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J Biol Chem, Vol. 274, Issue 43, 30370-30376, October 22, 1999
From the Acetylcholinesterase, a polymorphic enzyme,
appears to form amphiphilic and nonamphiphilic tetramers from a single
splice variant; this suggests discrete tetrameric arrangements where the amphipathic carboxyl-terminal sequences can be either buried or
exposed. Two distinct, but related crystal structures of the soluble,
trypsin-released tetramer of acetylcholinesterase from Electrophorus electricus were solved at 4.5 and 4.2 Å resolution by molecular replacement. Resolution at these levels is
sufficient to provide substantial information on the relative
orientations of the subunits within the tetramer. The two structures,
which show canonical homodimers of subunits assembled through
four-helix bundles, reveal discrete geometries in the assembly of the
dimers to form: (a) a loose, pseudo-square planar tetramer
with antiparallel alignment of the two four-helix bundles and a large
space in the center where the carboxyl-terminal sequences may be buried
or (b) a compact, square nonplanar tetramer that may expose
all four sequences on a single side. Comparison of these two structures points to significant conformational flexibility of the tetramer about
the four-helix bundle axis and along the dimer-dimer interface. Hence,
in solution, several conformational states of a flexible tetrameric
arrangement of acetylcholinesterase catalytic subunits may exist to
accommodate discrete carboxyl-terminal sequences of variable dimensions
and amphipathicity.
Differences in the molecular forms of the cholinesterases are the
primary determinants of their tissue distribution and disposition within a cell; association of subunits also may govern the turnover of
the enzyme (cf. Refs. 1 and 2). In mammals, the predominant form of acetylcholinesterase
(AChE)1 in the central
nervous system is an amphiphilic tetramer anchored to the membrane by a
hydrophobic, noncatalytic subunit; at the neuromuscular junction, it is
an asymmetric form containing 1-3 tetramers associated with the basal
lamina by a collagen-like, structural subunit. Collagen-tailed forms
are also predominant in the electric organ of the eel
Electrophorus electricus. In the form containing three
catalytic tetramers, three collagen-like subunits are disulfide-linked
together, and each is attached to a tetramer in which two catalytic
subunits forming a proximal dimer are
disulfide-linked to the tail subunit and
are associated with a peripheral dimer by quaternary interactions (3,
4) (Scheme 1).2 Apart from
the association as a dimer of disulfide-linked dimers, both amphiphilic
and nonamphiphilic tetramers appear to form from a single splice
variant. The eel AChET subunits (EeAChE) present the
capacity to form heteromeric quaternary associations (5). This suggests
discrete tetrameric arrangements where the amphipathic carboxyl-terminal sequences (T peptides) are either buried or exposed.
Tetrameric arrangements of AChE subunits were observed in
situ (6-8), and several models were proposed (4, 9-11); however, little structural information is available about the subunit
orientation in the tetramer and the association of tetramers with
anchor subunits. The crystal structure of recombinant mouse AChE
(mAChE) revealed a compact, pseudo-square planar tetrameric arrangement
of subunits (12); mAChE, however, lacks the carboxyl-terminal
hydrophobic glycophospholipid or the amphipathic helix found on natural
forms of AChE and is expressed as a monomer (13), a feature that poses the question of whether physiological forms of the enzyme would form a
similar tetramer. In fact, crystals of EeAChE and diffraction experiments were reported earlier (14-16), but solution of a structure was precluded by the limited resolution achieved and failure in obtaining heavy atom derivatives, along with unavailability of a
three-dimensional template for molecular replacement and of the primary
structure of the eel species. Since then, crystal structures of
Torpedo californica AChE (TcAChE) (17) and mAChE (18) were
solved, and the cDNA-derived primary structure of EeAChE was
determined (5).
Here we report two low resolution crystal structures solved from two
distinct crystal forms grown in different conditions from the soluble,
trypsin-released EeAChE tetramer. These structures reveal discrete but
related tetrameric arrangements of catalytic subunits that are
consistent overall with that found in the mAChE crystal and with
arrangements observed in situ. Moreover, comparison of these
arrangements suggests that, in solution, the AChE tetramer has
significant conformational flexibility.
Materials--
The prepacked Superdex-200 HiLoad 26/60 column
and the gel-filtration calibration markers were from Amersham Pharmacia
Biotech. N-Tosyl-L-phenylalanine chloromethyl
ketone-treated trypsin (EC 3.4.21.4) and the protein molecular weight
standards for SDS-PAGE were from Sigma. Other reagents and the salts
used for crystallization were of the highest grade available.
