From CNRS, Unité Propre de Recherche 9039, Architecture et Fonction des Macromolécules Biologiques, Institut
de Biologie et Microbiologie Structurale, F-13402 Marseille Cedex 20, France, the § Department of Pharmacology, University of
California at San Diego, La Jolla, California 92093-0636, and
¶ CNRS, Unité Mixte de Recherche 6560, Ingénierie des
Protéines, Institut Fédératif de Recherche Jean
Roche, Université de la Méditerranée, Faculté
de Médecine Secteur Nord,
F-13916 Marseille Cedex 20, France
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ABSTRACT |
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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 Acetylcholinesterase
(AChE),1 a member of the
family of proteins with an Differences in the molecular forms of the cholinesterases are the
primary determinants of their cellular disposition (3). Abnormal
associations of AChE are apparent in Alzheimer's dementia, where a
selective loss of the amphiphilic tetramers is observed (6, 7). AChE
was suggested to associate with or to nucleate the neuritic plaques
found in the disease (8-12); however, the mode of this association is unknown.
The active center of AChE, which consists of the triad
Ser203-Glu334-His447 in mammals
(13), is nearly centrosymmetric to the subunit and is located at the
bottom of a narrow gorge (14). 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 (15). Only slight differences were observed in
the conformation of Fas2-associated mAChE compared with uncomplexed
Torpedo californica AChE (TcAChE) (14). 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
(14) and the Fas2 complex (16). 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 (14, 17, 18) or
sealed with high surface complementarity by Fas2 (15, 16), 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- Purification, Crystallization, and Data Collection--
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 (19). 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 M
NaKPO4, 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) and scaled and merged with SCALA (21) (Table
I). Amplitude factors were generated with
TRUNCATE (22). 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 Vm 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 (23).
Structure Determination and Refinement--
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) (15) as a search model
with the AMoRe program package (24). 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 (25) 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, Rfree
was 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 (26).
The positions of misplaced amino and carboxyl termini and of a few side
chains were adjusted, and the Pro258-Gly264
portion, which was missing in the search model, was built into 2Fo
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. 1
and 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 (29) and WHATIF (30) (Table
I). Figures were generated with the programs RIBBON (31), GRASP (32),
and TURBO-FRODO (26).
Overall Description of the Structure
The mAChE molecule, which consists of a 12-stranded central-mixed
mAChE, which is devoid of the carboxyl-terminal amphipathic helix and
intersubunit disulfide-linking Cys, is monomeric in dilute solution
(19), but dimeric in the Fas2·mAChE crystals, where a homodimer
assembles through a tightly packed four-helix bundle (15). 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.
loop, composed of a cluster of hydrophobic residues conserved in
mammalian acetylcholinesterases along with flanking
-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.
INTRODUCTION
Top
Abstract
Introduction
References
/
-hydrolase fold (1, 2), rapidly
terminates cholinergic neurotransmission by hydrolysis of acetylcholine
(3, 4). 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 (5) and the other set is
formed between monomers (3, 4). However, there is currently little
structural information about the subunit orientation in the tetramer
and the association of tetramers with structural subunits.
1,4-GlcNAc-
1,6-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.
EXPERIMENTAL PROCEDURES
Data collection and refinement statistics
Fc and
Fo
Fc 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 (27), 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) (28). The conformations of some side chains were corrected
using mAChE coordinates from a 2.7-Å resolution structure of the
Fas2·mAChE
complex.2
RESULTS AND DISCUSSION
-sheet surrounded by 14
-helices, has the same overall conformation as found for the Fas2·mAChE complex (15): 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.
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Fig. 1.
Overall view of the compact,
pseudo-square planar mAChE tetramer. Panel A,
ribbon diagram of the mAChE dimer of dimers viewed perpendicular to the
four-helix bundle axis. Panel B, the dimer of dimers viewed
parallel to the four-helix bundle axis, 90° from panel A.
The italicized blue labels A and B refer to the
subunits in the left dimer (displayed in dark blue), and
labels C and D refer to the subunits in the right
dimer (light blue) for orientation shown in panels
A and C. The four-helix bundles formed by helices
37,8 and
10 from two subunits
are displayed in magenta. The short
loop
Cys257-Cys272 is displayed in red,
and helices
16,7 and
26,7 are in green. Panel
C, same orientation as in panel A showing the molecular
surface of dimer CD in blue, with the surfaces
buried to a 1.6-Å probe radius at the dimer-dimer interface in
gold. Helix
16,7 in the loop
region of subunit C is visible behind the ribbon trace of subunit A. The GlcNAc moiety and the GlcNAc-
1,4-GlcNAc-
1,6-Fuc trisaccharide
moiety linked to Asn350 in subunits A and D, respectively,
are displayed as gray bonds with colored spheres.
