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Crystal Structure of Human Renal Dipeptidase Involved in β-Lactam Hydrolysis
doi:10.1016/S0022-2836(02)00632-0 available online at http://www.idealibrary.com on
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J. Mol. Biol. (2002) 321, 177–184
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Crystal Structure of Human Renal Dipeptidase Involved in b-Lactam Hydrolysis Yasushi Nitanai1*, Yoshinori Satow1, Hideki Adachi2 and Masafumi Tsujimoto2 1 Faculty of Pharmaceutical Sciences, University of Tokyo Hongo, Bunkyo-ku, Tokyo 113 Japan 2
Laboratory of Cellular Biochemistry, RIKEN (The Institute of Physical and Chemical Research) Wako-shi, Saitama 351-0198 Japan
Human renal dipeptidase is a membrane-bound glycoprotein hydrolyzing dipeptides and is involved in hydrolytic metabolism of penem and carbapenem b-lactam antibiotics. The crystal structures of the saccharidetrimmed enzyme are determined as unliganded and inhibitor-liganded forms. They are informative for designing new antibiotics that are not hydrolyzed by this enzyme. The active site in each of the (a/b)8 barrel subunits of the homodimeric molecule is composed of binuclear zinc ions bridged by the Glu125 side-chain located at the bottom of the barrel, and it faces toward the microvillar membrane of a kidney tubule. A dipeptidyl moiety of the therapeutically used cilastatin inhibitor is fully accommodated in the active-site pocket, which is small enough for precise recognition of dipeptide substrates. The barrel and active-site architectures utilizing catalytic metal ions exhibit unexpected similarities to those of the murine adenosine deaminase and the catalytic domain of the bacterial urease. q 2002 Published by Elsevier Science Ltd
*Corresponding author
Keywords: crystal structure; human renal dipeptidase; metabolism of b-lactam antibiotics; cilastatin complex; binuclear metal ions
Human renal dipeptidase (hrDP) (EC 3.4.13.19, membrane dipeptidase and microsomal dipeptidase are synonyms) is a membrane-bound glycoprotein involved in the hydrolysis of dipeptides, and shows activity only toward dipeptides.1,2 In the renal cortex, it is bound to microvillar membranes in the brush-border region of proximal tubules via phosphatidylinositol.3 Although the dipeptidases lack the His-Glu-X-X-His amino acid sequences identified commonly in zinc proteases,4 and has no homology to other metallopeptidase, zinc ions are essential to their hydrolytic functions. Previous mutagenesis studies indicated that Glu125,5 His152,6 His198,6 and His2197 are involved in catalysis. Furthermore, His152 was shown as playing the important role of substrate/ inhibitor binding.6 But, the motif consisting of the
Present address: Y. Nitanai, Laboratory for Structural Biochemistry, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki, Sayo, Hyogo 679-5148, Japan. Abbreviations used: hrDP, human renal dipeptidase; PTE, phosphotriesterase; PEG, polyethylene glycol. E-mail address of the corresponding author: [email protected]
active site and the catalytic mechanism have not been clear. Although general roles in the organism are still unknown, the dipeptidase is well-known as the sole metabolic enzyme for penem and carbapenem b-lactam antibiotics in mammals.8 – 10 It hydrolyzes S(substitute)-L -cysteinyl-glycine adducts such as L -cystinyl-bis-glycine and S-N-ethylmaleimide-L cysteinyl-glycine, and is responsible for conversion of leukotriene D4 to E4.11 These facts suggest that the enzyme plays important roles in the renal metabolism of glutathione, its conjugates, and some drugs. The antibiotics are presently prescribed in clinical practice with the dipeptidyl inhibitor cilastatin.8 The hrDP is a homodimer, each subunit consisting of a 369 amino acid residue peptide (42 kDa), which shows sequence identities of about 75% to mammalian enzymes,12 – 16 and a low value of 23% to the prokaryotic enzyme of Acinetobacter calcoaceticus.17 The subunit has four possible N-glycosylation sites and, consequently, is obtained as a highly glycosylated form of 63 kDa.1 Furthermore, it has a glycosylphosphatidylinositol anchor to microvillar membranes in the brush-border region of the proximal tubules of the renal cortex.1
0022-2836/02/$ - see front matter q 2002 Published by Elsevier Science Ltd
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Crystal Structure of Human Renal Dipeptidase
Figure 1. (legend opposite)
Crystal Structure of Human Renal Dipeptidase
