The First Structure of Dipeptidyl-peptidase III Provides Insight into the Catalytic Mechanism and Mode of Substrate Binding
2008; Elsevier BV; Volume: 283; Issue: 32 Linguagem: Inglês
10.1074/jbc.m803522200
ISSN1083-351X
AutoresPravas Kumar Baral, Nina Jajčanin-Jozić, Sigrid Deller, Peter Macheroux, Marija Abramić, Karl Gruber,
Tópico(s)Signaling Pathways in Disease
ResumoDipeptidyl-peptidases III (DPP III) are zinc-dependent enzymes that specifically cleave the first two amino acids from the N terminus of different length peptides. In mammals, DPP III is associated with important physiological functions and is a potential biomarker for certain types of cancer. Here, we present the 1.95-Å crystal structure of yeast DPP III representing the prototype for the M49 family of metallopeptidases. It shows a novel fold with two domains forming a wide cleft containing the catalytic metal ion. DPP III exhibits no overall similarity to other metallopeptidases, such as thermolysin and neprilysin, but zinc coordination and catalytically important residues are structurally conserved. Substrate recognition is accomplished by a binding site for the N terminus of the peptide at an appropriate distance from the metal center and by a series of conserved arginine residues anchoring the C termini of different length substrates. Dipeptidyl-peptidases III (DPP III) are zinc-dependent enzymes that specifically cleave the first two amino acids from the N terminus of different length peptides. In mammals, DPP III is associated with important physiological functions and is a potential biomarker for certain types of cancer. Here, we present the 1.95-Å crystal structure of yeast DPP III representing the prototype for the M49 family of metallopeptidases. It shows a novel fold with two domains forming a wide cleft containing the catalytic metal ion. DPP III exhibits no overall similarity to other metallopeptidases, such as thermolysin and neprilysin, but zinc coordination and catalytically important residues are structurally conserved. Substrate recognition is accomplished by a binding site for the N terminus of the peptide at an appropriate distance from the metal center and by a series of conserved arginine residues anchoring the C termini of different length substrates. A number of bioactive peptides that play crucial roles in the nervous and endocrine systems have been identified. These peptides interact with corresponding receptors and subsequently modulate downstream pathways. The consequences of such processes are terminated by the degradation of those active peptides either by peptidases or through internalization by other mechanisms. A number of peptidases that act on physiologically important peptides have been identified and are classified into different groups according to their mode of action (exo- versus endopeptidases), catalytic signature motifs, or metal content (1Rawlings N.D. Morton F.R. Kok C.Y. Kong J. Barrett A.J. Nucleic Acids Res. 2008; 36: D320-D325Crossref PubMed Scopus (519) Google Scholar). Because of their role in the organism, inhibition of peptidases has therapeutic implications, such as the treatment of hypertension in the case of inhibition of angiotensin I-converting enzyme (2Waeber B. Nussberger J. Brunner H.S. Laragh J.H. Brenner B.M. Hypertension: Pathophysiology, Diagnosis and Management. Raven Press, New York1990: 2209-2232Google Scholar). Dipeptidyl-peptidase III (DPP III 2The abbreviations used are: DPP IIIdipeptidyl-peptidase IIIMADmultiple wavelength anomalous dispersionMIRmultiple isomorphous replacementTLStranslation/libration/screw. ; EC 3.4.14.4) enzymes form a group of aminopeptidases with molecular masses of ∼80-85 kDa. They specifically cleave dipeptides from the N termini of their substrates and have restricted specificity on dipeptidyl-arylamide substrates, preferring Arg-Arg-2-naphthylamide (3Chen J.