Structural Basis for Substrate Recognition and Hydrolysis by Mouse Carnosinase CN2
2008; Elsevier BV; Volume: 283; Issue: 40 Linguagem: Inglês
10.1074/jbc.m801657200
ISSN1083-351X
AutoresHideaki Unno, Tetsuo Yamashita, Sayuri Ujita, Nobuaki Okumura, Hiroto Otani, Akiko Okumura, Katsuya Nagai, Masami Kusunoki,
Tópico(s)Enzyme Structure and Function
Resumol-Carnosine is a bioactive dipeptide (β-alanyl-l-histidine) present in mammalian tissues, including the central nervous system, and has potential neuroprotective and neurotransmitter functions. In mammals, two types of l-carnosine-hydrolyzing enzymes (CN1 and CN2) have been cloned thus far, and they have been classified as metallopeptidases of the M20 family. The enzymatic activity of CN2 requires Mn2+, and CN2 is inhibited by a nonhydrolyzable substrate analog, bestatin. Here, we present the crystal structures of mouse CN2 complexed with bestatin together with Zn2+ at a resolution of 1.7Å and that with Mn2+ at 2.3Å. CN2 is a homodimer in a noncrystallographic asymmetric unit, and the Mn2+ and Zn2+ complexes closely resemble each other in the overall structure. Each subunit is composed of two domains: domain A, which is complexed with bestatin and two metal ions, and domain B, which provides the major interface for dimer formation. The bestatin molecule bound to domain A interacts with several residues of domain B of the other subunit, and these interactions are likely to be essential for enzyme activity. Since the bestatin molecule is not accessible to the bulk water, substrate binding would require conformational flexibility between domains A and B. The active site structure and substrate-binding model provide a structural basis for the enzymatic activity and substrate specificity of CN2 and related enzymes. l-Carnosine is a bioactive dipeptide (β-alanyl-l-histidine) present in mammalian tissues, including the central nervous system, and has potential neuroprotective and neurotransmitter functions. In mammals, two types of l-carnosine-hydrolyzing enzymes (CN1 and CN2) have been cloned thus far, and they have been classified as metallopeptidases of the M20 family. The enzymatic activity of CN2 requires Mn2+, and CN2 is inhibited by a nonhydrolyzable substrate analog, bestatin. Here, we present the crystal structures of mouse CN2 complexed with bestatin together with Zn2+ at a resolution of 1.7Å and that with Mn2+ at 2.3Å. CN2 is a homodimer in a noncrystallographic asymmetric unit, and the Mn2+ and Zn2+ complexes closely resemble each other in the overall structure. Each subunit is composed of two domains: domain A, which is complexed with bestatin and two metal ions, and domain B, which provides the major interface for dimer formation. The bestatin molecule bound to domain A interacts with several residues of domain B of the other subunit, and these interactions are likely to be essential for enzyme activity. Since the bestatin molecule is not accessible to the bulk water, substrate binding would require conformational flexibility between domains A and B. The active site structure and substrate-binding model provide a structural basis for the enzymatic activity and substrate specificity of CN2 and related enzymes. l-Carnosine (β-alanyl-l-histidine) and structurally related dipeptides, such as homocarnosine (γ-aminobutyryl-l-histidine) and anserine (β-alanyl-l-1-methylhistidine) are distributed in a wide variety of vertebrate tissues (1.Bonfanti L. Peretto P. De Marchis S. Fasolo A. Prog. Neurobiol. 1999; 59: 333-353Crossref PubMed Scopus (175) Google Scholar). l-Carnosine is present at particularly high concentrations in mammalian skeletal muscles and the brain, and it has been implicated in neuroprotection (2.Tabakman R. Lazarovici P. Kohen R. J. Neurosci. Res. 2002; 68: 463-469Crossref PubMed Scopus (113) Google Scholar), the olfactory system (1.Bonfanti L. Peretto P. De Marchis S. Fasolo A. Prog. Neurobiol. 