30 Pharmacia column (diameter, 5 cm; ¯ow rate of 1 Láh)1) The column was washed with 20 mM potassium phosphate, pH 7.0, 1 mM

EDTA (until D280 0) and then with 0.1, 0.2 and 0.3M

NaCl in buffer The BChE activity was eluted with buffer containing 0.3M NaCl and 0.1M N(Me)4Br The eluted enzyme was 21% pure as judged from speci®c activity Then, the 4sugOff17/455/481/486BChEDwas dialyzed against

20 mM Tris/HCl pH 7.4, and loaded onto 400 mL of DE52 anion exchanger (Whatman; catalog no 4057200, purchased from Fisher Scienti®c) packed in Pharmacia C26/100 column The column was washed with 20 mM

Tris/HCl pH 7.4 until D280 0 BChE was eluted with a NaCl gradient (0±0.5MNaCl in 1 L buffer); 80% of the BChE activity was recovered The cleanest fractions ( 80% pure) were loaded directly onto a 10-mL procainamide±Sepharose column packed in Pharmacia C10/20 (0.9 cm diameter ´ 16 cm) The column was washed with 2 L of 20 mM Tris/HCl pH 7.4 4sugOff17/455/481/486

BChED (9.3 mg, 6740 U; 98% pure) was eluted with

400 mL of 0.6M NaCl in 20 mM Tris/HCl pH 7.4, then dialyzed against 5 mM Mes pH 6.5 and concentrated to

10 mgámL)1 (7200 UámL)1) in an Amicon Dia¯o appa-ratus with a PM10 membrane The dialyzed, concentrated samplewas ®lteredthrougha0.2-lm®lterandstoredat4 °C Determination of kinetic parameters

Hydrolysis of butyrylthiocholine iodide at 25 °C was measured at concentrations ranging from 0.010 to 50 mM

according to the method of Ellman [27] The buffer was 0.1M sodium phosphate at pH 7.0 and contained 0.1 mgámL)1 Nbs2 and 0.1% BSA The active sites were titrated by the method of residual activity using diisopropyl phosphoro ¯uoridate (DFP) as titrant [28] Kinetic parameters (kcat, Km, Kss, b factor) were deter-mined by nonlinear ®tting of the apparent rate vs [S] using the equation described by Radic et al [29]

A home-made sparse matrix kit similar to the one described

by Jancarik & Kim [30] was used to screen for initial

crystallization conditions in a hanging drop system [20]

BChE crystallized at a concentration of 6.6 mgámL)1from a

0.1-M Mes buffer solution, pH 6.5 at 20 °C, containing

2.05±2.15M (NH4)2SO4 (Fluka) and using drops of 3 lL

and a protein to reservoir ratio of 1 : 2 (v/v) Crystals grew

in about 1 week Their quality was improved using the

recrystallization procedure described by Kryger [31]

Catalytic activity in the crystals

A recombinant BChE crystal grown at pH 6.5 was washed

twice for 5 min in a 100 lL drop of 0.1MMes pH 6.5 buffer

containing 2.4M(NH4)2SO4 Then the crystal was soaked in

a 20-lL drop of the same buffer containing 0.1 mgámL)1

Nbs2 and 5 mM butyrylthiocholine iodide (Sigma) The

change in crystal coloration (turning yellow) was followed

under a binocular magnifying glass No spontaneous

hydrolysis of the substrate in the soaking liquor was

observed when monitored by spectrophotometry at

412 nm

Data collection

Diffraction data were collected at k ˆ 0.932 AÊ wavelength

to 2.0 AÊ resolution at the ID14-eh2 beamline of the

European Synchrotron Radiation Facility with a

MAR-Research CCD detector To prevent ice formation, crystals

were soaked for a few minutes in a 2.4-M(NH4)2SO4, 15%

glycerol, 0.1MMes pH 6.5 buffer just before ¯ash-cooling

at 100 K in a nitrogen stream Collected data were indexed,

integrated and reduced using MOSFLM and SCALA from

the CCP4 suite [32]