-M. Barrett A.J. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Elsevier Science Publishers B.V., Amsterdam2004: 809-812Crossref Scopus (41) Google Scholar). The cDNAs for human, rat, and fruit fly DPP III were cloned, and their respective amino acid sequences (737, 738, and 786 amino acids long) were deduced (3Chen J.-M. Barrett A.J. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Elsevier Science Publishers B.V., Amsterdam2004: 809-812Crossref Scopus (41) Google Scholar, 4Fukasawa K. Fukasawa K.M. Kanai M. Fujii S. Hirose J. Harada M. Biochem. J. 1998; 329: 275-282Crossref PubMed Scopus (68) Google Scholar, 5Mazzocco C. Fukasawa K.M. Auguste P. Puiroux J. Eur. J. Biochem. 2003; 270: 3074-3082Crossref PubMed Scopus (27) Google Scholar). Based on complete genome sequence data, orthologs have also been identified in >50 species, including lower eukaryotes (yeasts) and some specific bacteria (6Abramić M. Špoljarić J. Šimaga Š. Period. Biol. 2004; 106: 161-168Google Scholar). In rat, DPP III is found to be expressed in various tissues with high levels in brain, liver, small intestine, and kidney (7Abramić M. Šimaga Š. Osmak M. Čičin-Šain L. Vukelić B. Vlahoviček K. Dolovcak L. Int. J. Biochem. Cell Biol. 2004; 36: 434-446Crossref PubMed Scopus (37) Google Scholar, 8Ohkubo I. Li Y.H. Maeda T. Yamamoto Y. Yamane T. Du P.G. Nishi K. Biol. Chem. 1999; 380: 1421-1430Crossref PubMed Scopus (30) Google Scholar). It is generally found to be a cytosolic protein, but membrane-associated DPP III in rat brain has been reported as well (9Lee C.-M. Snyder S.H. J. Biol. Chem. 1982; 257: 12043-12050Abstract Full Text PDF PubMed Google Scholar). DPP III from human seminal plasma (10Vanha-Perttula T. Clin. Chim. Acta. 1988; 177: 179-195Crossref PubMed Scopus (23) Google Scholar), lens tissue (11Swanson A.A. Davis R.M. McDonald J.K. Curr. Eye Res. 1984; 3: 287-291Crossref PubMed Scopus (17) Google Scholar), and erythrocytes (12Abramić M. Zubanović M. Vitale L. Biol. Chem. 1988; 369: 29-38Crossref PubMed Scopus (75) Google Scholar) has been purified and biochemically characterized, as were the enzymes from several other mammalian tissues, including bovine pituitary (13Ellis S. Nuenke J.M. J. Biol. Chem. 1967; 242: 4623-4629Abstract Full Text PDF PubMed Google Scholar), rat brain (9Lee C.-M. Snyder S.H. J. Biol. Chem. 1982; 257: 12043-12050Abstract Full Text PDF PubMed Google Scholar), and liver (8Ohkubo I. Li Y.H. Maeda T. Yamamoto Y. Yamane T. Du P.G. Nishi K. Biol. Chem. 1999; 380: 1421-1430Crossref PubMed Scopus (30) Google Scholar). Regarding non-mammals, DPP III has been partially purified and characterized from the slime mold Dictyostelium discoideum (14Huang J. Kim J. Ramamurthy P. Jones T.H.D. Exp. Mycol. 1992; 16: 102-109Crossref Scopus (13) Google Scholar) and Drosophila melanogaster (15Mazzocco C. Gillibert-Duplantier J. Neaud V. Fukasawa K.M. Claverol S. Bonneu M. Puiroux J. FEBS J. 2006; 273: 1056-1064Crossref PubMed Scopus (12) Google Scholar). dipeptidyl-peptidase III multiple wavelength anomalous dispersion multiple isomorphous replacement translation/libration/screw. The exact physiological roles of these enzymes are not yet clear. However, some pharmacological experiments link DPP III with pain regulation mechanisms because low levels of DPP III activity have been observed in the cerebrospinal fluid of individuals suffering from acute pain (16Sato H. Kimura K. Yamamoto Y. Hazato T. Masui. 2003; 52: 257-263PubMed Google Scholar). Similarly, high concentrations of DPP III found in the superficial laminae of rat spinal cord dorsal horn (17Chiba T. Li Y.H. Yamane T. Ogikubo O. Fukuoka M. Arai R. Takahashi S. Ohtsuka T. Ohkubo I. Matsui N. Peptides. 