In order to obtain diffraction-quality crystals, the human enzyme was expressed in the Pichia pastoris yeast, and its oligosaccharide moieties were trimmed off by treatment with endoglycosidase.18 Here, the structures of the unliganded native and cilastatin-liganded crystals are reported. The hrDP has an elongated ellipsoidal shape of ˚ £ 45 A ˚ £ 45 A ˚ (Figure 1(a) and (b)). The sub90 A units of (a/b)8 barrel architecture (Figure 1(c) and (d)) are related to each other by a non-crystallographic twofold axis. Their barrel axes are twisted about 408 around the long axis of the ellipsoid. The subunits are linked by a disulfide bond between Cys361 side-chains, and interfaced with each other by extensive molecular contacts located mainly at helices a2 and a3. Barrel helices and strands facing the dimer interface are long and in parallel with the barrel axis, and those located away from the interface are short and nearly perpendicular to the axis. Therefore, the barrel is highly distorted; for example, helix a3 proximal to the interface is tilted with respect to distal helix a7 by about 1108. The N-terminal ends of the barrel strands are capped by four helices, aa, a2a, ad, and ae, and the Cterminal ends face the membrane (Figure 1(d)). Ser369, which is modifiable with the glycosylphosphatidylinositol anchor, is located on the surface facing the membrane near the twofold axis. The active site, also located on this surface, is suitable for receiving substrates transported from the membrane. The loop regions downstream of barrel strands b2 and b4 are connected by a disulfide bond between Cys71 and Cys154, and those of b6 and b7 are connected by a disulfide bond between Cys226 and Cys258. Those disulfide bonds correspond precisely to the result of previous mutagenesis studies of cysteine residues.19 It was shown that the Cys361 alone was involved in disulfidelinked dimerization among six cysteine residues, and the others made two disulfide bonds within a subunit. Two of the four possible glycosylation sites, Asn41 and Asn316, show electron densities corresponding to the N-acetylglucosamine structure. These N-glycosylation sites are located on the flanking side of the barrel. Viewed toward the C-terminal ends of the barrel strands, the active site appears as a rectangular ˚ long, 6 A ˚ wide, and 6 A ˚ deep. pocket about 10 A Binuclear zinc ions Zn1 and Zn2 at a distance of ˚ are bridged by the carboxyl oxygen atoms of 3.3 A Glu125 at the bottom of the pocket (Figure 2(a)
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and (b)). In the native structure, Zn1 is liganded by Glu125 O11, Asp22 Od1, His20 N12, and two water molecules that form a disordered trigonal bipyramid. Zn2 is liganded to Glu125 O12, His198 N12, His219 N12 and two water molecules that form a disordered trigonal bipyramid. In the cilastatin-liganded structure, Zn1 is liganded to those three protein atoms and one water molecule, Wat421, which form a triangular pyramid, and Zn2 is liganded to those three protein atoms, Wat421 and one of the carboxyl oxygen atoms of the cilastatin molecule. These liganded atoms form a square pyramid. Wat421 of the complex structure seems not to correspond to any water molecule in the native structure, but the oxygen atom of the cilastatin molecule seems to correspond to one water molecule in the native structure. A longer sidewall of the pocket is constructed by the carbonyl oxygen atom of Gly291 and the carboxyl side-chains of Asp22 and Asp288 (Figure 2(c)). The opposite sidewall is lined by the basic side-chains of His152, His198, and Arg230. Surfaces of shorter sidewalls are constructed by hydrophobic residues Trp25, Tyr68, and Pro70 on the side near the dimer interface, and by Tyr252, Tyr255, and Pro293 on the other side. The construction of the pocket seems to be reinforced by two adjacent disulfide bonds of Cys71-Cys154 and Cys226-Cys258, and by three proline residues, Pro24, Pro70, and Pro293 (Figure 1(a) and (c)). Although the existence of one zinc ion per one monomer has been reported,20 the structure clearly shows that one monomer needs two zinc ions. Comparing the average B-factor of zinc ions ˚ 2 for the complex) ˚ 2 for the native and 16.2 A (21.1 A with the average B-factor of all protein atoms ˚ 2 for the complex), ˚ 2 for the native and 15.8 A (21.2 A the B-factor of the zinc ions seem to be very reasonable. Then, they should fully occupy their sites and coexist in the structure. Moreover, each zinc ion has its own ligands, just the same as the other binuclear metallo-enzymes (Figures 2(a), (b), and 3(b); and see below). Those are reasons why the active-site structure of binuclear zinc ions is not an artifact. The previous report20 itself suggested that the simple model of the active site as a conventional zinc proteinase was not adequate, because the dipeptidase did not show a linear relationship between the gram atoms of zinc present in the enzyme and the percentage of activity restored.