R E S U L T S A N D D I S C U S S I O N

Engineering of a low-glycosylated truncated BChE Glycosylation in¯uences the folding, secretion, stability, and solubility of ChE as well as the clearance of their plasmatic forms [22,33±35] Heavy glycosylation of BChE contributes to its long residence time in blood circulation and protects it against proteolysis For example, the glycosylation patterns may change with the tissue localiza-tion, but do not seem to play a critical role in the catalytic properties of the enzyme [36] Our goal was to favor the crystallization of BChE by designing an enzyme with the fewest possible glycosylation sites, while preserving its solubility, stability and functional properties Amino-acid sequences of AChE and BChE from different species were aligned to pinpoint the conserved N-glycosylation sites (Table 1) BChEs are generally more glycosylated than AChEs AChEs from different species contain three to six N-glycosylation sites, three of which are conserved in BChE Therefore, our ®rst attempt was to construct a recombinant BChE containing only these three glycosyla-tion sites (posiglycosyla-tions 256, 341 and 455) This was achieved by mutating six Asn residues in Asn-X-Ser/Thr recognition sites into Gln residues

These studies overlooked the possibility that a muta-tion of Asn486 might unmask a glycosylamuta-tion site at Asn485 Three glycosylation recognition sites are present

in the sequence N481ETQNNSTS489, but the peptide sequencing of human BChE showed that positions 481 and 486 were glycosylated, and position 485 was not [18] Because Asn485 and Asn486 are adjacent, the nongly-cosylation of Asn485 may be due to steric hindrance Therefore, we assume that all of the constructs with the double mutation N481Q/N486Q should be glycosylated

at position 485

Table 1 Comparison of the N-glycosylation positions for various cholinesterases.

Enzyme

Potential N-glycosylation sites (human BChE numbering)

17 57 106 241 256 341 455 481 486 Others BChE

AChE

A recombinant BChE containing the three conserved

glycosylation sites, N256, N341 and N455 plus the one

unmasked at position 485, was transiently expressed in 293T

cells Unfortunately, the expression level in the culture

medium was  10-fold lower than for the native enzyme

(Table 2, six sites off) The suppression of seven or nine sites

yielded poor expression levels as well (Table 2, seven and

nine sites off) due to retention of the protein inside the cell,

as shown by Western blotting As the expression level of

these clones was not high enough to produce large amounts

of BChE, new constructs were tested in which the

N-glycosylation sites were suppressed empirically

Suppression of sites N481 and N486 (Table 2, two sites

off) led to 45% higher expression levels than native BChE

Suppression of sites N455, N481 and 486 led to a 15%

greater expression level than the native enzyme (Table 2,

three sites off) When an additional site was suppressed at

position N256, the expression level was similar to that of the native enzyme (Table 2, four sites off; oligomeric domain: ÔyesÕ) The additional N341Q mutation resulted in a ®vefold lower active enzyme (Tables 2, ®ve sites off) Consequently, the ®ve glycosylation sites mutant was not used any further Interestingly, the N341 site is also conserved in Candida rugosa lipase, where it plays an important role in the stabilization of the open conformation of the enzyme [37] Such a role has not yet been observed in cholinesterases The tetramerization domain is located at the C-termini of AChE and BChE In human BChE, this domain comprises

40 amino acids, encoded by exon 4 Its deletion leads to higher levels of secretion into the culture medium and expression of monomers [25] Crystallization of monomeric cholinesterases is more favorable than for oligomeric forms, even if they form a noncovalent dimer by association of a four-helix bundle (helices 383±372 and 526±543; human

Table 2 In¯uence of the number and position of N-glycosylation sites on the expression level of secreted human BChE The presence or absence of the oligomerization domain at the C-terminus is indicated by yes or no Transient transfection in 293T cells was repeated in ®ve dishes The relative expression unit corresponds to 0.2 lmol butyrylthiocholine hydrolyzed per minute.

Number

sites o€ Oligomericdomain

Potential N-glycosylation sites

17 57 106 241 256 341 455 481 485 486 Relativeexpression level

a Wild-type human BchE b Clone chosen for crystallization trials.

Fig 1 Alignment of the amino-acid sequences of 4sugO€ 17/455/481/486 BChE D , human BChE, and crystallized forms of human AChE, mouse AChE and Torpedo californica AChE 4sugO€ 17/455/481/486 BChE D (Rec BChE), human BChE [18], human AChE [44], mouse AChE [38] and T californica AChE (Torca AChE) [45] were aligned using CLUSTALW Asterisks denote identity, and full stops show high similarity.