2003; 24: 773-778Crossref PubMed Scopus (38) Google Scholar), as well as the high in vitro affinity shown by the human enzyme toward important neuropeptides, such as enkephalins and endomorphins (18Barsun M. Jajčanin N. Vukelić B. Spoljarić J. Abramić M. Biol. Chem. 2007; 388: 343-348Crossref PubMed Scopus (60) Google Scholar), also indicate that this hydrolase could play a role in the mammalian pain modulatory system. These findings make DPP III a potential drug target, and efforts toward inhibitor design and synthesis are under way (19Agić D. Hranjec M. Jajčanin N. Starčević K. Karminski-Zamola G. Abramić M. Bioorg. Chem. 2007; 35: 153-169Crossref PubMed Scopus (19) Google Scholar). Recently, DPP III has also obtained much attention because of its overexpression in ovarian malignant tissues, and this property can be further exploited as a potential biomarker for carcinoma (20Šimaga Š. Babić D. Osmak M. Sprem M. Abramić M. Gynecol. Oncol. 2003; 91: 194-200Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Furthermore, a role for DPP III has been implied in cataractogenesis and endogenous defense mechanisms against oxidative stress (21Liu Y. Kern J.T. Walker J.R. Johnson J.A. Schultz P.G. Luesch H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5205-5210Crossref PubMed Scopus (169) Google Scholar, 22Zhang H. Yamamoto Y. Shumiya S. Kunimatsu M. Nishi K. Ohkubo I. Kani K. Histochem. J. 2001; 33: 511-521Crossref PubMed Scopus (37) Google Scholar). Biochemical investigations showed DPP III to be a zinc metallopeptidase containing 1 mol of zinc/mol of protein with a dissociation constant of 2.5 × 10-13 m at pH 7.4 (4Fukasawa K. Fukasawa K.M. Kanai M. Fujii S. Hirose J. Harada M. Biochem. J. 1998; 329: 275-282Crossref PubMed Scopus (68) Google Scholar). Commonly, cobalt ions significantly activate DPP III from various origins (3Chen J.-M. Barrett A.J. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Elsevier Science Publishers B.V., Amsterdam2004: 809-812Crossref Scopus (41) Google Scholar, 23Hirose J. Iwamoto H. Nagao I. Enmyo K. Sugao H. Kanemitu N. Ikeda K. Takeda M. Inoue M. Ikeda T. Matsuura F. Fukasawa K.M. Fukasawa K. Biochemistry. 2001; 40: 11860-11865Crossref PubMed Scopus (28) Google Scholar). All known DPP III sequences contain the unique motif HEXXGH, which enabled the recognition of the dipeptidyl-peptidase III family as a distinct evolutionary metallopeptidase family (family M49, MEROPS, the Peptidase Database (1Rawlings N.D. Morton F.R. Kok C.Y. Kong J. Barrett A.J. Nucleic Acids Res. 2008; 36: D320-D325Crossref PubMed Scopus (519) Google Scholar), www.merops.ac.uk). Mutagenesis data on the HEXXGH motif in the rat enzyme imply that the histidine residues coordinate the metal ion and that the glutamic acid acts as a general base in peptide hydrolysis (24Fukasawa K. Fukasawa K.M. Iwamoto H. Hirose J. Harada M. Biochemistry. 1999; 38: 8299-8303Crossref PubMed Scopus (53) Google Scholar). A second conserved linear motif within the DPP III family, EEXR(K)AE(D), is found 22-55 amino acids toward the C terminus from the first one (6Abramić M. Špoljarić J. Šimaga Š. Period. Biol. 2004; 106: 161-168Google Scholar, 7Abramić M. Šimaga Š. Osmak M. Čičin-Šain L. Vukelić B. Vlahoviček K. Dolovcak L. Int. J. Biochem. Cell Biol. 2004; 36: 434-446Crossref PubMed Scopus (37) Google Scholar), and the involvement of the second glutamic acid residue in the coordination of the active-site zinc was shown (24Fukasawa K. Fukasawa K.M. Iwamoto H. Hirose J. Harada M. Biochemistry. 