Figure 1. The overall view of the hrDP. (a) The dimeric structure of the hrDP viewed from the membrane-binding side. Ser369 anchored to the membrane is located on the C-terminal end, and the active sites are located on this side. The a-helices and the b-strands composing (a/b)8 barrels are shown in green and yellow, respectively, and the a-helices capping the barrels are shown in magenta. Zinc ions are drawn as red spheres; cysteine residues forming disulfide bonds are drawn as yellow ball-and-sticks; N-linked N-acetylglucosamine molecules are drawn as pink ball-and-sticks. (b) The dimer viewed from a 908 rotation along the long axis of the dimer. (c) Stereo representation of a monomer subunit. (d) A drawing of the folding of the monomer subunit. The strands or helices are drawn to reflect their relative lengths. The color scheme and labels are consistent with those in (a) to (c). All Figures except Figure 2(a) and (c) were produced with MOLSCRIPT30 and Raster3D.31
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Crystal Structure of Human Renal Dipeptidase
Figure 2. The hrDP active site with the cilastatin ligand. (a) The Fo 2 Fc electron density maps superimposed on the structural model. The map in white was calculated with phases from a model without the cilastatin and Wat421 molecules, and contoured at 3.0 times the rms values of electron densities. The map in violet was calculated without the zinc ions, and contoured at 12 times the rms values. The cilastatin ligand is shown as a purple stick. The cysteinyl moiety of the ligand is not discernible in the map and is omitted from the drawing. Hydrogen bonds are drawn as broken green lines, and coordinate bonds to zinc ions as broken cyan lines. The Figure was produced with XtalView32 and Raster3D.31 (b) A drawing of the interactions among the cilastatin molecule, the zinc ions and the residues comprising the active site. R1 stands for – (CH2)3SCH2CH(NHþ3 )COO2.The hydrogen bonds are indicated as broken green lines, and the coordinate bonds as broken blue lines. The distances in the subunit A are also shown in the Figure ˚ ). The distance between binuclear Zn ions is 3.3 A ˚ for both the subunits A and B. The distance from Wat421 to the (in A carbonyl carbon atom is shown. The carbon atom of the peptide bond should be attacked by the nucleophile in the case of a substrate. (c) The pocket of the active site viewed from the entrance. The residues constricting the pocket are drawn in CPK model and proximal ones are labeled. Acidic residues are drawn in red; basic in blue; aromatic in purple. The cilastatin molecule is shown in ball-and-sticks. The polar residues are not located uniformly (see the text). (d) The molecular surface is colored by the electrostatic potential calculated by GRASP.33 The view is from the same direction as that of (c). The active site consists of the interface of negative and positive potentials, which is consistent with the non-uniform distribution of the polar residues shown in the (c). The cilastatin molecule is sited on the interface. The Figure was produced with GRASP.33
Crystal Structure of Human Renal Dipeptidase
Figure 3. Comparison of the architectures of the metallo-hydrolases having disordered (a/b)8 barrels. (a) Arrangement of b-strands composing the barrels. Corresponding main-chain atoms of the b-strands are superimposed by least-squares fitting. The strands of subunit A of the hrDP are shown as yellow arrows; urease in sky-blue, phosphotriesterase in orange, and adenosine deaminase in red. The strands labeled b1 through b8 are sequentially arranged from the N-terminal side in each polypeptide. (b) Superimposition of the metal ions and ligands through least-squares fitting. The metal ions and ligand groups of the hrDP subunit A are drawn in yellow, urease in sky-blue, and phosphotriesterase in orange. Carbamoyllysine ligands are represented with Carb-K labels. The fittings in (a) and (b) were calculated independently.