1999; 59: 333-353Crossref PubMed Scopus (175) Google Scholar), and hypothalamic neuronal networks (3.De Marchis S. Modena C. Peretto P. Giffard C. Fasolo A. J. Comp. Neurol. 2000; 426: 378-390Crossref PubMed Scopus (17) Google Scholar). Our recent observations suggest that central and peripheral administration of l-carnosine at low doses attenuates 2-deoxyglucose-induced hyperglycemia (4.Yamano T. Niijima A. Iimori S. Tsuruoka N. Kiso Y. Nagai K. Neurosci. Lett. 2001; 313: 78-82Crossref PubMed Scopus (60) Google Scholar) and suppresses peripheral sympathetic nerve activity (5.Niijima A. Okui T. Matsumura Y. Yamano T. Tsuruoka N. Kiso Y. Nagai K. Auton. Neurosci. 2002; 97: 99-102Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 6.Tanida M. Niijima A. Fukuda Y. Sawai H. Tsuruoka N. Shen J. Yamada S. Kiso Y. Nagai K. Am. J. Physiol. 2005; 288: R447-R455Crossref PubMed Scopus (75) Google Scholar). These effects of l-carnosine are suppressed by central administration of thioperamide, a histamine H3 blocker. This suggests that l-carnosine regulates the autonomic nervous system via the hypothalamic histaminergic neurons (4.Yamano T. Niijima A. Iimori S. Tsuruoka N. Kiso Y. Nagai K. Neurosci. Lett. 2001; 313: 78-82Crossref PubMed Scopus (60) Google Scholar, 5.Niijima A. Okui T. Matsumura Y. Yamano T. Tsuruoka N. Kiso Y. Nagai K. Auton. Neurosci. 2002; 97: 99-102Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 6.Tanida M. Niijima A. Fukuda Y. Sawai H. Tsuruoka N. Shen J. Yamada S. Kiso Y. Nagai K. Am. J. Physiol. 2005; 288: R447-R455Crossref PubMed Scopus (75) Google Scholar). In addition, the dipeptide exhibits antioxidant and free radical scavenger properties via complexation of transition metals, such as zinc and copper, suggesting that it is also involved in neuroprotection from oxidative stress (2.Tabakman R. Lazarovici P. Kohen R. J. Neurosci. Res. 2002; 68: 463-469Crossref PubMed Scopus (113) Google Scholar, 8.Baran E.J. Biochemistry (Mosc.). 2000; 65: 789-797PubMed Google Scholar, 9.Trombley P.Q. Horning M.S. Blakemore L.J. Biochemistry (Mosc.). 2000; 65: 807-816PubMed Google Scholar). l-Carnosine is synthesized from β-alanine and l-histidine by carnosine synthetase and is degraded by intra- and extracellular dipeptidases known as carnosinases. Their enzymatic activities are regulated under various physiological conditions (10.Nagai K. Niijima A. Yamano T. Otani H. Okumra N. Tsuruoka N. Nakai M. Kiso Y. Exp. Biol. Med. 2003; 228: 1138-1145Crossref PubMed Scopus (102) Google Scholar). Carnosinase was first isolated (11.Hanson H.T. Smith E.L. J. Biol. Chem. 1949; 179: 789-801Abstract Full Text PDF PubMed Google Scholar) from the porcine kidney in 1949 and was subsequently found to be widely distributed in tissues of rodents and higher mammals (12.Wood T. Nature. 1957; 180: 39-40Crossref PubMed Scopus (22) Google Scholar, 13.Margolis F.L. Grillo M. Grannot-Reisfeld N. Farbman A.I. Biochim. Biophys. Acta. 1983; 744: 237-248Crossref PubMed Scopus (38) Google Scholar, 14.Wolos A. Piekarska K. Glogowski J. Koieczka I. Int. J. Biochem. 1978; 9: 57-62Crossref PubMed Scopus (16) Google Scholar, 15.Kunze N. Kleinkauf H. Bauer K. Eur. J. Biochem. 1986; 160: 605-613Crossref PubMed Scopus (46) Google Scholar). Recently, two types of carnosinases were identified in humans and mice: human CN1 (also known as CNDP1 or CNDP dipeptidase 1), human CN2 (CNDP2 or CNDP dipeptidase 2) (16.Teufel M. Saudek V. Ledig J.P. Bernhardt A. Boularand S. Carreau A. Cairns N.J. Carter C. Cowley D.J. Duverger D. Ganzhorn A.J. Guenet C. Heintzelmann B. Laucher V. Sauvage C. Smirnova T. J. Biol. Chem. 2003; 278: 6521-6531Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar), and mouse CN2 (17.Otani H. Okumura N. Hashida-Okumura A. Nagai K. J. Biochem. (Tokyo). 