Structure of Human Cytosolic X-prolyl Aminopeptidase
2008; Elsevier BV; Volume: 283; Issue: 33 Linguagem: Inglês
10.1074/jbc.m710274200
ISSN1083-351X
AutoresXin Li, Zhiyong Lou, Xuemei Li, Weihong Zhou, Ming Ma, Youjia Cao, Yunqi Geng, Mark Bartlam, Xuejun C. Zhang, Zihe Rao,
Tópico(s)Neuropeptides and Animal Physiology
ResumoX-prolyl aminopeptidases catalyze the removal of a penultimate prolyl residue from the N termini of peptides. Mammalian X-prolyl aminopeptidases are shown to be responsible for the degradation of bradykinin, a blood pressure regulator peptide, and have been linked to myocardial infarction. The x-ray crystal structure of human cytosolic X-prolyl aminopeptidase (XPN-PEP1) was solved at a resolution of 1.6Å. The structure reveals a dimer with a unique three-domain organization in each subunit, rather than the two domains common to all other known structures of X-prolyl aminopeptidase and prolidases. The C-terminal catalytic domain of XPNPEP1 coordinates two metal ions and shares a similar fold with other prolyl aminopeptidases. Metal content analysis and activity assays confirm that the enzyme is double Mn(II) dependent for its activity, which contrasts with the previous notion that each XPNPEP1 subunit contains only one Mn(II) ion. Activity assays on an E41A mutant demonstrate that the acidic residue, which was considered as a stabilizing factor in the protonation of catalytic residue His498, plays only a marginal role in catalysis. Further mutagenesis reveals the significance of the N-terminal domain and dimerization for the activity of XPNPEP1, and we provide putative structural explanations for their functional roles. Structural comparisons further suggest mechanisms for substrate selectivity in different X-prolyl peptidases. X-prolyl aminopeptidases catalyze the removal of a penultimate prolyl residue from the N termini of peptides. Mammalian X-prolyl aminopeptidases are shown to be responsible for the degradation of bradykinin, a blood pressure regulator peptide, and have been linked to myocardial infarction. The x-ray crystal structure of human cytosolic X-prolyl aminopeptidase (XPN-PEP1) was solved at a resolution of 1.6Å. The structure reveals a dimer with a unique three-domain organization in each subunit, rather than the two domains common to all other known structures of X-prolyl aminopeptidase and prolidases. The C-terminal catalytic domain of XPNPEP1 coordinates two metal ions and shares a similar fold with other prolyl aminopeptidases. Metal content analysis and activity assays confirm that the enzyme is double Mn(II) dependent for its activity, which contrasts with the previous notion that each XPNPEP1 subunit contains only one Mn(II) ion. Activity assays on an E41A mutant demonstrate that the acidic residue, which was considered as a stabilizing factor in the protonation of catalytic residue His498, plays only a marginal role in catalysis. Further mutagenesis reveals the significance of the N-terminal domain and dimerization for the activity of XPNPEP1, and we provide putative structural explanations for their functional roles. Structural comparisons further suggest mechanisms for substrate selectivity in different X-prolyl peptidases. X-prolyl aminopeptidases (aminopeptidase P or AP-P; E.C. 3.4.11.9) are found in a variety of organisms including mammals, yeasts, and bacteria. There are two forms of mammalian AP-P in terms of their cellular locations: a cytosolic form (XPNPEP1) and a membrane-bound form (XPNPEP2). Both forms can degrade bradykinin, a blood pressure-regulating peptide, and are inhibited by the specific peptide inhibitor apstatin (1Prechel M.M. Orawski A.T. Maggiora L.L. Simmons W.H. J. Pharmacol. Exp. Ther. 1995; 275: 1136-1142PubMed Google Scholar, 2Lloyd G.S. Hryszko J. Hooper N.M. Turner A.J. Biochem. Pharmacol. 1996; 52: 229-236Crossref PubMed Scopus (23) Google Scholar, 3Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar). The cytosolic form of AP-P has been identified in human leukocytes (4Rusu I. Yaron A. Eur. J. Biochem. 1992; 210: 93-100Crossref PubMed Scopus (51) Google Scholar), platelets (5Vanhoof G. De Meester I. Goossens F. Hendriks D. Scharpe S. Yaron A. Biochem. Pharmacol. 