AChE numbering) under the protein concentrations used

for crystallization [31,38] Therefore, a truncated BChE

lacking both the tetramerization domain and the N256,

N455, N481 and N486 N-glycosylation sites was

con-structed Deletion of the tetramerization domain was

achieved by introducing a stop codon at position 530

according to Blong et al [25] As expected, activity

measured in culture media was about sevenfold higher for

the monomeric form (BChED) than for the oligomeric form

(Tables 2, four sites off; oligomeric domain: ƠnoÕ) In

another effort, a second truncated clone also lacking four

N-glycosylation sites (N17, N455, N481 and N486) was

constructed The activity level of this enzyme was slightly

lower than that of the previous clone but suf®ciently high to

produce signi®cant amounts of enzyme Consequently, this

clone (4sugOff17/455/481/486 BChED) was chosen for large

scale expression Figure 1 shows how the amino-acid

sequence of 4sugOff17/455/481/486 BChED compares to the

native human BChE enzyme and to the crystallized forms of

Torpedo californica, human and mouse AChEs According

to this alignment, the X-ray structure of Torpedo californica

AChE should provide a good probe model to solve the

structure of 4sugOff17/455/481/486 BChED by a

molecular-replacement procedure

Preparation of 4sugOff17/455/481/486BChED

The mutated BChEDcloned into the pGS expression vector,

that expresses Gln-synthethase for selection purposes, was

transfected into CHO cells Stable clones secreting high

levels of recombinant BChEDwere selected for large-scale

production Puri®cation was carried out by anion-exchange

and af®nity chromatography Axelsen et al reported that decamethonium, used during the last af®nity chromatogra-phy step of T californica AChE, was present in the crystals despite extensive dialysis of the puri®ed enzyme [39] Thus,

to avoid contamination by a ligand, NaCl was used for elution of BChE from af®nity chromatography gels The purity of the ®nal enzyme preparation was estimated to be greater than 98% based on its speci®c activity and the presence of a single band on SDS/PAGE

Characterization of 4sugOff17/455/481/486BChED

The kinetics of butyrylthiocholine hydrolysis by recombi-nant BChE under standard conditions (0.1M phosphate buffer, pH 7.0) can be described by the model of Radic [29] The kinetic parameters are very close to the values reported previously for the native BChE [40], with

kcatˆ 28 000 min)1 and Kmˆ 25.6 ‹ 0.4 lM (n ˆ 3) The native enzyme and recombinant BChE display similar substrate activation with Kssˆ 510 ‹ 35 lM

(n ˆ 3) and b factor ˆ 2.85 ‹ 0.15 (n ˆ 3) Thus, the catalytic properties of the recombinant enzyme can be considered to be the same as the plasma enzyme SDS/PAGE analysis of the puri®ed recombinant BChE monomer displayed a single broad band in the 70±75 kDa molecular mass range In contrast, the puri®ed plasma BChE showed a faint band at 170 kDa (nonreducible dimer) and a major broad band at 85 kDa (monomer) under reducing conditions (Fig 2A) The apparent molec-ular mass of the recombinant monomer is consistent with the expected molecular mass for the truncated BChE after the deletion of 45 residues at the C-terminal sequence and

Fig 2 Gel electrophoresis analysis of 4sugO€ 17/455/481/486 BChE D (Rec) and human native BChE (Nat) (A) SDS/PAGE (4.5% stacking/10% separating) was carried out under reducing conditions according to Laemmli [46] using the Biorad MiniProtean II gel system and Coomassie blue staining (B) Isoelectrofocusing gel was carried out on a Pharmacia Phast System using Phast gel (4±6.5; pH range) and silver staining [47].