2005; 37: 167-175Crossref Scopus (44) Google Scholar). The biochemical properties of these types were investigated in detail. CN2 is present in mammalian brain and is especially abundant in the tuberomammillary nucleus of the hypothalamus, the thalamic parafascicular nucleus, neuronal fibers, and the mitral cell layer of the olfactory bulb in the nervous system (17.Otani H. Okumura N. Hashida-Okumura A. Nagai K. J. Biochem. (Tokyo). 2005; 37: 167-175Crossref Scopus (44) Google Scholar). The cell bodies of histaminergic neurons localize in the tuberomammillary nucleus, suggesting that CN2 is involved in histamine synthesis in the histaminergic neurons, possibly by supplying l-histidine as the substrate for the histamine-synthesizing enzyme histidine decarboxylase. Sequence-based alignments of human CN1 and human CN2 with mouse CN2 show sequence identities of 53 and 91%, respectively. Human CN1 was identified as a dipeptidase that hydrolyzes Xaa-His dipeptides, including those with first residues β-Ala (carnosine), γ-aminobutyric acid (homocarnosine), N-methyl-β-Ala, Ala, and Gly. On the other hand, CN2 has a broader specificity than CN1, but it does not hydrolyze homocarnosine and is sensitive to inhibition by bestatin (IC50 = 7 nm) (16.Teufel M. Saudek V. Ledig J.P. Bernhardt A. Boularand S. Carreau A. Cairns N.J. Carter C. Cowley D.J. Duverger D. Ganzhorn A.J. Guenet C. Heintzelmann B. Laucher V. Sauvage C. Smirnova T. J. Biol. Chem. 2003; 278: 6521-6531Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Unlike most other metallopeptidases, CN2 requires Mn2+ for complete activity, and Zn2+ alone cannot activate this enzyme. Based on the similarity in primary sequences, CN1 and CN2 have been classified as metallopeptidases belonging to the M20 family of clan MH (18.Barrett A.J. Rawlings N.D. Woessner J.F. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, London1998: 1412-1416Google Scholar). Of these, the crystal structures of PepV from Lactobacillus delbrueckii (19.Vongerichten K.F. Klein J.R. Matern H. Plapp R. Microbiology. 1994; 140: 2591-25600Crossref PubMed Scopus (47) Google Scholar) and CPG2 (carboxypeptidase G2) from Pseudomonas sp. (20.Rowsell S. Pauptit R.A. Tucker A.D. Melton R.G. Blow D.M. Brick P. Structure (Camb.). 1997; 5: 337-347Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), which share 17 and 18% sequence identities with mouse CN2, respectively, have been reported thus far. PepV and CPG2 are composed of two domains: one catalytic domain with two Zn2+ ions at the active center and one noncatalytic domain known as the lid domain or the dimerization domain. The dimerization domain of CPG2 provides the surface for the same interaction to form a homodimer structure, whereas PepV is present as a monomer due to the different structural features of the lid domain. Furthermore, the crystal structure of a member of the M28 family of dinuclear zinc aminopeptidases from Aeromonas proteolytica (AAP) 8The abbreviation used is: AAP, aminopeptidase(s) from A. proteolytica. 8The abbreviation used is: AAP, aminopeptidase(s) from A. proteolytica. (21.Chevrier B. Schalk C. D'Orchymont H. Rondeau J.M. Moras D. Tarnus C. Structure (Camb.). 