1992; 44: 479-487Crossref PubMed Scopus (35) Google Scholar), and rat and guinea pig brains (6Harbeck H.T. Mentlein R. Eur. J. Biochem. 1991; 198: 451-458Crossref PubMed Scopus (92) Google Scholar, 7Gilmartin L. O’Cuinn G. Neurosci. Res. 1999; 34: 1-11Crossref PubMed Scopus (14) Google Scholar). The membrane-bound form, first purified from porcine kidney (8Dehm P. Nordwig A. Eur. J. Biochem. 1970; 17: 372-377Crossref PubMed Scopus (22) Google Scholar) and later purified from bovine and rat lungs (1Prechel M.M. Orawski A.T. Maggiora L.L. Simmons W.H. J. Pharmacol. Exp. Ther. 1995; 275: 1136-1142PubMed Google Scholar, 9Simmons W.H. Orawski A.T. J. Biol. Chem. 1992; 267: 4897-4903Abstract Full Text PDF PubMed Google Scholar), is attached to the lipid bilayer through a glycosylphosphatidylinositol anchor (10Hooper N.M. Hryszko J. Turner A.J. Biochem. J. 1990; 267: 509-515Crossref PubMed Scopus (87) Google Scholar). A previous study has shown that injection of apstatin into mice can reduce myocardial infarction severity (11Wolfrum S. Richardt G. Dominiak P. Katus H.A. Dendorfer A. Br. J. Pharmacol. 2001; 134: 370-374Crossref PubMed Scopus (34) Google Scholar), suggesting a pathological role of X-prolyl aminopeptidases in mammals. Human XPNPEP1 consists of 623 amino acid residues with a calculated molecular mass of 69,886 Da. The enzyme purified from human leukocytes exists as a dimer of 140 kDa (4Rusu I. Yaron A. Eur. J. Biochem. 1992; 210: 93-100Crossref PubMed Scopus (51) Google Scholar). The catalytic activity of the enzyme is enhanced in the presence of Mn2+ (3Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar). Each 70-kDa subunit of the enzyme was thought to contain only one Mn2+ ion in a previous study (3Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar). A Blast search against the NCBI sequence data base revealed similarity in the C-terminal catalytic domains among X-prolyl peptidases of known structures. In contrast, the overall sequence homology between human XPNPEP1 and other X-prolyl peptidases is low. In particular, XPNPEP1 is significantly larger in size than other members of the X-prolyl peptidases (about 40-50 kDa). Here we report the crystal structure of human XPNPEP1 at 1.6-Å resolution. Whereas other X-prolyl peptidases with known structures all contain two domains, the XPNPEP1 structure possesses a novel three-domain organization with a conserved C-terminal catalytic domain. In contrast to previous reports, we identified the presence of a double Mn2+ binding site in the catalytic domain, both in the crystal structure and in solution. Cloning and Expression—The cDNA of wild type (WT) XPN-PEP1 and that of a domain I-truncated mutant (residues 162-623) were cloned into the pET28a vector (Novagen) between SalI and HindIII sites. E41A (i.e. Glu41 to Ala substitution) and W477E point mutants were produced from the constructed pET28a-wild type XPNPEP1 plasmid with a one-step overlap extension PCR method by using the Easy Mutagenesis System kit (Transgen). All XPNPEP1 variants were expressed as an N-terminal His6-tagged protein in Escherichia coli BL21(DE3) in LB medium supplemented with 1 mm MnCl2 (manganese-rich LB). Selenomethionine-substituted WT 3The abbreviations used are:WTwild typeICP-MSinductively coupled plasma mass spectrometryAUCanalytical ultracentrifugation. protein was expressed in a metE- E. coli host strain B834 (Novagen) in the M9 minimal medium supplemented with 50 mg of selenomethionine per liter and 1 mm MnCl2. wild type inductively coupled plasma mass spectrometry analytical ultracentrifugation. For expression, E. coli cells cultured overnight were diluted 100-fold in fresh medium and cultured at 37 °C to an optical density of about 0.8 at 600 nm. The cell culture was then cooled down to 16 °C and induced with 0.5 mm isopropyl β-d-1-thiogalactopyranoside. It was grown for another 20 h at 16 °C with shaking at 220 rpm, and then the cells were harvested by centrifugation. Protein Purification—The harvested cells were resuspended in buffer A (20 mm Tris-HCl (pH 7.9), 500 mm NaCl, and 10% (v/v) glycerol) and lysed by sonication. The released His6-tagged