1999; 38: 8299-8303Crossref PubMed Scopus (53) Google Scholar). Apart from that, little is known about the molecular mechanism of action of this hydrolase because no experimental three-dimensional structure of DPP III enzymes is available. Homology modeling was not possible because of the low sequence identity to proteins with known structure, already indicating a novel fold. Because of the more facile accessibility, we chose the ortholog from Saccharomyces cerevisiae, sharing ∼40% sequence identity with mammalian DPP III, for our structural studies. The enzyme was cloned and expressed in Escherichia coli, and we present here its high resolution crystal structure and discuss the implications of this first DPP III structure for the catalytic mechanism and substrate recognition. Cloning of Yeast DPP III for Large-scale Expression in E. coli—The open reading frame YOL057W encoding yeast DPP III was amplified by PCR using genomic DNA isolated from S. cerevisiae (BY4741). The PCR product was inserted into the NdeI/XhoI restriction sites of pET21a (Novagen). Cloning into this vector using NdeI/XhoI and deletion of the stop codon allow expression of the protein with a hexahistidine affinity tag at the C terminus. Cysteine-to-serine point mutations in yeast DPP III were produced using the QuikChange XL site-directed mutagenesis kit (Stratagene) with pET21a-DPP III-His6 as template. Expression and Purification of S. cerevisiae DPP III-His6—A single colony of E. coli BL21-CodonPlus(DE3)-RIL was grown overnight in 5 ml of Luria broth supplemented with 100 μg/ml ampicillin and 36 μg/ml chloramphenicol, which was then used to inoculate a 500-ml culture. After 3 h at 37 °C, expression was induced by the addition of isopropyl 1-thio-β-d-galactopyranoside to a final concentration of 0.3 mm. Cells were allowed to grow overnight at 20 °C, harvested by centrifugation, and subsequently stored at -70 °C. For protein purification, 4 liters of E. coli cells expressing DPP III-His6 were grown in parallel, and the resulting bacterial paste was resuspended in 50 mm sodium phosphate (pH 8.0) containing 300 mm sodium chloride and 10 mm imidazole (buffer A) as the resuspension buffer. The cells were lysed by five cycles of sonication, 1 min each. Cell debris were removed by centrifugation at 25,000 × g for 20 min at 4 °C, and the supernatant was subjected to affinity chromatography on nickel-nitrilotriacetic acid resin essentially according to protocol number 11 provided by the supplier (Qiagen). In particular, buffer A containing 20 mm imidazole was used to wash the column, and the bound protein was eluted in buffer A containing 150 mm imidazole. The progress of DPP III-His6 purification was monitored by 12.5% SDS-PAGE. All fractions of high purity and an appropriate amount of enzyme (according to SDS-PAGE) were pooled and concentrated by centrifugation with the Amicon CentriPrep system (molecular mass cutoff of 10 kDa). Further analysis of recombinant wild-type DPP III by size-exclusion chromatography indicated that the protein exists in a mono- and dimeric form. The latter form could be resolved into monomers using dithiothreitol, suggesting formation of intermolecular disulfide bonds between monomers. Crystallization trials with wild-type DPP III were unsuccessful and prompted us to elucidate the nature of the dimerization process by generating five single cysteine-to-serine mutant proteins. This led to the identification of Cys-130 as the cause for dimerization. A biochemical characterization of these five single mutant proteins will be presented elsewhere. 