1994; 2: 283-291Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar) is similar to the catalytic domain of PepV and CPG2. However, AAP does not have a noncatalytic domain and is present as a monomer in solution. In an attempt to determine the structure of CN2, we obtained two types of CN2 crystals: one complexed with Zn2+ (Zn2+ complex) and the other with Mn2+ (Mn2+ complex). Here, we report the structures of the two forms of CN2 crystals. We identified the residues crucial for l-carnosine-specific binding and catalysis and gained a structural basis for explaining the differences in the substrate specificity of CN2 and the related enzymes. Then we discuss metal ion selectivity and conformational flexibility of CN2 based on the structures of CN2 and other members of the M20/M28 peptidase family. Purification of Mouse Carnosinase CN2—Otani et al. (17.Otani H. Okumura N. Hashida-Okumura A. Nagai K. J. Biochem. (Tokyo). 2005; 37: 167-175Crossref Scopus (44) Google Scholar) described the cloning, expression, and purification of CN2. For crystallization, the purification procedures were slightly modified from that described by Yamashita et al. (22.Yamashita T. Unno H. Ujita S. Otani H. Okumura N. Hashida-Okumura A. Nagai K. Kusunoki M. Acta Crystallogr. Sect. F. 2006; 62: 996-998Crossref PubMed Scopus (4) Google Scholar). The cDNA encoding CN2 was subcloned into the expression vector pGEX-4T3 (Amersham Biosciences). The CN2 protein fused with glutathione S-transferase was then overexpressed in the Escherichia coli strain BL21(DE3)pLysS in the presence of 1 mm isopropyl 1-thio-β-d-galactopyranoside and 2 mm MnCl2 for 12-16 h at 25 °C. The E. coli cells were collected and sonicated in a buffer containing 25 mm Tris-HCl (pH 7.4), 50 mm NaCl, 0.2 mm MnCl2, and 1 mm dithiothreitol. After centrifugation, the supernatant was mixed with glutathione-Sepharose beads (Amersham Biosciences), washed, and incubated with thrombin (Amersham Biosciences) at 25 °C for 12-16 h to obtain the full-length CN2. The soluble fraction was then separated on a gel filtration column (HiLoad 26/60 Superdex 200 pg; Amersham Biosciences) and an anion exchange column (Hi-trap Q; Amersham Biosciences). Proteins were eluted from the column with a linear gradient of 50-750 mm NaCl in the same buffer. Crystallization—Crystallization of CN2 complexed with Mn2+ was carried out using the hanging drop vapor diffusion method. Two microliters of a protein solution (20 mg/ml) containing 25 mm Tris-HCl (pH 7.4), 50 mm NaCl, 0.2 mm MnCl2, 1 mm dithiothreitol, and 30 mm bestatin were mixed with an equal volume of a reservoir solution and allowed to equilibrate against 0.5 ml of the reservoir solution at 293 K. Wing-shaped crystals were obtained using a reservoir solution containing 20% polyethylene glycol 3350 and 0.2 m KF. The crystal size and quality were further improved with a combination of the macroseeding and microseeding techniques (23.Stura E.A. Wilson I.A. Ducruix A. Giege R. Crystallization of Nucleic Acids and Proteins: A Practical Approach. 2nd Ed. Oxford University Press, New York1999: 177-208Google Scholar). A single crystal with dimensions of 0.4 × 0.2 × 0.1 mm was obtained in a hanging drop in 20% (w/v) polyethylene glycol 3350, 20% (w/v) glycerol, and 0.2 m KF. CN2 crystals complexed with zinc ions were prepared in the same way, but Mn2+ ions were removed by transferring the crystals into a harvest solution containing 20% polyethylene glycol 3350, 20% glycerol, and 0.2 m KF for 1 day before x-ray data collection. We obtained two types of crystals, Zn2+ and Mn2+ complexes, as confirmed by x-ray absorption fine structure spectra (supplemental Fig. S1). Since Zn2+ ions were not added during purification, crystallization, and crystal harvesting procedures, the Zn2+ ions in the Zn2+ complex crystal are thought to be derived from the culture medium and held by CN2 during the purification, crystallization, and crystal harvesting procedures. However, since zinc ions were not