protein was purified following standard protocols of nickel-nitrilotriacetic acid resin (Qiagen). It was eluted from the resin with buffer B (20 mm Tris-HCl (pH 7.9), 500 mm NaCl, 10% (v/v) glycerol, and 300 mm imidazole) and dialyzed against a salt-free buffer (20 mm Tris-HCl (pH 8.0)). Further purification was performed with a Hitrap Q HP affinity column (Amersham Biosciences) and the final protein sample was dialyzed against a buffer of 20 mm Tris-HCl (pH 8.0) and 20 mm NaCl. Crystallization—Crystals of native or selenomethionyl-labeled protein were grown by the hanging-drop vapor-diffusion method. The reservoir contained 20% (v/v) polyethylene glycol (PEG) 400, 0.15 m CaCl2, and 100 mm HEPES (pH 7.5). A typical hanging drop consisted of 2 μl of protein solution (20 mg/ml) mixed with 2 μl of the reservoir solution. Large (over 0.5 mm) colorless block-shaped crystals suitable for diffraction were grown within a week at 16 °C. Data Collection, Phasing, and Model Refinement—Crystallographic data from the crystals of native and selenomethionyllabeled protein were collected on beamlines BL5A and BL17A of the Photon Factory synchrotron facility (KEK, Tsukuba, Japan). The diffraction images were integrated and scaled using HKL2000 (12Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). A 3.5-Å resolution structure of selenomethionyl-labeled XPNPEP1 was solved by the multiwavelength anomalous diffraction phasing method at the selenium absorption edge using SOLVE/RESOLVE (13Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). The 3.5-Å resolution phases were extended to 1.6 Å using the native data set and the program ARP/WARP (14Blanc E. Roversi P. Vonrhein C. Flensburg C. Lea S.M. Bricogne G. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2210-2221Crossref PubMed Scopus (604) Google Scholar). The program automatically built about 80% of residues, and the remainder were built manually with COOT (15Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar). The structure was refined with REFMAC5 (16Number Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). Figures were drawn with the program PYMOL (DeLano Scientific, San Carlos, CA). Enzyme Activity Assays—The enzyme activity was assayed with bradykinin (Phoenix Pharmaceuticals, Inc.) or the Arg-Pro-Pro tripeptide (synthesized by SBS Genetech, Beijing, China) as the substrate in 100 mm Tris-HCl (pH 8.0) and 100 mm NaCl at 37 °C for 5 min (100 μl final volume). The free amino acid released by the enzyme was detected with the o-phthalaldehyde, 5-mercaptoethanol reagent as previously described (9Simmons W.H. Orawski A.T. J. Biol. Chem. 1992; 267: 4897-4903Abstract Full Text PDF PubMed Google Scholar). Fluorescence of the o-phthalaldehyde derivate was measured with a microplate reader (Type 374, Thermo Electron Corporation). For kinetic analysis, assays were prepared with a range of concentrations of bradykinin (0.01-0.10 mm) or Arg-Pro-Pro (0.02-0.20 mm) and 1 μg of the purified WT enzyme. To examine the effects of different factors on the enzyme activity, 0.09 mm Arg-Pro-Pro and 1 μg of purified enzyme variant were used in a 100-μl assay. To verify the effect of EDTA, purified WT enzyme was incubated in 100 mm Tris-HCl (pH 8.0) and 100 mm NaCl with 50 mm EDTA for 10 min, and dialyzed against 100 mm Tris-HCl (pH 8.0) and 100 mm NaCl prior to measuring its relative activity. Other Assays—Analysis of the total metal content was carried out using inductively coupled plasma mass spectrometry (ICP-MS, Thermo) at the Tsinghua University Analysis Center (Beijing, China). Purified protein samples without crystallization trial were extensively dialyzed against 100 mm Tris-HCl (pH 8.0) and 100 mm NaCl before ICP-MS analysis. Analytical ultracentrifugations (AUC) were performed with the sedimentation velocity method at 58,000 rpm at the Institute of Biophysics, Chinese Academy of Sciences (Beijing, China). The protein samples for AUC were prepared at a concentration of about 0.5 mg/ml in a buffer of 100 mm Tris-HCl (pH 8.0) and 100 mm NaCl. The AUC data were processed as a c(M) distribution model (17Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3089) Google Scholar). Structure