Download Crystal Structures of Human Glutaminyl Cyclase, an Enzyme

Crystal Structures of Human Glutaminyl Cyclase, an
Enzyme Possibly Involved in Alzheimer's Disease
and Osteoporosis
Kai-Fa Huang1,2, Yi-Liang Liu1, Wei-Ju Cheng3, Tzu-Ping Ko2, and
Andrew H.-J. Wang1,2,3,4*
Abstract
N-terminal pyroglutamate (pGlu) formation from its glutaminyl (or glutamyl) precursor is
required in the maturation of numerous bioactive peptides. The aberrant formation of pGlu
may be related to several pathological processes, such as osteoporosis and amyloidotic diseases. This N-terminal cyclization reaction, once thought to proceed spontaneously, is greatly
facilitated by the enzyme glutaminyl cyclase (QC). To probe this important but poorly understood modification, we have solved the crystal structures of human QC in free form and bound
to a substrate and three imidazole-derived inhibitors. The structure reveals an α/β scaffold
akin to that of two-zinc exopeptidases but with several insertions and deletions, particularly in
the active-site region. The relatively closed active site displays alternate conformations due to
the different indole orientations of Trp-207, resulting in two substrate (glutamine t-butyl ester)binding modes. The single zinc ion in the active site is coordinated to three conserved
residues and one water molecule, which is replaced by an imidazole nitrogen upon binding of
the inhibitors. Together with structural and kinetic analyses of several active-site-mutant
enzymes, a catalysis mechanism of the formation of protein N-terminal pGlu is proposed. Our
results provide a structural basis for the rational design of inhibitors against QC-associated
disorders.
Introduction
N-terminal pyroglutamate (pGlu) formation from its glutaminyl precursor (Figure 1) is an important posttranslational or cotranslational event in
the processing of numerous bioactive neuropeptides, hormones, and
cytokines during their maturation in the
secretory pathway. These regulatory
peptides require the N-terminal pGlu to
develop the proper conformation for
Figure 1. QC catalyzes the N-terminal pyroglutamate formation of numerous bioactive proteins from their glutaminyl (left) or glutamyl (right) precursors.
binding to their receptors and/or to protect the N termini of the peptides from
Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
3
National Core Facility of High-Throughput Protein Crystallography, Academia Sinica, Taipei, Taiwan
4
Department of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan
1
2
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exopeptidase degradation. Previously, this N-termi-
and pGlu3-Aβ(3-42/43), are major fractions of the
nal cyclization reaction was thought to proceed
Aβ peptides within the core of neuritic plaques (7).
spontaneously, until the glutaminyl cyclases (QCs)
The N-terminal pGlu could enhance the hydropho-
were identified as catalysts that are responsible for
bicity, proteolytic stability, and neurotoxicity of
this posttranslational modification (1,2). To date,
these peptides (8), probably causing a profused
QCs have been identified in both animal and plant
accumulation of pGlu-Aβ peptides in several
sources, particularly in mammalian neuroendocrine
senile plaques and thus accelerating the progression
tissues, such as hypothalamus and pituitary (2,3).
of neurodegenerative disorders.
In humans, several genetic diseases, such as
osteoporosis, a multifactorial hormonal disease that
is characterized by reduced bone mass and microarchitectural deterioration of bone tissue, appear to
result from mutations of the QC gene (4). The gene
encoding QC (QPCT) lies on chromosome 2p22.3.
Within the region, 13 SNPs were analyzed and
showed a striking correlation with osteoporosis susceptibility in adult women. These genetic mutations
were proposed to affect the pathogenesis of osteo-
Overall Structure of Human QC
In this report, we describe the first high-resolution (1.56-1.68 Å) crystal structures of human QC
in free form and bound to a substrate and three imidazole-derived inhibitors (9) (Figure 2). The globular structure of the enzyme reveals a mixed α/β
fold with a size of 63 x 58 x 41 Å3 (Figure 3A). The
structure has an open-sandwich topology comprising a central six-stranded β-sheet surrounded by
two (cyan) and six (magenta) α-helices on opposite
porosis by alterating
QC activity and subsequent gonadotropinreleasing hormone and
estrogen homeostasis
through the hypothalamus-pituitary-gonadal
axis.