Purification and Analysis of the Tetramer--
EeAChE, as a
mixture of asymmetric forms, was isolated from homogenized electric
organs by affinity chromatography and subjected to tryptic cleavage as
described previously (19). The released tetramer was purified by
size-exclusion fast performance liquid chromatography (Amersham
Pharmacia Biotech) on a Superdex-200 column equilibrated and eluted
with 100 mM NaKPO4, pH 7.5, 400 mM
NaCl, 0.01% NaN3 (w/v) at a flow rate of 0.5 ml
min
AChE activity measurements were conducted spectrophotometrically (20)
with 0.5 mM acetylthiocholine and 0.33 mM
dithiobis(2-nitrobenzoic acid) in 100 mM NaPO4,
pH 7.0, 0.1 mg ml
SDS-PAGE was performed on homogeneous 7.5% gels using a PhastSystem
apparatus (Amersham Pharmacia Biotech). The samples were boiled for 5 min in the presence of 2.5% (w/v) SDS with (reducing conditions) or
without (nonreducing conditions) 5% (v/v) Crystallization and Data Collection--
Crystallization was
achieved by vapor diffusion using hanging drops of 4 µl and a
protein-to-well solution ratio of 1:1. Form A crystals were grown at
4 °C using 1.1-1.5 M NaKPO4, pH 8.0-9.5, as the well solution; form B crystals were grown at 20 °C using 1.4 M ammonium sulfate, pH 5.5-6.0, as the well solution. Data were collected at 20 °C on mounted crystals and using a 300 mm MarResearch imaging plate detector equipped with a Siemens rotating anode (50 kV × 80 mA). Oscillation images were integrated with DENZO (21) and scaled and merged with SCALA (22) (Table
I). Amplitude factors were generated with
TRUNCATE (23). Form A crystals belonged to the orthorhombic space group
F222 with unit cell dimensions a = 118 Å, b = 215.9 Å,
c = 229.4 Å, giving a Vm value of 4.5 Å3/Da (73% solvent) for one EeAChE subunit (~80 kDa) in
the asymmetric unit (24). Form B crystals belonged to the monoclinic
space group C2 with unit cell dimensions a = 211 Å, b = 129.7 Å, c = 195.4 Å ( Structure Solution and Refinement--
Initial phases for
structure A were obtained by molecular replacement using the AChE
subunit from the mAChE structure (Protein Data Bank code 1MAA) (12) as
a search model with the AMoRe program package (25) (Table I). The
phases calculated from a positioned catalytic subunit were improved by
solvent flattening using program DM (23) and a mask built around the
subunit. The same procedure with additional averaging was used for
structure B. For the two structures, rigid body refinement was applied
to the whole subunit using the program CNS (26). Accuracy of the structures was further checked by omitting from the starting model and
before rigid body calculation the entire 17-residue helix
Figs. 2, 3, and 4 were generated with the ALSCRIPT (27), RIBBON (28),
and TURBO-FRODO (29) programs, respectively.
Characterization of the Purified EeAChE Tetramer
The trypsin-released tetramer eluted from the gel filtration
column as a single, symmetric absorbance peak of apparent mass of
~360 kDa; analysis of the specific AChE activity throughout the peak
suggested functional homogeneity of the tetramer (data not shown).
However, electrophoretic analysis suggested structural heterogeneity.
Indeed, although SDS-PAGE performed in reducing conditions yielded the
three broad bands of ~80, 50, and 30 kDa, characteristic for reduced
EeAChE (30, 31), SDS-PAGE performed in nonreducing conditions yielded a
weak, thin band of ~320 kDa representing residual tetramer and two
pairs of closely migrating, intense bands in a ratio of about 1:1 and
average apparent masses of ~160 and 80 kDa, values consistent with
dimers and monomers, respectively (Fig.
1). The purified tetramer thus appears
not only to be composed of two equal populations of dimers differing slightly in their masses (as expected from a dimer of dimers where one
set of disulfides links with the residual tail and the other set forms
between monomers; cf. Scheme 1) but also to contain, in the
same proportion, two equal populations of slightly different monomers.
Trypsin cleaves peptides and proteins at the ester linkages of
arginines and lysines, two residues that are found upstream of the
linking cysteines not only in the amino-terminal end of the tail (32,
33) but also in the carboxyl-terminal end of the EeAChE catalytic
subunit (T peptide, Fig. 2) (5). In the presence of SDS, dimers that lack the disulfide dissociate into monomers. Hence, the EeAChE tetramer subjected to crystallization is
composed of two equal populations of covalent and noncovalent dimers,
either proximal or distal to the tail.