Secondary structure elements are specified according to Ref. 2.
In the tetramer, a short surface loop and flanking -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
-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
-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 (33). Including the
area buried at the four-helix bundle interface within each dimer (15),
the total mAChE surface area buried within the tetramer encompasses
6000 Å2.
Detailed Description of the Structure (Structure A)
At the dimer-dimer interface, mAChE loop 257-272, a short loop (34) 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
-helices
16,7 and
26,7 (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. 1-3). 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
16,7, 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 (15); only the Pro
doublet is seen in the Fas2·mAChE complex at 2.7-Å
resolution.2
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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 10 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
10; 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
10
ends at Thr543, the last residue encoded by exon 4 before
the region of alternative splicing (13, 19).
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 (15), and Asn265 in
subunits A and D, although the density is weaker. In addition, a
GlcNAc-1,4-GlcNAc-
1,6-Fuc trisaccharide moiety is linked to Asn350 in subunit D (Figs. 1 and 2) (35, 36).
Comparison of the mAChE and Fas2·mAChE Structures
The structure of mAChE at 2.9-Å resolution provides greater
delineation of the positions of the -carbon and side chains than the
Fas2·mAChE complex (3.2 Å) (15) and establishes several distinctive
features of mAChE compared with TcAChE (Protein Data Bank 2ACE) (28).
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
(15).
The most prominent feature arising upon superimposition of the
structures is the close resemblance of the two peripheral
site-occluding loops, the short 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
16,7 of mAChE subunit A also adopt positions
similar to residues in bound Fas2: the side chain of Arg253
in mAChE mimics, in both position and orientation, the side chain of
Arg27 in Fas2, and mAChE Gly256 forms the same
van der Waals contacts with His287 as Fas2
Leu48. Hence, in the tetramer, the loops that arise from
two of the subunits appear to occlude substrate access to the catalytic
sites of the two other subunits, similar to steric occlusion observed in the Fas2·mAChE complex. In contrast, Fas2 loop I interacts with
the large
loop Cys69-Cys96 on one side of
the gorge entrance, whereas mAChE helix
16,7
fits into a furrow extending between the end of helix
36,7 and helix
27,8, on the opposite side of the mAChE
gorge entrance. Since the intersubunit and Fas2-mAChE interactions
differ substantially in affinity, complexation of a disulfide-linked
tetramer with fasciculin would be expected to reposition residues at
the interface, a rearrangement consistent with observation of
equivalent affinities of the four AChE subunits for fasciculin
(37).
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The mAChE Gorge and Peripheral Anionic Site
Despite extensive dialysis (19), a DECA molecule is still present, approaching full occupancy in the active site gorges of all four mAChE subunits (Fig. 5). Superimposition of subunit A, the peripheral site of which is free of an occluding loop and totally accessible to the solvent, with TcAChE in the DECA·TcAChE complex (Protein Data Bank 1ACL) (17) shows that the proximal (N-1) and distal (N-2) quaternary ends of DECA are in van der Waals contact with the indole rings of Trp286 in the peripheral site and Trp86 in the active center and superimpose in the two structures. In mAChE, however, the 10-carbon methylene chain adopts an inverted curvature and follows the opposite wall of the gorge, a difference that involves a rotated C-5-C-6 bond and a positioning of the C-7-C-8 bond nearly 5 Å away from its position in TcAChE; hence, the C-7-C-8 portion of the methylene chain winds behind the Tyr124 ring in mAChE. The conformation of DECA in mAChE likely results from the 90° rotation of the Tyr337 phenol ring, which lies perpendicular to the gorge axis, whereas the homologous Phe330 ring in TcAChE lies parallel to the gorge axis (17, 28). The tyrosine hydroxyl, unique to mammalian AChEs, forces a different pathway for the methylene chain by its steric and polar contributions. Also, the Tyr341 ring is shifted toward the center of the gorge, ~1.5 Å removed from its position in TcAChE; hence, the hydroxyl moieties on Tyr341 and Tyr337 are separated by 3.8 Å, compared with 3 Å in TcAChE. The orientation of Tyr337, also observed in the Fas2·mAChE complex (15), could either be intrinsic to the mouse enzyme or arise from complexation with DECA or Fas2; it is, however, identical to that of Phe330 in TcAChE in complexes with various inhibitors other than DECA (17, 28). Hence, fluctuations of configuration of the active center gorge of AChE permit accommodation of a large set of active center ligands.