completely removed from buffers for purification and soaking, we could not completely exclude the possibility that the Zn2+ ions were derived from these buffers. Data Collection, Structure Solution, and Refinement—A CN2 crystal was mounted in a nylon cryoloop (Hampton Research) and placed directly into a nitrogen stream at 100 K. The metal type in a crystal was determined by x-ray absorption fine structure spectra. A crystal containing only Mn2+ ions was used for Mn2+ multiple-wavelength anomalous dispersion, and that containing only Zn2+ ions was for Zn2+ multiple-wavelength anomalous dispersion data collection. Data collection was carried out using synchrotron radiation at beamline NW-12 of the Photon Factory (KEK, Tsukuba, Japan). For the Mn2+ complex, two wavelengths at the Mn-K absorption edge (1.8941 Å) and peak (1.8926 Å) and one remote wavelength (1.7926 Å) were used. For the Zn2+ complex, wavelengths at the Zn-K absorption edge (1.2834 Å), peak (1.2827 Å), and one remote wavelength (1.2573 Å) were used. High resolution data for the Zn2+ complex were collected at 1.000 Å. A complete data set was collected through contiguous rotation ranges at a particular wavelength before proceeding to the next wavelength. Rotation data were recorded in frames of 1° oscillation. The data collected at different wavelengths were processed with the program HKL2000 (24.Otwinowski Z. Minor W. Macromol. Crystallogr. Pt. A. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar). The phases were calculated using the program SHARP (25.de La Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1796) Google Scholar). After solvent flattening and density modification, protein models for the Mn2+ and Zn2+ complexes were constructed independently by the program ARP/wARP (26.Perrakis A. Morris R.M. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2561) Google Scholar), and the models were improved with the program O (27.Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar). Each model was refined using the program Refmac5 (28.Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13712) Google Scholar). All figures were produced using MOLSCRIPT (29.Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), POVSCRIPT+ (30.Fenn T.D. Ringe D. Petsko G.A. J. Appl. Crystallogr. 2003; 36: 944-947Crossref Scopus (293) Google Scholar), PyMOL (31.DeLano W.L. PyMOL. DeLano Scientific, San Carlos, CA2002Google Scholar), and RASTER3D (32.Merritt E.A. Murphy M.E. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2854) Google Scholar). Enzyme Assay—cDNA encoding a CN2 mutant carrying a His228 to Ala substitution was synthesized using QuikChange (Stratagene), and the nucleotide sequence was verified. The protein was then expressed as described above and purified by glutathione-Sepharose. After thrombin digestion, it was further purified by ion exchange chromatography on a Hitrap-Q column (Amersham Biosciences). The purified protein (20 mg/ml) was incubated in a buffer containing 50 mm Tris-HCl (pH 8.8), 10 mm MnCl2, 5 mm dithiothreitol, and 10 mm l-carnosine for 30 min at 37 °C. The reaction was terminated by the addition of bestatin to yield a final concentration of 1 mm, and a 1-μl aliquot was spotted onto a cellulose plate for thin layer chromatography. Amino acids were separated by an ascending solvent composed of 15% water, 10% formic acid, 75% isopropyl alcohol and detected by spraying 10 mg/ml ninhydrin solution in ethanol. For quantitative measurement of carnosinase activity, histidine was derivatized with o-phthalaldehyde at alkaline pH after enzyme reaction (33.Bando K. Shimotsuji T. Toyoshima H. Hayashi C. Miya K. Ann. Clin. Biochem. 