The apparent mass of the EeAChE tetramer estimated from chromatography is ~12% higher than that estimated from electrophoresis, a difference that suggests either dimensional asymmetry or a high level of hydration of the tetramer (or both). Substantial dimensional asymmetry was reported earlier for the EeAChE tetramer (3), the homologous 11S TcAChE species (34), and the fetal bovine serum AChE tetramer (35). Alternatively, two highly solvated crystal forms were grown from the purified EeAChE tetramer (cf. "Experimental Procedures"). Tetrameric Arrangements of EeAChE Subunits For each of the two EeAChE structures herein reported, the low resolution achieved does not reveal details in the positions of the side chains in the catalytic subunit nor in those located at the subunit interfaces. As well, structure elements that are unique to the eel enzyme, peptide Ile418-Gln446 and the carboxyl-terminal T peptide Glu571-Leu610 (5) (Fig. 2), are not resolved. Yet, each of the structures provides substantial information on the arrangement of the subunits within the tetramer. A single solution was found for the positions and orientations of the subunits in pairs whether molecular replacement used an isolated subunit or a dimer of subunits as a search model. Indeed, dimers of subunits related by four-helix bundles and identical to dimers observed in structures of TcAChE (17, 36) and mAChE (12, 18) were observed. The two EeAChE structures, however, differ in the geometry of the two dimers within the tetramer. Because of the current resolutions, the EeAChE backbone was not modeled, and the backbone shown is that of mAChE. Accordingly, the EeAChE residues are referred to relative to the mAChE backbone based on sequence homology (Fig. 2), and the number in parentheses that follows the EeAChE residue number (5) denotes the corresponding position in mAChE (13, 37). Structure A--
In EeAChE structure A, the two dimers arrange as
a loose, pseudo-square planar tetramer of a marked dimensional
asymmetry (Fig. 3). The two four-helix
bundle axes are aligned antiparallel, and the main axes of the two
dimers are tilted by ~40° from each other, a geometry reminiscent
of that observed in the mAChE structure (12). At the dimer-dimer
interface, which extends over 75 Å in a direction roughly
perpendicular to the four-helix bundle axis, loop
Cys256(257)-Cys267(272), which is located
between helices
In tetramer A, the four gorges that lead to the EeAChE active centers
are oriented roughly antiparallel in a direction perpendicular to the
tetramer plane. All four peripheral sites, of which two, from
diagonally opposed subunits, are exposed on one face of the tetramer
and two on the other face, are freely accessible to the solvent, a
geometry consistent with the binding of four fasciculin or inhibitory
antibody molecules/AChE tetramer (38-40). The EeAChE-specific peptide
Ile418-Gln446, which is located between the
very long helix Structure B--
In EeAChE structure B, the two dimers arrange as
a compact, square nonplanar tetramer, also of a marked dimensional
asymmetry (Fig. 3). Compared with structure A, the tetramer folds as to position the two four-helix bundle axes 60° from each other, and one
dimer rotates relative to the second one by ~40° as to reorient the
main axes of the two dimers antiparallel. At the dimer-dimer interface,
which now extends in a direction perpendicular to the plane made by the
two four-helix bundle axes, the facing loops Cys256(257)-Cys267(272) are still separated by
13 Å, consistent with bridges possibly imparted by the facing
oligosaccharide moieties linked to Asn161(162) and
Asn260(265), and the four Thr570(543) residues,
which are terminal to helices In tetramer B, the axes of the EeAChE active center gorges are oriented antiparallel within a dimer but are tilted by 120° from a dimer to the second one. Of the four peripheral sites, two, from diagonally opposed subunits, are exposed at the surface of the assembly and are freely accessible to the outside solvent; the two other peripheral sites, from the second pair of diagonally opposed subunits, face the tetramer internal space. A symmetric situation applies to the four peptides Ile418-Gln446, of which two are exposed to the outside solvent and are disordered and two face the internal space. As a result, residue Val447, at the same position as mAChE-Ala420 in subunit A in the first dimer, is separated by 17 Å from peripheral site residues Tyr336(341) and Trp281(286) at the gorge entrance of subunit C in the second dimer. The presence of a weak, loop-shaped electron density in this region between subunits A and C suggests that, in the internal space, the peptide undergoes stabilizing interactions. The same weak density is observed between the peptide of subunit D and the peripheral site region of subunit B. Hence, in tetramer B, peptides Ile418-Gln446 of two subunits may interact with the peripheral site regions of the facing two subunits, a situation reminiscent of the recently reported intersubunit interaction of mAChE loop Cys257-Cys272, also rich in Gly residues, with the facing peripheral site region (12). As a result, the surface area buried at the dimer-dimer interface is larger, and the space between the two dimers is 2-fold narrower than in tetramer A. The Carboxyl-terminal T Peptides The EeAChE carboxyl-terminal T peptide,