All of the critical residues that were reported play an important role in the active site, and it is clear why mutation of these residues causes reduced enzyme activity (Figure 2(a) and (b)). Glu125, His198 and His219 are ligands to the zinc ions and the mutation of these residues should result in removal of the zinc ion(s). His152 is not a ligand to the zinc ion, but it is responsible for the recognition of a substrate or an inhibitor because of making a hydrogen bond, and it would play an important role in the catalytic process (see below). The results of the mutagenesis studies are consistent with the active-site structure, and the active-site structure reveals why mutations lead to reduced activity. Even if cilastatin lacks an N-terminal amino group, it is regarded as a dipeptide analogue that has a dimethylcyclopropyl group and a heptenoic group followed by a cysteinyl moiety as sidechains. The former group is located at the N-terminal end of cilastatin, and the latter group at the C-terminal end. The dipeptidyl moiety of cilastatin is sandwiched between the negatively charged and positively charged sidewalls (Figure 2(c) and (d)). Both ends of the moiety are clamped tightly by the hydrophobic sidewalls. The interactions between cilastatin and hrDP are characterized by the following three observations (Figure 2(a) –(c)). First, the carboxyl terminus of the dipeptidyl moiety has four hydrogen bonds; a pair of hydrogen bonds with the side-chain of Arg230, one with Asp288, and one with Tyr255. Second, the oxygen and nitrogen atoms of the central peptide unit are hydrogen-bonded to His152 N12 and to Gly291 O, respectively. That is the reason why the result of
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mutation of His152 to Leu lacks the ability of substrate/inhibitor binding.6 Third, the dimethylcyclopropyl group is surrounded by the hydrophobic sidewall of Trp25, Tyr68, and Pro70. The cysteinyl moiety linking to the heptenoic group protrudes into the solvent region, and seems not to be essential to the binding. Wat421 liganded to Zn2 is located immediately above the carbonyl plane ˚ from the of the peptide unit, at a distance of 2.8 A carbonyl carbon atom. Virtually no space is left when the active-site pocket is fully occupied by the dipeptidyl moiety (Figure 2(c)). The bound cilastatin structure explains the substrate specificity of the hrDP with preference for a dipeptide having a hydrophobic bulky group at its N-terminal side.21 When the structures of imipenem and glycyldehydrophenylanine are superimposed on the structure of the cilastatin ligand, their dipeptide backbones are built into the pocket without structural alterations. In the case of the imipenem structure, which was calculated using the semi-empirical molecular orbital method in the Discover/InsightII suite of programs (Biosym, San Diego, California, USA), the backbone atoms are superimposable with ˚ . If a small rms positional difference of 0.25 A the dipeptide substrates are superimposed in the active site, their N-terminal amino groups are placed near the dimethylcyclopropyl carbon atom of the cilastatin, and then the location of the groups will be suitable for interacting with Zn1. According to this structure, Wat421 is possibly activated by Asp288 on the negatively charged sidewall, which is conserved among the mammalian dipeptidase (Figure 2(a) and (b)). The deprotonated Wat421 will be able to function as a nucleophilic reagent and to attack the carbonyl carbon atom of the scissile peptide bond. As a consequence, a tetrahedral adduct is formed. In this intermediate state, the oxyanion of the adduct is stabilized by the positive charge of the conserved His152. This role of His152 in the hydrolysis is comparable to that of the oxyanion holes of serine proteases. The peptide conformation of the bound cilastatin is highly constrained by the dimethylcyclopropyl group, which is in contact with Zn1, Trp25 and Gly291 (Figure 2(a) and (c)). This constrained conformation seems to displace the carbonyl carbon and oxygen atoms of the peptide slightly away from optimum locations in the active-site pocket. It prevents the carbon atom from undergoing nucleophilic attack and hence prevents formation of the tetrahedral adduct. This subtle displacement gives us a clue to understanding how cilastatin works as an inhibitor, not as a substrate. Further analysis of the action of cilastatin as an inhibitor is definitively required (T. P. Smyth, J. G. Wall & Y.N., unpublished results). The murine adenosine deaminase,22 bacterial Klebsiella aerogenes urease,23 and Pseudomonas diminuta phosphotriesterase (PTE)24 are classified as metallo-hydrolases,25 and have a common
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Crystal Structure of Human Renal Dipeptidase
Table 1. Statistics on the data collections and crystallographic refinements Native
Liganded
30– 2.3 174,691 30,657 92.3 0.093
20– 2.0 185,396 46,290 96.1 0.087
CH3HgCl 9183 0.181 0.62/0.69 1.16/1.73 0.60 (0.59/0.78)
Mersalyl acid 11,830 0.209 0.64/0.70 1.13/1.64
Native
Liganded
C. Crystallographic refinement No. protein atoms No. water molecules No. zinc ions/sugar residues No. cilastatin R Free R (10% data)
5776 213 4/4 – 0.181 0.258
5776 607 4/4 2 0.185 0.254
rms deviations from ideal values ˚) Bond length (A Bond angle (deg.)