Determination and Refinement—The structure of human XPNPEP1 was solved at 1.6-Å resolution using the multiwavelength anomalous diffraction method. Refinement of the XPNPEP1 structure resulted in a final model with a crystallographic R-factor (Rcryst) of 0.154 and a free R-factor (Rfree) of 0.195. One asymmetric unit of this C2221 crystal form contained a single protein molecule composed of 623 amino acid residues. The polypeptide chain was complete with the exception of the N-terminal His6 tag, the N-terminal residues 1-2, and the C-terminal residues 620-623, which were not included in the final model because of poor electron density. For the same reason, Asn553, Arg554, and the side chain of Phe509 were assigned zero occupancy. All other residues had excellent electron density, and the final average temperature factor (B) was 27.2 Å2. An asymmetric unit also contained 1,055 ordered water molecules, one partial PEG molecule containing six ethylene glycol residues, and four metal ions. Of the four metal ions, two major ones localized at the active site were refined as full occupancy Mn2+ with temperature factors of 14.8 Å2 (Mn1 in Fig. 1A) and 17.7 Å2 (Mn2), respectively. The two minor ions were refined as full occupancy Ca2+ and Na+, respectively. Although crystals were grown in 0.15 m CaCl2, native XPNPEP1 treated with EDTA (followed by dialysis before crystallization) yielded no crystal but thick precipitation under the same condition, suggesting that the two active-site metal ions observed in the native crystal protein were unlikely to be calcium ions replacing Mn2+ during crystallization. Besides the mobile Asn553, only Glu434, affected by the inter-molecule Ca2+, localizes in the Ramachandran unfavorable region. Experimental structure factors and the coordinates of the refined model have been deposited in the Protein Data Bank (PDB) with access code 3CTZ. Crystallographic statistics are summarized in Table 1.TABLE 1Crystallographic data and refinement statisticsStatisticNativePeakEdgeRemoteData collectionSpace groupC2221Unit-cell parameters (Å)a = 71.4b = 131.4c = 169.1Wavelength (Å)1.00000.97920.97940.9600Resolution range (Å)19.6-1.620-3.520-3.520-3.5Measured reflections741,39475,36675,17274,950Unique reflections104,75310,42410,42310,417Completeness (%)99.0 (95.8)100 (100)100 (100)100 (100)RmergeaRmerge = ∑h∑i|Ii(h) - |/∑h∑i , where Ii(h) is the intensity of an individual measurement of the reflection, and is the mean intensity of the reflection.0.039 (0.209)0.076 (0.098)0.077 (0.102)0.081 (0.113)Redundancy7.1 (6.4)7.2 (7.1)7.2 (7.1)7.2 (7.0)I/σ47.1 (6.7)32.3 (14.6)30.3 (13.3)28.4 (11.3)RefinementRcrystbRcryst = ∑(||Fobs| - |Fcalc||)/∑|Fobs|, where Fobs and Fcalc are the observed and calculated structure-factor amplitudes, respectively./No. of ref. used0.154/98,520RfreecRfree was calculated as Rcryst using the reflections in a test set not used for structure refinement, which is a randomly selected subset containing 5% of unique reflections./No. of ref. in test set0.196/5,155Number of ion atoms4Number of water molecules1,055Overall figure of merit0.88Root mean square deviationBond length (Å)0.011Bond angles (degrees)1.28Average B factor (A2)27.2Ramachandran plot (%)dCalculated using MolProbity. Numbers reflect the percentage of residues in the preferred, allowed, and disallowed regions, respectively.96.84/2.81/0.35a Rmerge = ∑h∑i|Ii(h) - |/∑h∑i , where Ii(h) is the intensity of an individual measurement of the reflection, and is the mean intensity of the reflection.b Rcryst = ∑(||Fobs| - |Fcalc||)/∑|Fobs|, where Fobs and Fcalc are the observed and calculated structure-factor amplitudes, respectively.c Rfree was calculated as Rcryst using the reflections in a test set not used for structure refinement, which is a randomly selected subset containing 5% of unique reflections.d Calculated using MolProbity. Numbers reflect the percentage of residues in the preferred, allowed, and disallowed regions, respectively. Open table in a new tab Monomer Structure—The crystal structure unveiled a novel three-domain subunit for XPNPEP1. It includes an N-terminal domain (domain I, residues 1-161), a middle domain (domain