1984; 21: 510-514Crossref PubMed Scopus (37) Google Scholar), and absorbance at 405 nm was determined. CN2 is a metallopeptidase that hydrolyzes a variety of dipeptides, including l-carnosine, to form the corresponding amino acids (Fig. 1A). The enzymatic activity of CN2 is inhibited by bestatin (Fig. 1B), which is considered to act as a nonhydrolyzable substrate analog for a wide variety of peptidases. At first, we tried to obtain CN2 crystals without bestatin but could not obtain a crystal suitable for structural analysis. Next, we tried to obtain crystals of CN2 complexed with bestatin. After optimizing crystallization conditions, we obtained two forms of crystals of CN2-bestatin complex. One included Mn2+ ions (Mn2+ complex), and the other included Zn2+ ions (Zn2+ complex), as determined by x-ray absorption fine structure (supplemental Fig. S1). Both types were isomorphs to each other and crystallized in space group P21 with one homodimer in a crystallographic asymmetric unit. The structures of these two forms of CN2 were determined by the multiple-wavelength anomalous dispersion method independently, and they were refined to resolutions of 1.7 Å (Zn2+ complex) and 2.3 Å (Mn2+ complex). The data collection and refinement statistics are listed in Table 1.TABLE 1Data collection and refinement statisticsParametersValuesMn2+ complexZn2+ complexData collection and processing statisticsUnit cell parameters (Å, degrees)a = 54.49, b = 199.18, c = 55.21, β = 118.92a = 54.41, b = 199.77, c = 55.49, β = 118.52Space groupP21P21Mn2+ complexZn2+ complexEdgePeakRemoteHigh resolutionEdgePeakRemoteWavelength (Å)1.89411.89261.79261.0001.28341.28271.2573Resolution (Å)50.0-2.8 (2.91-2.81)50.0-2.8 (2.90-2.80)50.0-2.3 (2.38-2.30)50.0-1.7 (1.76-1.70)50.0-1.8 (1.86-1.80)50.0-1.8 (1.86-1.80)50.0-1.8 (1.86-1.80)Redundancy7.3 (6.3)7.4 (6.9)6.6 (3.1)5.0 (3.4)7.0 (4.4)6.9 (4.4)7.0 (4.6)Measured183799186077264160545738617481618712638696Completeness (%)99.9 (99.1)99.3 (94.4)86.8 (30.9)96.1 (74.4)92.8 (56.1)93.2 (57.5)94.7 (63.8)I/∑(I)31.8 (6.4)45.2 (15.6)35.3 (3.4)38.4 (2.8)46.2 (5.4)47.1 (6.3)45.5 (5.0)RmergeaRmerge = 100∑|I - |/∑I, where I is the observed intensity and is the average intensity of multiple observations of symmetry-related reflections. (%)6.9 (27.8)5.9 (14.2)5.8 (23.5)3.8 (34.2)4.0 (19.9)4.1 (18.0)4.2 (22.1)Refinement statisticsResolution43.0-2.339.3-1.7Protein atoms74587458Bestatin4444Manganese4Zinc4Water molecules472937Rwork/Rfree (%)19.2/23.919.6/23.9Root mean square deviationsBond length (Å)0.0130.015Bond angle (degrees)1.4241.583a Rmerge = 100∑|I - |/∑I, where I is the observed intensity and is the average intensity of multiple observations of symmetry-related reflections. Open table in a new tab The asymmetric unit is composed of one homodimer shaped like a curved cylinder having dimensions of ∼45 × 55 × 115 Å. The overall crystal structure of the Mn2+ complex is shown in Fig. 2. The two polypeptide chains were related by noncrystallographic 2-fold symmetry in a dimer, and a total of 478 amino acid residues were observed in the structure; of the 478 residues, 473 were residues of CN2 protein, and 5 N-terminal residues (Gly-Ser-Pro-Asn-Ser) were derived from the expression vector pGEX-4T3. The two subunits are tightly associated with each other, and each has an active site structure containing two metal ions and one bestatin molecule. Overall Structure—The structure of each subunit is divided into two domains, A (residues 1-203 and 416-476) and B (residues 204-415) (Fig. 3). Each domain has an α/β fold structure consisting of β-strands and α-helices that were numbered as shown in Fig. 3. Domain A essentially comprises 12 α-helices (α1-α9, α17-α19) and a large twisted β-sheet in which the β-strands are arranged in the order of