0.007 1.40
0.007 1.46
A. Diffraction data collection ˚) Spacing (A No. measured reflections No. unique reflections Completeness (%) Rmerge(I ) B. Isomorphous phasing statistics Heavy-atom derivatives ˚) No. reflections (up to 3.0 A RF versus native data RCullisa Phasing powera Overall figure-of-meritsb
a b
K2PtCl4 11,611 0.160 0.63/0.72 0.98/1.62
The values are for the acentric and centric reflections. The values in the parentheses are for the acentric and centric reflections.
distorted (a/b)8 barrel architecture. When the main-chain atoms of the barrel b-strands of the hrDP are superimposed on the corresponding atoms of these structures, the b-strands show an unexpected and remarkable superposition (Figure ˚ from the 3(a)), with an rms difference of 1.29 A ˚ adenosine deaminase; 1.56 A from the urease, and ˚ from the PTE. The urease and the PTE have 1.77 A binuclear metal ions, each bridged by the carboxyl group of carbamoyllysine, Ni ions in the urease and Zn ions in the PTE, in a spatial arrangement very similar to that of the hrDP (Figure 3(b)). The residues liganding to these binuclear ions are also superimposable in a common sequential order of the liganded groups. These metallo-hydrolases with the (a/b)8 barrel architecture possibly have a common ancestral origin or have been evolved to adopt binuclear catalysis.
Experimental details The recombinant hrDP was expressed as a 369 residue polypeptide in the yeast P. pastoris, and its oligosaccharide moieties were trimmed off by treatment with the endoglycosidase Hf.18 The saccharide-trimmed hrDP retained full activity and its monomeric subunit showed a molecular mass of 42 kDa. Crystallization conditions were as described.18 Briefly, droplets containing 10 mg/ml of protein and 7.5% (w/v) PEG 8000 (pH 5.6) were equilibrated against reservoir solutions of 15%
PEG 8000 (pH 5.6). Box-shaped crystals were obtained after a few days. The macro-seeding technique was adopted for obtaining lager crystals. The diffraction data from unliganded native crystals were recorded at 277 K on Fuji Imaging Plates using Cu Ka X-rays, and were integrated into intensities using the DENZO and SCALEPACK program suite.26 The cilastatin-liganded crystals were prepared by soaking the unliganded crystals in a solution of 30% (w/w) PEG 8000, 15% (v/v) glycerol, 2 mM ZnCl2, and 25 mM cilastatin at pH 7.6, and their diffraction intensities were collected at 120 K. The structure was determined by multiple isomorphous replacement phasing from three kinds of derivative crystals, combined with the solventflattening and non-crystallographic symmetry (NCS) molecular averaging proceeded under the CCP4 suite.27 In the CH3HgCl2 derivative, four heavy-atom sites were found near His90 and Cys93 of both subunits. In the mersalyl acid derivative, there were six sites, near Asn32, His90 and Cys93 of both subunits. In the K2PtCl4 derivative, four of five sites were found near Met30 and Met95 of both subunits, and the last one seemed to be on the NCS axis of the dimer, near both Cys361. Structural models were constructed and refined using the TRUBO-FRODO28 and X-PLOR suite.29 To avoid the phase bias, omit maps and difference Fourier maps were used to build and check the models. The initial models were constructed
Crystal Structure of Human Renal Dipeptidase
without any N-acetylglucosamine molecules, because there was no information about which possible N-glycosylation sites (Asn41, Asn263, Asn316 and Asn342) are linked to the sugar moiety. In the course of the refinement, the extra electron densities extending from the side-chains of Asn41 and Asn316 of both subunits appeared, then the sugars were constructed in the models. The other sites did not show any clear extra density. Finally, omit refine maps were calculated to check all of the models. A Ramachandran plot of the native structure shows that 86.4% of the residues belong to the most favored regions, 12.8% to the additionally allowed regions, 0.8% to the generously allowed regions, 0% to the disallowed regions; in the complex structure, the values were 86.6%, 12.6%, 0.8% and 0%, respectively. Statistics on the data collection and crystallographic refinements are given in Table 1.