II, residues 162-322), and a C-terminal domain (domain III, residues 323-623) (Fig. 1A). Secondary structure elements in each domain were defined by the DSSP program (18Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (12421) Google Scholar) (supplemental materials Table S1). Domain I is composed of a six-stranded (strands β1-β6) mixed β-sheet flanked by six α-helices (helices α1-α6). The topological order of the β-sheet is β4-β3-β2-β1-β5-β6, where strand β2 points in the opposite direction from the rest. Four helices (α1, α2, α3, and α6) are localized on one side of the sheet, and the remaining two (α4 and α5) on the other. The structure of domain II is similar to that of domain I. The core of domain II is also made up of a six-stranded (strands β8 and β10-β14) mixed β-sheet flanked by six α-helices (helices α8-α13). In addition, domain II contains a small antiparallel β-sheet (β7 and β9) and a short helix (α7) outside the core. Fig. 2A shows the result of secondary-structure matching superposition (19Krissinel E. Henrick K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2256-2268Crossref PubMed Scopus (3189) Google Scholar) between domains I and II, with 118 residues aligned to give a root mean square deviation of 2.6 Å. The two domains are related by a pure rotation of 150°. Domain III contains one strongly curved five-stranded antiparallel β-sheet (sheet I, β16-β17-β20-β26-β25), and two additional antiparallel β-sheets (sheet II, β15-β18-β19, and sheet III, β23-β22-β24-β27-β28-β21). On the outer face of the three sheets lie six α-helices (α14-α19). Of these, helices α14-α17 are oriented roughly parallel to the strands in sheet I, and α18 and α19 are near-perpendicular to the former helices. Strands β27 and β28 form a short hairpin structure that protrudes from the core. Domains I and II are primarily held together by hydrophobic interactions. Domains II and III are linked by the residues between helix α13 and helix α14 (residues 321-323). Homodimer—Our solution studies, including gel filtration and AUC, revealed that XPNPEP1 proteins primarily exist as 140-kDa dimers (Fig. 3), which is consistent with previous reports (3Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar, 4Rusu I. Yaron A. Eur. J. Biochem. 1992; 210: 93-100Crossref PubMed Scopus (51) Google Scholar). In the crystal structure, two symmetry-related XPNPEP1 molecules (named as subunits A and B) are related by a dyad to form a homodimer (Fig. 1B). The two subunits are mainly held together by hydrophobic interactions (Fig. 1C). The side chains of Tyr439, Leu481, Leu484, and Tyr526 in subunit A and Tyr549, Phe551 in the β27-β28 hairpin of subunit B form one hydrophobic core. The symmetry equivalent hydrophobic residues form the second hydrophobic core of the dimer interface. Residue Pro460 and the side chains of Phe459, Leu468, Phe471, and Trp477 in subunit A, together with their symmetry equivalents, form a third hydrophobic core. In addition to these hydrophobic interactions, a salt bridge between Glu442A (i.e. Glu442 of subunit A) and Lys548B and their symmetric counterparts, together with two pairs of hydrogen bonds between Glu442A/B and Tyr549B/A, and between Leu467A/B and Ser470B/A, also help to stabilize the interaction between the two subunits. Approximately 1,600 Å2 (6%) of the solvent accessible surface area from each subunit is buried upon dimer formation. Among the above discussed residues, Trp477 plays a vital role for dimerization. Mutating this Trp to Glu abolished the capability of the enzyme to form a native dimer in our AUC studies (Fig. 3). Nevertheless, a small peak appeared at the position of 120 kDa in the AUC studies on the W477E mutant. We speculate that it represents a fraction of monomer or dimer with abnormal molecule shapes. Active Site—The putative active site is located in the inner (concave) surface of the curved β-sheets of domain III (Fig. 1A) on the basis of comparison with homologous structures, such as the structure of E. coli AP-P (20Wilce M.C. Bond C.S. Dixon N.E. Freeman H.C. Guss J.M. Lilley P.E. Wilce J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3472-3477Crossref PubMed Scopus (167) Google Scholar, 21Graham S.C. Maher M.J. Simmons W.H. Freeman H.C. Guss J.