Interestingly, QC
also catalyzes the Nterminal glutamate
cyclization into pGlu
(Figure 1) (5). This
reaction is probably
related to the formation of several plaqueforming peptides, such
as amyloid-β (Aβ)
peptides, which play a
pivotal
role
in
Alzheimer ’s disease
(6). Peptides containing N-terminal pGlu,
Figure 2. Crystal structures of human QC, revealing alternate conformations of the active site (yellow
e.g., pGlu3-Aβ(3-40)
(purple, blue, and green).
and cyan), and showing its binding modes with a substrate (red) and three imidazole-derived inhibitors
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Figure 3. Overall structure of human QC. (A) A ribbon diagram. The zinc-coordinated residues and Arg-54 (genetic mutation to Trp
residue occurred frequently in adult women with osteoporosis) are depicted with a ball-and-stick model. (B) A topology diagram. The
color codes for secondary structural elements are identical to those in A.
sides and flanked by two (yellow) α-helices at one
Active-Site Structure of Human QC
edge of the β-sheet. This twisted β-sheet is
The active site of the enzyme is mainly creat-
formed by two antiparallel and four parallel strands,
ed by six loops (Figure 3B). The catalytic pocket is
constituting the hydrophobic core of the molecule.
near the C-terminal edge of the central parallel
The coil and loop regions of the structure represent
strands (Figure 3A). It is relatively narrow but
42% of the total residues; about half of them (green)
accessible to the bulk solvent by means of a solvent
are major components of the active site (Figure 3B).
channel. The single zinc ion (10) of human QC lies
The structure of an osteoporosis-related genet-
at the bottom of the active-site pocket and is tetra-
ic mutant of human QC, R54W (4), shows only a
hedrally coordinated to Asp-159, Glu-202, His-330,
slight movement of the residues adjacent to Trp-54,
and a water molecule. In addition, several other
which is ~34 Å away from the active site. We found
highly conserved residues abut the zinc environ-
that this mutant retains ~70% of the catalytic activi-
ment (Figure 4), suggesting some roles in catalysis.
ty of the wild-type enzyme (Table 1), and its associ-
The acidic Glu-201, Asp-248, and Asp-305 are
ation with osteoporosis may be attributed to some
pointing to each other, likely forming hydrogen
unknown interactions with other molecules rather
bonds between them. The peptide bond between
than its activity.
Figure 5. Comparison of conf-A and conf-B of human QC bound
Figure 4. A stereoview of the human QC catalytic region.
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to the substrate glutamine t-butyl ester.
Figure 6. (A) Human QC (left) shares a conserved core structure
with the double-zinc amino- (central) and carboxy- (right) peptidases. (B) Comparison of the active-site structures of human QC and
an aminopeptidase from A. proteolytica.
the γ-amide group of the substrate N-terminal Gln
residue to direct toward the zinc-catalytic center in
favor of an intramolecular cyclization.
Asp-159 and Ser-160 adopts a cis-configuration sta-
Structures of Human QC in Complex
bilized by a network of hydrogen bonds.
with Substrate and Inhibitors
The active-site pocket is lined by several
The bound substrate (glutamine t-butyl ester)
hydrophobic residues, having approximate dimen-
in conf-A and conf-B of human QC adopts two
sions of 13 x 11 x 7 Å3. There are six water mole-
binding modes (Figure 5), likely due to the different
cules located inside the pocket, including the water
indole orientations of Trp-207. In conf-A, the t-
coordinated to the zinc ion. It is noteworthy that the
butyl group of the substrate is embedded between
two independent QCs in the asymmetric unit have
Glu-202 and Trp-207 of the enzyme with few spe-
different active-site conformations (denoted as
cific interactions. In conf-B, the t-butyl group is ori-
“conf-A” and “conf-B”), particularly at the segment
ented toward the surface of the enzyme, likely due
of L205-H206-W207 (Figure 5). The Trp-207
to the crowding of the bulky indole ring of Trp-207.
indole ring in conf-A is directed toward the surface
In contrast, the binding mode of the inhibitors
of the molecule, whereas that in conf-B is oriented
has no obvious difference between conf-A and
closer to the zinc ion.
Human QC Shares a Conserved Core
Structure
with
the
Double-Zinc
Exopeptidases
Human QC bears some degrees of structural
similarity to the two-zinc exopeptidases (Figure
6A). The key structural difference is in the coil and
loop regions attributed to several insertions and
deletions, especially the loops surrounding the
active-site pocket. Interestingly, the specific S1
pocket in the active site of the two-zinc aminopeptidases was not found in the structure of human QC
(Figure 6B). Consequently, human QC active site
has a more closed conformation, probably inducing
Figure 7. Structures of human QC in free form (A) and bound to
the imidazole-derived inhibitors 1-vinylimidazole (B), 1-benzylimidazole (C), and N-ω-acetylhistamine (D).