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The mAChE structure provides an initial opportunity to compare exposed and occluded peripheral sites within an asymmetric unit. However, superimposition of loop-donor subunit A with loop-acceptor subunit C does not reveal large differences in conformations of the two sites. r.m.s. deviations are 0.44 and 0.77 Å for the main chain and all atoms, respectively; only the side chain of Trp286 at the gorge entrance of subunit C undergoes a 20° tilt, which may arise from the steric constraint induced by the interacting loop. However, the presence of DECA in the catalytic site of non-occluded subunit A might mask structural rearrangements expected from the unliganded peripheral site. Yet in subunit C, the bound loop partially dissociates DECA, which, although disordered, seems to adopt two discrete conformations with a hinge point near the C-4-C-5 bond. In one conformation, the proximal quaternary end of DECA interacts weakly with the indole ring of Trp286. In the second conformation, this quaternary end is displaced and flips toward the Phe338 and Ile294 side chains, some 5 Å away (Fig. 5). Residual inhibitor was observed in structures of native TcAChE, perhaps even with a tetramethylammonium-eluted enzyme (14, 17, 28). In contrast, DECA is not found in the gorge of the Fas2·mAChE complex (15), consistent with the inhibitor molecule being displaced by bound Fas2 and eliminated during dialysis of the complex prior to crystallization (19).
Other Crystal Forms of mAChE
Structures B and C are essentially identical to structure A. However, structure B contains residual EDR instead of DECA in the active site of all four subunits (data not shown). Comparison of mAChE in this structure with TcAChE in the EDR·TcAChE complex (Protein Data Bank 1ACK) (17) shows similar overall positioning of the EDR molecule, located between His447, Ser203, Tyr337, and Trp86 in mAChE; however, a 30° tilt of the EDR ring is observed, which may arise from accommodation to the Tyr337 orientation found in mAChE. There is no difference between subunits A and C, indicating that the occluding loop, which is too short to sterically destabilize the associated EDR, does not perturb it allosterically. Also, bound Fas2 does not dissociate residual EDR from the active site of the complexed enzyme.2
Structure C originates from a crystal grown in conditions different
from those used for the structure A crystal and contains no bound
phosphate. In subunit A, a sialic acid moiety, Sia-2,6-Gal, was
fitted into an unaccounted density proximal to His287 and
appears to be terminal to the oligosaccharide chain
GlcNAc-
1,4-GlcNAc-
1,4-Man-
1,3-Man, linked to
Asn350 (data not shown) (36); the junction between the last
Man and Gal moieties was not resolved, but Asn350 is
located 25 Å away from His287, a value consistent with the
mean distance between the two ends of an N-linked
carbohydrate chain. A similar carbohydrate chain may be linked to
Asn350 in subunit D, although a weaker density is observed.
mAChE is composed of two species of roughly equal populations proposed to differ in their carbohydrate composition or some other
post-translational modification (19). Combined gel-filtration,
polyacrylamide gel electrophoresis, and isoelectric focusing
experiments showed that the population of a higher mass is of a lower
pI,3 an observation
consistent with sialic acid in only two subunits in the mammalian
tetramer (35).
Significance of the Structure
Quaternary Structure of mAChE-- A square planar model for association of catalytic and structural subunits was recently proposed based on sequence alignments, computational predictions, and mutagenesis and biochemical data (38). The tetrameric structure of mAChE (Fig. 1) largely agrees with this model. Recombinant mAChE, however, is truncated after residue 548; hence, it lacks the carboxyl-terminal sequences encoded by the alternatively spliced exons and is expressed as a monomer (19), a feature that poses the question of whether physiological forms of the enzyme would form a similar tetramer. In mouse, the alternatively spliced sequences are: (a) a 30-amino acid polar sequence encoded by the retained intron, (b) a glycophospholipid signal sequence that is cleaved at selected positions upon addition of the glycophospholipid, or (c) a 40-amino acid peptide containing an amphipathic helix and a free cysteine. Only the last splicing option allows for tetrameric assembly in situ (4, 39); assembly may be facilitated by the amphipathic helix- and polyproline-containing peptides (5). Nevertheless, truncated mAChE associates as dimers in concentrated solution and as dimers and tetramers in the crystals. Upon gel filtration at protein concentrations used for crystallization and low ionic strength (10-12 mg/ml; µ = 50 mM), mAChE elutes as a dimer, whereas at concentrations <100 µg/ml, it elutes as a monomer; tetramers were not detected under these conditions or at high ionic strengths (µ = 3 M) approaching the crystallization conditions.3 Since protein concentrations exceeding those for dimer formation are apparently required to detect tetramers, it seems unlikely that occlusion of internal sites as seen in the structure would influence kinetic studies, typically conducted at low enzyme concentrations.