Glu571(544)-Leu610, located in the extension
of helix The amphipathic character of the T peptide precludes its full exposition to the solvent and total disorder. In addition, in contrast to the large surface area buried at the mAChE dimer-dimer interface (12), the limited surface areas buried at the dimer-dimer interfaces of the EeAChE tetramers appear insufficient for cohesion of the two dimers (42). These constraints, along with the large internal space observed in each of the two tetramers, raise the questions of the positions of the four T peptides in the EeAChE structures and of their contribution to the dimer-dimer interface. In the loose tetramer A, the four carboxyl-terminal T peptides could either be buried at the center of the arrangement, as proposed based on a square planar model for association of nonamphiphilic AChE subunits (10), or pair off and exit on either side of the tetramer plane, as recently suggested from analysis of the compact, pseudo-square planar tetramer of crystalline mAChE (12). In the folded, compact tetramer B, the four T peptides could be exposed on the same side of the arrangement, roughly similar to a recent model for tetrameric human butyrylcholinesterase (9). Either option would be expected to stabilize the four T peptides and provide the tetramer with the locking points, internal or external to the arrangement, that are required for dimer-dimer cohesion. Actually, an additional density is apparent in the EeAChE structures,
which is made of a loop-shaped portion located in the extension of
helix
A Flexible AChE Tetramer? Overall, each of EeAChE tetramers A and B is consistent not only
with the tetrameric arrangement of subunits of crystalline mAChE (12)
but also with tetrameric arrangements observed in situ
(cf. Fig. 2c in Ref. 6). Also, a structure similar to structure A but showing a larger tilt (by ~12°) of the two dimers in the tetramer was recently solved by Raves et al. (43)
(Protein Data Bank code 1EEA) from a data set earlier collected on a crystal of the same space group as our form A crystals but different unit cell dimensions and grown from polyethylene glycol (16). The
existence of two extreme conformational states of the EeAChE tetramer
could be pH or/and temperature-dependent because the EeAChE
form A crystals (as the form grown by Schrag et al. (16) and
the mAChE crystals) were grown at pH Conformational flexibility of the homologous 11S TcAChE species,
evidenced by fluorescence polarization spectroscopy, was proposed to
reflect motion of discrete segments of the tetramer molecule (segmental
motion) rather than global rotation of the tetramer as a rigid
macromolecule (44). Hence, structures A and B may correspond to
distinct conformational states of an EeAChE tetramer undergoing, in
solution, significant flexibility about the four-helix bundle axis and
along the dimer-dimer interface axis. Most importantly, the possibility
for several conformations of an overall tetrameric but malleable
arrangement of AChE subunits would make the tetramer able to fit either
of the carboxyl-terminal sequences, differing in length and amphipathic
character, which characterize the diverse AChE molecular forms. Whether
this flexibility is also related to regulation of catalysis is unknown.
Because the high solvent content of the crystal forms used in this
study precludes achievement of a higher resolution structure, even if synchrotron radiation were used, efforts to grow crystal forms of a
higher diffraction potency are underway.
We thank Drs. Stéphanie Simon and Jean Massoulié (Ecole Normale Supérieure, Paris) for providing us with the cDNA-encoded sequence of EeAChE prior to publication and Dr. Palmer Taylor (UCSD, La Jolla) for critical review of the manuscript and fruitful discussions.
* This work was supported by the Association Française contre les Myopathies (to P. E. B. and P. M.) and the Commissariat à l'Energie Atomique (to J. G.). A preliminary report of these structures was presented during the Sixth International Meeting on Cholinesterases and Related Proteins (La Jolla, CA, 1998).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates (code 1C2B for structure A and code 1C2O for structure B) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
2 Scheme 1 is not intended to describe the crystal structures reported in this study.
3 P. Marchot, unpublished data.
The abbreviations used are: AChE, acetylcholinesterase; EeAChE, E. electricus AChE; mAChE, recombinant mouse AChE; PAGE, polyacrylamide gel electrophoresis; TcAChE, T. californica AChE.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. This article has been cited by other articles:
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