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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 ACADEMIA SINICA 82 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 83 ACADEMIA SINICA 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. ACADEMIA SINICA 84 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). 85 ACADEMIA SINICA 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. ACADEMIA SINICA 86 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. References: 1. Fischer, W.H. & Spiess, J. (1987) Proc. Natl. Acad. Sci. 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