Deviation of the mAChE carboxyl termini out from the tetramer plane (Fig. 1) suggests that the twisted square planar structure could accommodate, on each side and with minimal perturbation, alternatively spliced sequences serving either as attachment sites for structural subunits or as membrane-anchoring domains. Since both amphiphilic and non-amphiphilic tetramers appear to form from a single splice variant (40), discrete tetrameric species are likely where the amphipathic carboxyl-terminal sequences are either buried or exposed. Related tetrameric arrangements of catalytic subunits were recently observed in two 4.5-Å resolution crystal structures determined from two distinct crystal forms of trypsin-released tetrameric AChE from Electrophorus electricus: (a) a loose pseudo-square planar arrangement with free space in the center where the carboxyl-terminal sequences might be buried and (b) a compact square non-planar arrangement in which a folding of the tetramer along the dimer-dimer interface axis exposes the four sequences on the same side.2 Comparison of these two arrangements with that of mAChE indicates significant flexibility of the tetramer about the four-helix bundle axis and along the dimer-dimer interface axis. Hence, more than a single tetrameric arrangement may exist to accommodate the discrete carboxyl-terminal sequences.
Conformational Mobility of mAChE--
loops are often
important for protein stability and function and are so positioned for
molecular recognition;
loop lids, which are flexible until
substrate or inhibitor is bound, are thought to play a role in
enzymatic catalysis (34). Conformational gating provides a mechanism to
recruit substrates to the active site, to exclude solvent, to sequester
reactive intermediates, or to enhance substrate specificity.
Conformational mobility of loop Cys69-Cys96,
the large
loop that is structurally homologous to the lid that
sequesters the substrate in certain neutral lipases, which also show
the
/
-hydrolase fold (1, 2), has been proposed in gating
accessibility of small molecules to the active center and in allosteric
modulation of AChE catalysis (41-43). In the lipases, lid opening
uncovering the catalytic site is a key feature of interfacial
activation; however, the lid domain may be formed by one or more
helices or surface loops that differ not only in length and sequence,
but also in location relative to the
/
-hydrolase core and the
active site (44-49). In several
/
-hydrolase proteins, the domain
located between strands
6 and
7 is quite
independent of the
/
-hydrolase core and can accommodate large
insertions such as a regulatory module (50), a putative helical lid
(45), or a capping domain for the active site (51), without perturbing the core structure. The flexible short
loop
Cys257-Cys272, which is also located between
strands
6 and
7 of mAChE and protrudes to
the surface of the molecule not far from the gorge entrance (Fig. 2),
and helix
16,7, where several residues
interact with the facing subunit in the tetramer (Figs. 1 and 3), are
virtually conserved in the entire
/
-hydrolase family
(cf. alignment in Refs. 2, 50, and 52-55). Yet a
conformational rearrangement of helix
16,7
or
26,7 (or both) would be required for the
mAChE helix-loop-helix region to fold back and interact with the
peripheral anionic site of the same mAChE subunit. Partial melting of
-helices into
-sheets participates in structural reorganization
of the human pancreatic lipase lid (49). Unwinding of a helical turn,
refolding of a flexible connection, and
cis,trans-isomerization of a Pro residue acting as a hinge
point accompany positional shift of the flap in Candida
rugosa lipase (47). The mobile surface loop of cytochrome c peroxidase mutant W191G contains the
Pro190-Gly-Gly-Ala-Ala-Asn-Asn196 sequence,
which shows striking similarity to the mAChE short
loop, and is
followed by an
-helix starting at Asn196, similar to
mAChE helix
26,7. Movement of the peroxidase
loop involves a double-hinged rotation about Pro190
and Asn195 with a cis,trans-isomerization of
Pro190 and an interchange in the positions of the
Asn195 side and main chain atoms (56, 57). Hence, the
sequence of the short
loop and the cis-conformation of
Pro258 at its base should confer conformational flexibility
to the helix-loop-helix domain of mAChE.