3N. Jajčanin-Jozić, S. Deller, T. Pavkov, P. Macheroux, and M. Abramić, manuscript in preparation. Here, we have continued our structural work with the C130S mutant protein, which exhibited comparable enzymatic activity to the wild-type protein. Selenomethionine-labeled DPP III was expressed from the met- E. coli strain B834(DE3). The purification of the selenomethionine-containing DPP III followed the same protocol as for the recombinant C130S mutant protein. X-ray Crystallography—For crystallization, the C130S mutant of DPP III from S. cerevisiae was concentrated to 18 mg/ml (100 mm Tris-HCl (pH 7) and 100 mm NaCl). Initial conditions were identified from the Hampton Index Screen using the microbatch technique. After optimization, diffraction quality plate-like crystals (with a maximum dimension of 0.3 mm) were grown using sitting drop vapor diffusion in 20% (w/v) polyethylene glycol 3350 and 900 mm MgCl2 in 100 mm Tris-HCl (pH 7.0). The crystals were monoclinic, space group P21, with one DPP III molecule in the asymmetric unit. Crystals of selenomethionine-labeled protein were grown under the same conditions. In parallel, we tried to obtain "classical" heavy atom derivatives. Native PAGE shift experiments (25Boggon T.J. Shapiro L. Structure. 2000; 8: R143-R149Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) indicated that mercury and osmium compounds specifically bound to DPP III. On the basis of these results, we succeeded in preparing a mercury derivative by soaking the crystals for 2 h in a solution containing mercury p-hydroxybenzoate. All data sets were collected from flash-cooled crystals at 100 K without the use of any additional cryoprotectant. The structure was solved by a combination of MAD and MIR. Selenomethionine MAD data were collected at beamline X12 at the Deutsches Elektronen-Synchrotron/European Molecular Biology Laboratory Hamburg (λpeak = 0.9779 Å, λinfl = 0.9787 Å, λrem = 0.9180 Å). The mercury derivative data set was collected at the XRD beamline at Elettra Trieste (λ = 0.8298 Å), and the high resolution native data set was obtained at beamline ID23 at the European Synchrotron Radiation Facility in Grenoble (λ = 0.9537 Å). The data sets were processed using DENZO/SCALEPACK (26Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38609) Google Scholar) and MOSFLM (27Powell H.R. Acta Crystallogr. Sect. D. 1999; 55: 1690-1695Crossref PubMed Scopus (308) Google Scholar). Heavy atom sites (13 selenium and 1 mercury) were found independently with SOLVE/RESOLVE (28Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D. 1999; 55: 849-861Crossref PubMed Scopus (3219) Google Scholar) as well as SHELXD (29Schneider T.R. Sheldrick G.M. Acta Crystallogr. Sect. D. 2002; 58: 1772-1779Crossref PubMed Scopus (1580) Google Scholar). Phases (overall figure of merit of 0.65) were calculated and extended to 1.95-Å resolution using PHENIX (30Adams P.D. Grosse-Kunstleve R.W. Hung L.W. Ioerger T.R. McCoy A.J. Moriarty N.W. Read R.J. Sacchettini J.C. Sauter N.K. Terwilliger T.C. Acta Crystallogr. Sect. D. 2002; 58: 1948-1954Crossref PubMed Scopus (3664) Google Scholar). A first model was built using PHENIX and ARP/wARP (31Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2563) Google Scholar), yielding ∼600 of the 711 amino acids. The starting model was completed and refined against the high resolution data set using PHENIX and COOT (32Emsley P. Cowtan K. Acta Crystallogr. Sect. D. 2004; 60: 2126-2132Crossref PubMed Scopus (23605) Google Scholar). Rfree values (33Kleywegt G.J. Brunger A.T. Structure. 1996; 4: 897-904Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) were computed from 5% randomly chosen reflections not used for the refinement. In the later stages of refinement, a TLS analysis was performed (34Painter J. Merritt E.A. J. Appl. Crystallogr. 