β1-β4-β8-β5-β9-β17, with β4 representing the only antiparallel strand. In addition, a second smaller two-stranded β-sheet (β6-β7) and a third two-stranded β-sheet (β10-β18) are located above α-helix α6. Domain B also has a large twisted β-sheet (β13-β14-β11-β15-β16) and seven α-helices (α10 -α16) together with an additional small two-stranded β-sheet (β12, β16). Domain B is connected to domain A by two β-strands, namely, β11 and β17. Domains A and B constitute a continuous β sheet structure. The subunit topology is illustrated in supplemental Fig. S2. Structural Comparison of CN2 with Other M20/M28 Family Metallopeptidases—The structure of mouse CN2 was compared with related dinuclear metallopeptidases of the M20 and M28 families (supplemental Fig. S3, A-D). L. delbrueckii aminopeptidase PepV (34.Jozic D. Bourenkow G. Bartunik H. Scholze H. Dive V. Henrich B. Huber R. Bode W. Maskos K. Structure (Camb.). 2002; 10: 1097-1106Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and Pseudomonas sp. CPG2 (carboxypeptidase G2) (20.Rowsell S. Pauptit R.A. Tucker A.D. Melton R.G. Blow D.M. Brick P. Structure (Camb.). 1997; 5: 337-347Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) are M20 family peptidases, and both of these peptidases have two domains consisting of the catalytic domain and the lid or dimerization domain. In contrast, AAP (A. proteolytica aminopeptidase) (21.Chevrier B. Schalk C. D'Orchymont H. Rondeau J.M. Moras D. Tarnus C. Structure (Camb.). 1994; 2: 283-291Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar), which belongs to the M28 family, is a single domain protein having only one catalytic domain and is present in monomeric form in the crystal. Domain B of CN2 is topologically similar to the dimerization domain of CPG2. This domain provides the binding interface for homodimer formation. Structural alignment of the α-carbon atoms of 212 residues in CN2 domain B against those of the CPG2 dimerization domain revealed that 76 residues of CPG2 structurally corresponded to residues of CN2 with a root mean square deviation of 1.82 Å. Recently, a crystal structure of another M20 family member, Streptococcus pneumoniae metallopeptidase, was deposited with the Protein Data Bank (code 2pok). This protein also has a similar domain architecture, and its 204 residues corresponded to the 212 residues of CN2 domain B with a root mean square deviation of 3.1 Å for Cα atoms. Sequence identities of CN2 with CPG2 and Streptococcal peptidase for these peptide segments are 28 and 13.2%, respectively, and no structural similarity was found in other structures in a search by the Dali server (available on the World Wide Web). Despite the structural similarity, the three proteins show different orientations in the dimer architectures (supplemental Fig. 3, A and B). In CPG2 and the Streptococcal peptidase, the dimerization domain of one subunit interacts only with the dimerization domain of the other subunit but not with any residues of the catalytic subunits. Thus, the dimerization domain appears to be an independent structural unit that is responsible for dimer formation. In contrast, domain B of CN2 interacts not only with domain B of the other subunit but also with domain A of both subunits. In addition, several residues of domain B interact with the active site of the other subunit as described below. Subunit Interactions—Two subunits of CN2 are related by noncrystallographic 2-fold rotation symmetry with an interface area of 3500 Å2. These interactions are mediated mainly between four peptide segments; α11, β13, loops L1 (residues 224-237), and L2 (residues 323-331) (Figs. 2 and 3). The main interaction interface is constructed between α11 and β13 of one subunit and those