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 1770-1779Crossref PubMed Scopus (26) Google Scholar, 22Graham S.C. Lilley P.E. Lee M. Schaeffer P.M. Kralicek A.V. Dixon N.E. Guss J.M. Biochemistry. 2006; 45: 964-975Crossref PubMed Scopus (36) Google Scholar). Two well coordinated metal ions were observed in this active site (Fig. 4). ICP-MS data consistently indicated that the molar ratio between Mn2+ and the 70-kDa XPNPEP1 subunit was 1.79:1, whereas the content of other common metals was negligible (Table 2). Although our crystallized XPNPEP1 protein sample was expressed with manganese-rich LB media and appeared clear, we carried out similar expression of the protein in plain LB media (i.e. without manganese supplementation) and interestingly obtained some “red protein.” ICP-MS analysis on this red protein sample indicated that the protein contains mainly iron (0.73:1), manganese (0.70:1), and magnesium (0.41:1) ions (Table 2). Therefore, the molar ratio between the total metal ion content and the protein remained close to 2:1.TABLE 2Metal content of XPNPEP1 expressed from different mediaExpression conditionMolar ratio between metal and proteinaThe results are the mean of triplicate assays, and the amount of protein was regarded as 1. Standard deviations less than 0.0005 are not presented.Manganese-richMagnesiumCalciumManganeseIronLB mediabManganese-rich LB media was plain LB media with addition of MnCl2 salt, to a final concentration of Mn2+ 1 mm.0.012 ± 0.0020.017 ± 0.0041.791 ± 0.0060.019 ± 0.003CoCopperZincnTotalNDcND means no signal was detected.0.0020.0101.851 ± 0.015Plain LB mediaMagnesiumCalciumManganeseIron0.409 ± 0.0060.020 ± 0.0010.698 ± 0.0140.730 ± 0.011CobaltCopperZincnTotalND0.0170.040 ± 0.0011.914 ± 0.033a The results are the mean of triplicate assays, and the amount of protein was regarded as 1. Standard deviations less than 0.0005 are not presented.b Manganese-rich LB media was plain LB media with addition of MnCl2 salt, to a final concentration of Mn2+ 1 mm.c ND means no signal was detected. Open table in a new tab In our crystal structures, both Mn2+ ions are well coordinated. One of the Mn2+ ions (termed Mn1, see Fig. 4) is coordinated by the Oδ-1 atoms of Asp415 (2.15 Å) and Asp426 (2.14 Å), the Oϵ-1 atom of Glu537 (2.19 Å), and two water molecules (termed W1 and W2) with Mn2+-ligand distances of 2.23 and 2.27 Å, respectively. These Mn2+-ligands form an approximate trigonal-bipyramidal coordination geometry, with the Oδ-1 atoms of the two aspartate residues and W1 in the equatorial plane, and the Oϵ-1 atom of the glutamate residue and W2 on the axis. The coordination sphere of the second Mn2+ ion (termed Mn2) is comprised of the Oδ-2 atom of Asp426 (2.37 Å), the Oϵ-2 atoms of Glu523 (2.27 Å) and Glu537 (2.26 Å), Nϵ-2 atom of residue His489 (2.27 Å), and two water molecules (W1, 2.31 Å and W3, 2.17 Å), which complete a distorted octahedral coordination. Furthermore, W1 and the carboxylate groups of Asp426 and Glu537 act as bridges between the two Mn2+ ions. The side chains of His395, His485, His498, and Glu41 surrounding the two Mn2+ ions are likely to play roles in recognition and catalysis during the substrate hydrolysis, according to studies on the equivalent residues in the active site of E. coli AP-P (22Graham S.C. Lilley P.E. Lee M. Schaeffer P.M. Kralicek A.V. Dixon N.E. Guss J.M. Biochemistry. 2006; 45: 964-975Crossref PubMed Scopus (36) Google Scholar), which shares an almost identical active site with XPNPEP1. Activity Assay—Activity assays on the same enzyme protein sample used for the crystallization were performed with the tripeptide Arg-Pro-Pro and bradykinin as substrates. The assays were performed in Mn2+-free buffer and gave a Km value of 308 (±8) μm and a kcat of 7.7 s-1 on Arg-Pro-Pro, whereas Km was measured as 78 (±9) μm and kcat as 3.8 s-1 for bradykinin. Our Km and kcat values on bradykinin were comparable with previous reports (3Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar, 5Vanhoof G. De Meester I. Goossens F. Hendriks D. Scharpe S. Yaron A. Biochem. Pharmacol. 