2006; 39: 109-111Crossref Scopus (650) Google Scholar), and four TLS groups were defined (residues 2-310, 311-432, 433-672 plus a zinc ion, and 673-711) for refinement in PHENIX. The first refinement cycle including TLS led to a reduction in both R and Rfree of ∼2%. The final structure contains amino acid residues 2-711, one zinc ion, two magnesium ions, and 450 water molecules, resulting in R = 0.1923 and Rfree = 0.2283. Three residues (Ser-37, His-578, and Ser-680) were modeled with alternative conformations. The stereochemistry of the structure was checked using PROCHECK (35Morris A.L. MacArthur M.W. Hutchinson E.G. Thornton J.M. Proteins. 1992; 12: 345-364Crossref PubMed Scopus (1419) Google Scholar), showing 99.7% of the residues in the core and allowed regions and none in the disallowed regions of the Ramachandran plot (supplemental Fig. 5). Details of the data collection, processing, and structure refinement are summarized in Table 1. The coordinates and structure factors (code 3csk) have been deposited in the Protein Data Bank.TABLE 1Data collection, phasing, and refinement statisticsNativeSeMetHgData collectionSpace groupP21Cell dimensionsa, b, c (Å)60.62, 110.12, 67.9060.62, 110.21, 67.7359.35, 107.83, 67.78α, β, γ90°, 113.4°, 90°90°, 113.4°, 90°90°, 114.2°, 90°PeakInflectionRemoteWavelength (Å)0.95370.97790.97870.91800.8298Resolution (Å)30-1.95 (2.06-1.95)aValues in parentheses are for the highest resolution shell.25-2.20 (2.25-2.20)aValues in parentheses are for the highest resolution shell.30-2.35 (2.48-2.35)aValues in parentheses are for the highest resolution shell.Rsym0.081 (0.414)0.083 (0.283)0.085 (0.324)0.091 (0.437)0.098 (0.714)I/σI11.5 (2.5)41.5 (6.4)38.8 (4.7)35.2 (3.4)14.4 (2.1)Completeness (%)95.5 (99.5)98.0 (80.1)97.6 (75.7)98.6 (83.1)98.1 (87.0)Redundancy2.9 (2.9)7.0 (4.9)6.9 (4.0)6.8 (4.5)3.7 (2.9)RefinementResolution (Å)30-1.95No. reflections57,149Rwork/Rfree0.1923/0.2283No. atomsProtein5703Ligand/ion3Water450B-factorsProtein47.1Ligand/ion49.2Water47.0r.m.s.d.Bond length (Å)0.015Bond angle0.578°a Values in parentheses are for the highest resolution shell. Open table in a new tab Overview of the DPP III Structure—We determined the crystal structure of the C130S mutant of DPP III from S. cerevisiae at 1.95-Å resolution by a combination of selenomethionine MAD and MIR. (For a representative portion of the electron density map, see supplemental Fig. 1.) This specific variant was chosen because the mutation gave rise to homogeneous monomeric protein samples compared with the monomer/dimer mixtures usually obtained for the native enzyme. 3N. Jajčanin-Jozić, S. Deller, T. Pavkov, P. Macheroux, and M. Abramić, manuscript in preparation. The mutation site turned out to be on the surface of the protein and far away (56 Å) from the metal center. Not surprisingly, this particular amino acid exchange also did not significantly alter the catalytic activity of the enzyme. DPP III is an elongated molecule with overall dimensions of 75 × 60 × 50 Å. Two lobes can be identified that are separated by a cleft, ∼40 Å wide and 25 Å high. This cleft divides the whole enzyme into two domains: an upper mostly helical domain (residues 429-670) and a lower domain with mixed α- and β-secondary structures (Fig. 1). The lower domain has an extension (residues 346-383) that forms an α-helix followed by a long loop and embraces the upper domain. A five-stranded β-structure forms the core of the lower domain (β7, β8, β9, β10, and β11) (magenta in Fig. 1; see supplemental Fig. 2 for the numbering of secondary structure elements). On one side, this structure is flanked by five helices (α1, α8, α9, α10, and α12), whereas the other side is accessible and forms part of the "floor" of the cleft opposite to the catalytic metal center (Fig. 1). The C-terminal tail of the protein (residues 671-711) folds back and interacts with the lower domain mainly by forming a short, parallel, two-stranded β-sheet with residues 9-18 and by contacts of the C-terminal α-helix. The catalytic zinc ion is bound to the upper domain at the "roof" of the cleft. An extensive search for similar folds was carried out using MSDssm (36Krissinel E. Hentick K. Acta Crystallogr. Sect. D. 2004; 60: 2256-2268Crossref PubMed Scopus (3188) Google Scholar), DALI (37Holm L. Sander C. Trends Biochem. Sci. 1995; 20: 478-480Abstract Full Text PDF PubMed Scopus (1291) Google Scholar), VAST (38Shindyalov I.N. Bourne P.E. Protein Eng. 1998; 11: 739-747Crossref PubMed Scopus (1702) Google Scholar), and DEJAVU (39Kleywegt G.J. Jones A.T. Methods Enzymol. 