Heterologous Associations on AChE Surfaces--
Certain brain
regions contain large amounts of AChE, but are devoid of acetylcholine
and its biosynthesis capacity. This disparity in localization and the
appreciable secretion of AChE into extracellular space suggest that
AChE may have a signaling function independent of substrate catalysis
(58-60). Non-catalytic properties of AChE may also play a role in
neuropathology. Colocalization of AChE and amyloid -peptide deposits
in the brain of Alzheimer's disease patients indicates a presence, if
not an active role, in amyloid plaque formation (8-11). AChE was
demonstrated to accelerate the assembly of amyloid
-peptides into
Alzheimer's fibrils, with a possible involvement of the peripheral
anionic site (12, 61-63).
The solution structure of amyloid -peptide-(1-40) shows a
helix-loop-helix motif with a disordered central loop (64).
Superimposition of this structure with that of the helix-loop-helix
domain of mAChE (subunit A) brings several side chains in the
-peptide and mAChE in the same position (Fig.
6). In particular, Lys16,
Ala21, and Val24 in the
-peptide superimpose
with Arg247, Ala252, and Val255 in
mAChE, respectively. Moreover, the side chains of His14 in
the
-peptide and Arg245 in mAChE align, a position
suitable for hydrogen bonding with Glu292 in mAChE
(cf. subunit C). Finally, the side chains of
Glu22 in the
-peptide and Arg253 in mAChE
also align (as does the side chain of Arg27 in bound Fas2),
a substitution that likely disrupts polar interactions, but not van der
Waals contacts, with Pro290 and His287 in the
peripheral site of mAChE. In solution, the high mobility of the two
loops may bring dipeptides Val24-Gly25 and
Gly29-Ala30 in the
-peptide in the same
positions as Val255-Gly256 and
Gly260-Gly261 in mAChE. Hence, the amyloid
-peptide and the mAChE helix-loop-helix domain may interact in a
similar manner with the AChE peripheral anionic site, which could serve
as a nucleation site to promote aggregation.
|
Conformational plasticity of the amyloid -peptides permits not only
variable positioning of
-helices, but also local
-strand structures (64-66). In the amyloid
-peptides, as in the prion proteins, an
-helix to
-sheet conversion was proposed to
accompany aggregation (67). Sequence analysis revealed similarity of
the amyloid protein region Gln15-Gly37 to the
prion protein region Met120-Gly142 (64), a
region suggested to undergo
-helix to
-sheet transition in the
pathogenic states (68-70). In addition to its structural similarity to
the amyloid
-peptide, the mAChE helix-loop-helix domain with its
glycine cluster shows intriguing sequence similarity to the
Met120-Gly142 region of the prion proteins
(Fig. 6). This feature also makes rearrangement of the mAChE domain an
attractive consideration.
In summary, the crystal structure of mAChE shows a tetrameric assembly
of subunits; points to flexibility within the active center gorge; and
reveals the peripheral anionic site, at the rim of the gorge, to be the
site of association of surface loops not only from inhibitory peptides,
but also from adjacent subunits. Hence, this may also be a site for
heterologous protein association with AChE.
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ACKNOWLEDGEMENTS |
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We thank Joan Kanter (University of California at San Diego) for purification of the recombinant enzyme; Philippe Cantau and Didier Nurizzo (CNRS) for help in in-house and synchrotron data collection, respectively; and Christian Cambillau (CNRS) for interest and support. Expert assistance from Victor Lamzin (EMBL-Deutsches Elektronen-Synchrotron) and Javier Pérez (Laboratoire pour l'Utilisation du Rayonnement Electromagnétique) is much appreciated.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant GM18360 and Department of Army Medical Defense Grant 17-1-8014 (to P. T.); National Science Foundation-CNRS Collaborative Project 3906 (to P. M. and P. T.); the Association Française contre les Myopathies (to P. E.B. and P. M.); and the European Union (HCMP Access to Large Installations Project, Contract CHGE-CT93-0040) and CNRS (to Y. B. and P. M. for data collection at EMBL Hamburg and the Laboratoire pour l'Utilisation du Rayonnement Electromagnétic, respectively). Part of this work 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 of structure A (code 1MAA) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
To whom correspondence should be addressed. Fax:
33-4-9165-7595. E-mail: marchot.p{at}jean-roche.univ-mrs.fr.
The 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.
2 Y. Bourne, unpublished data.
3 P. Marchot, unpublished data.
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REFERENCES |
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