1992; 44: 479-487Crossref PubMed Scopus (35) Google Scholar). Meanwhile, the red enzyme (enzyme expressed from plain LB) showed 44% activity of the Mn2+-bound enzyme, and the activity of the enzyme dropped to 10% after treatment with 50 mm EDTA. Although not directly involved in metal binding, Glu41 is the only residue located outside of the catalytic domain but close to the active site in the three-dimensional structure (Fig. 4). To test its function in catalysis, we made a mutant substituting Glu41 with Ala. This mutant maintained 91% of the WT activity, suggesting that it only marginally affects the activity of the enzyme. In contrast, another mutant, W477E, designed to block dimer formation and a domain I truncation mutant showed only 6 and 2% of the WT activity, respectively (Fig. 5). XPNPEP1 Contains Two Mn2+ Ions in the Active Site—Prior to our structural study, Cottrell and colleagues (3Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar) had reported some significant work on the characterization of recombinant XPNPEP1. In particular, they assayed the effects of different metal ions and chelating agents on the activity of XPNPEP1, and identified the enzyme as Mn2+ dependent for its activity. It was based on this observation that we chose manganese-rich LB in our experiments to avoid Mn2+ depletion during protein expression. Nevertheless, their conclusion that each 70-kDa XPNPEP1 subunit contains only one metal ion was markedly different from the observation in our XPNPEP1 structure. The XPNPEP1 crystal structure displays two well coordinated metal ions with very strong electron density in each active site. The electron density map for the two metal ions can be observed even when contoured at the level of +20 standard deviations. To further investigate the metal content of the enzyme, an ICP-MS analysis on the XPNPEP1 recombinant protein expressed from manganese-rich LB was performed and revealed that the enzyme contains 1.79 Mn2+ ions per 70-kDa subunit and much lower levels of other metals. This result indicates the molar ratio between Mn2+ and the enzyme is nearly 2:1, which is entirely consistent with our structural observation. To study the effects of Mn2+ from the cell culture medium on the metal content of recombinant XPNPEP1, we expressed the enzyme in plain LB medium following the method of Cottrell and colleagues (3Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar). ICP-MS analysis of this sample revealed three major metal contents, i.e. magnesium, manganese, and iron ions in the enzyme with the molar ratio 0.41, 0.70, and 0.73 per 70-kDa subunit. Although the molar ratio between Mn2+ and protein dropped to a level comparable with the previous report (0.99:1), the molar ratio between the total metal content and protein remains nearly 2:1, which is consistent with our structural observation. The detailed differences in metal content between our study and that of Cottrell et al. (3Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar) may be due to different expression conditions, including temperature and induction time. For example, we used a “slow” expression method at 16 °C for 20 h. In contrast, their method was a quick one at 40 °C for 3 h. We believe that slow cell growth allows the recombinant protein to fold properly and to obtain the optimum metal ion content. Moreover, in our case, the difference in metal composition between XPNPEP1 samples expressed from plain and manganese-enriched LB medium was probably caused by the depletion of Mn2+ in the former, because the protein yields from both media were as high as 30 mg/liter of E. coli culture. The Mn2+ content of XPNPEP1 expressed from plain LB was 39% (0.70:1.79) of that from manganese-rich LB and is fairly close to their activity ratio (44%). This suggests that only the portion of XPNPEP1 in which two Mn2+ ions are coordinated possesses catalytic activity, whereas the remaining portion, which coordinates either two magnesium or iron ions, presents little or no catalytic activity. Here, we made an assumption that XPNPEP1 preferentially binds the same type of ions in the two metal-binding sites, based on avai
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