1997; 277: 525-545Crossref PubMed Scopus (303) Google Scholar) but did not yield any significant matches. Zinc Binding—The zinc-binding site is part of the upper domain and is built up by His-460, His-465, and Glu-517, which coordinate the metal ion through the N-ϵ atoms of the imidazole rings and the carboxylate group, respectively (Fig. 2A). The two His ligands belong to the conserved HEXXGH signature motif (24Fukasawa K. Fukasawa K.M. Iwamoto H. Hirose J. Harada M. Biochemistry. 1999; 38: 8299-8303Crossref PubMed Scopus (53) Google Scholar) of the M49 family of metallopeptidases (1Rawlings N.D. Morton F.R. Kok C.Y. Kong J. Barrett A.J. Nucleic Acids Res. 2008; 36: D320-D325Crossref PubMed Scopus (519) Google Scholar), which is part of helix α14 (Fig. 1 and supplemental Fig. 2). The third ligand (Glu-517) is part of the second conserved sequence motif of the DPP III family (516EECRAE521) (6Abramić M. Špoljarić J. Šimaga Š. Period. Biol. 2004; 106: 161-168Google Scholar, 7Abramić M. Šimaga Š. Osmak M. Čičin-Šain L. Vukelić B. Vlahoviček K. Dolovcak L. Int. J. Biochem. Cell Biol. 2004; 36: 434-446Crossref PubMed Scopus (37) Google Scholar) and is situated on helix α16. The two other glutamate residues in this motif (Glu-516 and Glu-521) are hydrogen-bonded to the zinc-coordinating histidine residues (His-465 and His-460, respectively). A water molecule completes the tetrahedral coordination of the zinc ion. This water molecule is also hydrogen-bonded to Glu-461 (Fig. 2B). Although the DPP III structure represents a novel fold, the mode of zinc binding closely resembles that observed in other structurally unrelated metallopeptidases, such as neprilysin (40Oefner C. D'Arcy A. Hennig M. Winkler F.K. Dale G.E. J. Mol. Biol. 2000; 296: 341-349Crossref PubMed Scopus (243) Google Scholar) and thermolysin (41Matthews B.W. Acc. Chem. Res. 1988; 21: 333-340Crossref Scopus (673) Google Scholar) (supplemental Fig. 3). These two enzymes also coordinate the metal ion through two histidines and one glutamate, and a structural superposition of the zinc-binding residues reveals a very similar coordination geometry (Fig. 2C). In all three cases, the two histidine residues are part of a conserved motif, which, in the case of DPP III and its homologs, contains an additional amino acid residue (HEXXGH compared with the conventional HEXXH motif). In neprilysin and thermolysin, this metal-binding motif is part of a helical structure in which the terminal histidine residues are located on the same side of the helix. Despite the insertion of an additional residue, an equivalent coordination geometry is feasible in DPP III because of a slight widening of the last turn of the helix (Fig. 2D and supplemental Fig. 4) and a concomitant distortion of the helical hydrogen bonding pattern (i to i+5 instead of i to i+4). In line with this observation is experimental evidence that the deletion of one leucine residue from the HELLGH motif in rat DPP III did not completely abolish its activity (24Fukasawa K. Fukasawa K.M. Iwamoto H. Hirose J. Harada M. Biochemistry. 