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with the active site of human QC, leaving a large
space in the catalytic pocket after its binding
(Figure 7B). However, the bulky hydrophobic
phenyl ring on 1-benzylimidazole is closely surrounded and stabilized by the phenyl and indole
groups of Phe-325 and Trp-329, respectively
(Figure 7C). In contrast, the substituent of N-ωacetylhistamine is oriented almost parallel to the
backbone of segment G301-Q304, stabilized mainly
by three additional hydrogen bonds (Figure 7D). In
general, the binding of the inhibitors does not
induce significant conformational changes in the
enzyme, likely because of their smaller sizes compared with those of the active-site pocket. From the
Figure 8. Proposed catalysis mechanism of human QC.
structural information, we conclude that an elec-
conf-B. Binding of the inhibitors results in the
tron-rich nucleophile with a good ability to ligate
removal of six water molecules within the active-
the zinc ion of human QC, combined with bulky
site pocket (Figure 7A), including the zinc-coordi-
hydrophobic substituents, is likely the structural
nated one, which is replaced by an imidazole nitro-
basis of a potent QC inhibitor.
gen of the inhibitors. The inhibitors adopt different
orientations because of their different modifications
Proposed Substrate-Binding and
Catalysis Mechanism of Human QC
on the imidazole ring. The small vinyl moiety of the
We proposed a plausible substrate-binding and
inhibitor 1-vinylimidazole shows no interaction
catalysis mechanism (Figure 8) of human QC based
Table 1. Kinetic parameters of wild-type and mutant human QC
Km (mM)
kcat (s-1)
kcat/Km (mM-1s-1)
Wild-Type
0.63 ± 0.01*
8.63 ± 0.48
13.663 ± 0.497
Mutant R54W
0.76 ± 0.04
7.35 ± 0.26
9.704 ± 0.824
K144A
1.47 ± 0.02
11.67 ± 0.34
7.944 ± 0.368
F146A
0.82 ± 0.16
7.91 ± 2.14
9.536 ± 0.769
E201D
12.62 ± 2.98
0.87 ± 0.28
0.068 ± 0.007
E201Q‡
ND
W207L
1.77 ± 0.07
0.43 ± 0.01
0.243 ± 0.002
W207F
0.59 ± 0.05
2.32 ± 0.07
3.943 ± 0.189
D248A‡
Q304L
ND
1.16 ± 0.09
9.39 ± 1.18
D305L‡
8.028 ± 0.386
ND
F325A
4.67 ± 0.24
12.91 ± 0.06
2.772 ± 0.132
W329A
29.53 ± 2.29
1.35 ± 0.07
0.046 ± 0.001
*Values are represented as mean ± S.D. (n = 2 or 3).
‡E201Q, D248A and D305L were shown to possess the ≈ 0.001%, ≈ 0.1% and ≈ 0.03% activity of the wildtype enzyme, respectively. ND: Not detectable.
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on the structures as well as some
active-site-mutant enzymes (Table
1). The conserved Glu-201 of
human QC may act as the general
base and acid to transfer a proton
from the α-amino group of the substrate to the leaving amino group on
the scissile γ-amide. The zinc ion
polarizes the γ-amide carbonyl
group of the substrate and simultaneously stabilizes the oxyanion
formed by the nucleophilic attack of
the α-nitrogen. Asp-248 probably
stabilizes the leaving γ-amide
amino group during the catalysis
process. In respect of the mechanism of glutamyl cyclase activity,
this leaving amino group is replaced
by a hydroxyl group, and the reaction is favored at pH 6.0 (5).
Conclusion
Figure 9. Glutaminyl (A) and glutamyl (B) cyclase activities of human QC.
Many peptides that are involved in the pro-
formation of N-terminal pGlu on several amyloid-
gression of amyloid plaque, such as Aβ peptides,
related peptides. We have solved the atomic-resolu-
have been reported to contain the N-terminal pGlu
tion structures of human QC and its complexes with
residue that is derived from its Glu precursor. We
substrate and inhibitors. Our studies thus provide
have demonstrated that human QC can convert the
insights into the mechanism of protein N-terminal
Glu-Aβ peptide into pGlu-Aβ, despite a signifi-
pGlu formation and form a firm basis for the ration-
cantly lower rate compared with the QC activity
al design of inhibitors against QC-associated disor-
(Figure 9). Because QCs are abundant in mam-
ders.
malian brain tissues, QC may be responsible for the
The original paper was published in Proceedings of the National Academy of Sciences 102, (2005):13117-13122.
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