four N-glycosylation sites The broadness of the band

suggests that the puri®ed 4sugOff17/455/481/486 BChED still

displays a signi®cant glycosylation-related heterogeneity

This issue was addressed using IEF analysis The

carbohy-drate chains of BChE are partly capped by sialic acids [22],

which directly in¯uence the pI of the enzyme Whereas

plasma BChE displayed a continuous smear extending from

pH 4.0 to 5.7, thus re¯ecting high sialylation heterogeneity,

4sugOff17/455/481/486 BChED displayed 10 well-resolved

bands between pH 5.0 and 6.5 (Fig 2B) This was a de®nite

improvement of the enzyme homogeneity, and encouraged

us to start BChE crystallization trials

Crystallization of 4sugOff17/455/481/486BChED

and data collection

Initial crystallization conditions were screened according to

Jancarick & Kim [30] using the hanging drop method

Tetragonal crystals appeared within 1 week in a pH 6.5

0.1M Mes buffer solution containing 2.1M (NH4)2SO4

(Fig 3A) Interestingly, these crystals appear

morphologi-cally similar to the crystals of fully glycosylated equine

serum BChE obtained in 1944 [41] However these BChE

crystals were not further characterized due to technical

limitations at that time

To check whether the crystallized recombinant BChE was

still active, one crystal was soaked in Ellman's buffer

containing 5 mMbutyrylthiocholine and 2.4M(NH4)2SO4

This higher concentration of precipitant was necessary to

avoid the dissolution of the crystal After a few minutes, the

colorless crystal turned yellow, the color of the product of

the Ellman's reaction (Fig 3B) The crystalline enzyme

seems to be suf®ciently ¯exible to display an observable

catalytic activity, and small molecules such as

butyrylthi-ocholine, Nbs2 and the product of the Ellman's reaction

may easily diffuse in a short period of time inside and

outside the crystals However we cannot rule out the

possibility that the substrate might have been hydrolyzed by

the protein located in the crystal surface, which is likely to

solubilize during the soaking experiment

The crystals that measured up to 0.3 mm in their longest

dimension diffracted to 2.2±2.3 AÊ resolution at 100 K, using

15% glycerol (v/v) as a cryoprotectant, and synchrotron

radiation at the ESRF ID14-eh1 beamline As

recrystalli-zation improved the quality of human AChE crystals [31],

we reproduced the procedure by transferring

crystal-containing drops over reservoirs of water until the crystals

dissolved The drops were then placed over the original

reservoir solution, or a solution with slightly lower precip-itant concentration, for recrystallization As reported for human AChE, these new crystals were fewer but larger with longest dimensions of up to 0.6 mm They diffracted to 2.0 AÊ at 100 K, using 15% glycerol (v/v) as a cryoprotec-tant, and synchrotron radiation at the ESRF ID14-eh2 beamline Analysis of the collected data (Table 3) indicated that BChE crystals belong to the tetragonal space group I422 with unit cell dimensions a ˆ b ˆ 154.7 AÊ,

c ˆ 127.9 AÊ, giving a Vm of 2.73 AÊ3 per Da (estimated 60% solvent) for a crystal containing a single molecule of recombinant BChE ( 70 kDa) per asymmetric unit [42] A total of 371 832 observations were obtained at 2.0 AÊ resolution giving  49 298 unique re¯ections (98.6% com-plete, Rsymˆ 0.073) The structure has been successfully solved by molecular replacement starting from the model of native T californica AChE, PDB code 2ace [43] The re®nement of the model is underway

In summary, a recombinant human butyrylcholinesterase suitable for crystallization has been constructed by sup-pressing four out of nine N-glycosylation sites and deleting its oligomerization domain Large amounts of pure recom-binant enzyme were obtained by expression in CHO cells and puri®cation by anion-exchange and af®nity chroma-tographies The recombinant enzyme showed less heteroge-neity than the natural form while conserving identical catalytical properties Crystals were grown at pH 6.5 using (NH4)2SO4 as the precipitant After their quality was improved by recrystallization, they diffracted to 2.0 AÊ resolution The ®rst three-dimensional structure of a butyrylcholinesterase is expected to improve our knowledge regarding ChE mechanism, such as allosteric modulation, product clearance outside the active site gorge and motion

of water molecules Moreover, the three-dimensional struc-ture of human BChE should provide a template for the design of new mutants capable of hydrolyzing nerve agents and drugs such as cocaine with increased ef®ciency

A C K N O W L E D G E M E N T S

This work was supported by the US Army Medical Research and Materiel Command under contract DAMD 17-97-1-7349 to O L and

Fig 3 Tetragonal crystals of 4sugO€ 17/455/481/486 BChE D (A) The

larger crystal has dimension of 0.5 ´ 0.5 ´ 0.3 mm 3 (B) Crystal after a

10-min soaking in Ellman's bu€er with precipitant and 5 mM

butyryl-thiocholine.

Table 3 Data collection and processing Values for the highest reso-lution shell are given in parentheses.

Space group I422 Unit-cell parameters a ˆ b ˆ 154.66 AÊ,

c ˆ 127.89 AÊ

a ˆ b ˆ c ˆ 90°

X-ray source ESRF Beamline ID14-eh2 Wavelength 0.933 AÊ Di€raction limit 2.0 AÊ

No of measured re¯ections 371 832

No of unique re¯ections 49 298 Highest resolution shell 2.1 ® 2.0 AÊ Completeness 98.6% (99.1%) Multiplicity 7.1 (6.4)

R sym (on I) 0.073% (0.431%)

b Factor average 30.65 AÊ 2

the DeÂleÂgation GeÂneÂrale de l'Armement under contract DGA/DSP/

STTC-PEA 990802/99 CO 029 (ODCA, Washington, DC,

00-2-032-0-00) to P M We thank, respectively, Hassan Belrhali and Joanne

McCarthy for the opportunity to collect data at the ID14-eh1 and

ID14-eh2 beamline at the ESRF in Grenoble.

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