1999; 38: 8299-8303Crossref PubMed Scopus (53) Google Scholar), indicating that this truncated motif is still able to build up a functional zinc coordination site. Active-site Cleft—The central cleft region extending on both sides of the zinc ion is the most plausible area for substrate binding. This region is quite open and appears to be easily accessible to peptide substrates. The roof part of the cleft is formed by the helical structure of the upper domain, whereas the floor is formed mainly by the central five-stranded β-sheet. Helix α13 (connecting the two domains) forms the backside of the cleft. On the left side, the cleft is confined by the C-terminal tail going from the upper to the lower domain, whereas it opens up toward the right side of the cleft, which is formed by the loop region of the arm-like extension of the lower domain (Fig. 1). A TLS analysis (using four rigid groups) indicated considerable flexibility of the protein and identified libration modes of the upper and lower domains, which lead to an opening and closing of the substrate-binding cleft. A sequence alignment of eukaryotic DPP III enzymes reveals a high sequence identity between human, rat, and mouse DPP III (>90% identity), whereas these enzymes share only ∼40% sequence identity with yeast DPP III. Previously, five conserved regions were identified in such alignments (supplemental Fig. 2) (6Abramić M. Špoljarić J. Šimaga Š. Period. Biol. 2004; 106: 161-168Google Scholar, 7Abramić M. Šimaga Š. Osmak M. Čičin-Šain L. Vukelić B. Vlahoviček K. Dolovcak L. Int. J. Biochem. Cell Biol. 2004; 36: 434-446Crossref PubMed Scopus (37) Google Scholar). In our structure, two of these five regions involve zinc-binding residues, whereas the others are located in close proximity to this site (supplemental Fig. 2). In addition, we performed an AMAS analysis (42Livingstone C.D. Barton G.J. Comput. Appl. Biosci. 1993; 9: 745-756PubMed Google Scholar) on a sequence alignment of DPP III enzymes from human, rat, mouse, and yeast and calculated a conservation score that takes into account physicochemical properties of amino acid side chains. This score was then mapped onto the molecular surface of yeast DPP III, clearly indicating a high degree of conservation among eukaryotic DPP III enzymes especially around the zinc-binding site and the proposed active-site cleft (Fig. 3A). Specific conserved residues in the vicinity of the metal center include Glu-461, His-578, and Arg-582. Structurally equivalent residues are also found in the active sites of thermolysin (Glu-143, His-231, and Arg-203) and neprilysin (Glu-584, His-711, and Arg-717). In the latter enzymes, the glutamic acid residue has been shown to act as a general base in the deprotonation of the water molecule that attacks the scissile amide bond. The role of the histidine is to stabilize the tetrahedral intermediate, whereas the arginine residue binds to the P1′ carbonyl oxygen (41Matthews B.W. Acc. Chem. Res. 1988; 21: 333-340Crossref Scopus (673) Google Scholar). A superposition of all three enzyme structures (based on the zinc-binding residues, His-His-Glu) reveals a very similar arrangement of these important residues (Fig. 2C). This close structural resemblance with thermolysin and neprilysin and the full conservation of these residues in DPP III enzymes indicate similarly important roles in catalysis. Predicted Mode of Substrate Binding—As yet, no structural information on DPP III-substrate complexes is available. We tried to identify possible modes of substrate binding based on comparisons with structures of inhibitor complexes of neprilysin and thermolysin. Specifically, we chose the complexes of thermolysin with phosphonamidate benzyloxycarbonyl-Phe-P-Leu-Ala (Protein Data Bank code 4tmn) and of neprilysin with N-α-l-rhamnopyranosyloxy(hydroxyphosphinyl)-l-leucyl-l
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