of the other subunit. Loop L2 consists of 9 amino acid residues between β13 and β14 in domain B of one subunit, which interacts with β13 in domain B of the other subunit. The other side of loop L2 constructs part of the bestin-binding cleft of the other subunit. Loop L1 is located between β11 and α11 and consists of a loop, a short β-strand β12, and a short α-helix (α10). Loop L1 of one subunit interacts not only with α14 and α15 in domain B of the other subunit but also with a loop (residues 415-418) between β16 and α18 and a loop (residues 436-449) between β1 and α19 of domain A. These domain B-domain B and domain A-domain B interactions enhance the interface surface area between the two subunits in CN2 (3500 Å2) compared with that of that in CPG2 (1273 Å2). Active Site Structure—A CN2 homodimer contains two active sites, each of which has one bestatin and two Mn2+ ions that were observed to have well defined electron densities (Fig. 5, A and B). Crystal structures of peptidases in complexes with bestatin for AAP (35.Stamper C.C. Bienvenue D.L. Bennett B. Ringe D. Petsko G.A. Holz R.C. Biochemistry. 2004; 43: 9620-9628Crossref PubMed Scopus (30) Google Scholar) and leucine aminopeptidase have been reported (36.Burley S.K. David P.R. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6916-6920Crossref PubMed Scopus (128) Google Scholar), where bestatin-binding sites were located on the protein surface of one subunit. In contrast, each active site of CN2 is located in a cleft constructed by domains A and B of one subunit together with domain B of the other subunit. In this cleft, bestatin interacts with Mn2+ ions and amino acid residues of several different peptide segments (Fig. 5C). Of these, 6 are residues on domain A (i.e. Glu166 on α8; Asp195 and Tyr197 on a loop between β9 and β10; and Glu414, Gly416, and Ser417 on a loop between β16 and α18). On the other hand, His380 is from β15 in domain B and contributes to connect the two domains. Moreover, it should be noted that bestatin in one subunit also interacts with several residues on the other subunit. These include His228′ and Val231′ on loop L1 and Thr330′ on loop L2 of the other subunit, where primes refer to residues on the other subunit. To confirm that His228 of domain B is involved in substrate recognition, we synthesized a recombinant protein carrying a mutation at His228 to Ala (H228A), and its enzymatic activity was examined (Fig. 5, D and E). Incubation of l-carnosine with recombinant wild-type CN2 resulted in l-carnosine hydrolysis, yielding β-alanine and l-histidine, as detected by thin layer chromatography. On the other hand, the activity was not detected in the H228A mutant protein. CD spectra of wild type and H228A mutant proteins had almost the same shape in the range of 198-250 nm, implying that the H228A mutant protein had no aberration in the stability and folding (supplemental Fig. S4). These results indicate that His228 is really involved in the enzymatic reaction on the active site of domain A of the other subunit. In the CN2 complex, a bestatin molecule "rides" on top of the line connecting the two metal ions in the same way as phosphinate complexed with PepV (33.Bando K. Shimotsuji T. Toyoshima H. Hayashi C. Miya K. Ann. Clin. Biochem. 1984; 21: 510-514Crossref PubMed Scopus (37) Google Scholar). P1 and P1′ side chains of bestatin are accommodated in hydrophobic pockets (S1 and S1′ pockets) adjacent to the dinuclear metal ions in the active site. The S1 pocket, which is composed of Leu210, Gly416, Tyr197, Glu414, Val231′, and His228′, is the possible N-terminal side chain pocket, as judged by the structural similarity between bestatin and
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