Protein Data Bank accession codes The coordinate set have been deposited with the RCSB Protein Data Bank, with the accession code of 1ITQ for the unliganded crystal and 1ITU for the liganded crystal.
Acknowledgments We specially thank Timothy P. Smyth on comments and suggestions on the manuscript. This study was supported by the JSPS-RFTF program 96L00505 grant to Y.S.
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7. Keynan, S., Hooper, N. M. & Turner, A. J. (1997). Identification by site-directed mutagenesis of three essential histidine residues in membrane dipeptidase, a novel mammalian zinc peptidase. Biochem. J. 326, 47 –51. 8. Kahan, F. M., Kropp, H., Sundelof, J. G. & Birnbaum, J. (1983). Thienamycin: development of imipenen– cilastatin. J. Antimicrob. Chemother. 12, 1 – 35. 9. Kropp, H., Sundelof, J. G., Hajdu, R. & Kahan, F. M. (1982). Metabolism of thienamycin and related carbapenem antibiotics by the renal dipeptidase, dehydropeptidase. Antimicrob. Agents Chemother. 22, 62 – 70. 10. Kim, H. S. & Campbell, B. J. (1982). beta-Lactamase activity of renal dipeptidase against N-formimidoylthienamycin. Biochem. Biophys. Res. Commun. 108, 1638 –1642. 11. Adachi, H., Kubota, I., Okamura, N., Iwata, H., Tsujimoto, M., Nakazato, H. et al. (1989). Purification and characterization of human microsomal dipeptidase. J. Biochem. (Tokyo), 105, 957–961. 12. Igarashi, P. & Karniski, L. P. (1991). Cloning of cDNAs encoding a rabbit renal brush border membrane protein immunologically related to band 3. Sequence similarity with microsomal dipeptidase. Biochem. J. 280, 71 –78. 13. Rached, E., Hooper, N. M., James, P., Semenza, G., Turner, A. J. & Mantei, N. (1990). cDNA cloning and expression in Xenopus laevis oocytes of pig renal dipeptidase, a glycosyl-phosphatidylinositolanchored ectoenzyme. Biochem. J. 271, 755– 760. 14. An, S., Schmidt, F. J. & Campbell, B. J. (1994). Molecular cloning of sheep lung dipeptidase: a glycosyl phosphatidylinositol-anchored ectoenzyme that converts leukotriene D4 to leukotriene E4. Biochim. Biophys. Acta, 1226, 337 –340. 15. Adachi, H., Ishida, N. & Tsujimoto, M. (1992). Primary structure of rat renal dipeptidase and expression of its mRNA in rat tissues and COS-1 cells. Biochim. Biophys. Acta, 1132, 311 – 314. 16. Satoh, S., Keida, Y., Konta, Y., Maeda, M., Matsumoto, Y., Niwa, M. & Kohsaka, M. (1993). Purification and molecular cloning of mouse renal dipeptidase. Biochim. Biophys. Acta, 1163, 234– 242. 17. Adachi, H. & Tsujimoto, M. (1995). Cloning and expression of dipeptidase from Acinetobacter calcoaceticus ATCC 23055. Biochemistry, 118, 555– 561. 18. Nitanai, Y., Satow, Y., Adachi, H. & Tsujimono, M. (1996). Crystallization and preliminary X-ray investigation of a glycoprotein, human renal dipeptidase. J. Cryst. Growth, 168, 280–283. 19. Keynan, S., Habgood, N. T., Hooper, N. M. & Turner, A. J. (1996). Site-directed mutagenesis of conserved cysteine residues in porcine membrane dipeptidase. Cys361 alone is involved in disulfide-linked dimerization. Biochemistry, 35, 12511 –12517. 20. Armstrong, D. D., Mukhopadhyay, S. K. & Campbell, B. J. (1974). Physicochemical characterization of renal dipeptidase. Biochemistry, 13, 1745– 1750. 21. Campbell, B. J., Shih, Y. D., Forrester, L. J. & Zahler, W. L. (1988). Specificity and inhibition studies of human renal dipeptidase. Biochim. Biophys. Acta, 956, 110 – 118. 22. Wang, Z. & Quiocho, F. A. (1998). Complexes of adenosine deaminase with two potent inhibitors: X-ray structures in four independent molecules at pH of maximum activity. Biochemistry, 37, 8314 –8324.
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Edited by R. Huber (Received 11 March 2002; received in revised form 19 June 2002; accepted 19 June 2002)
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J. Mol. Biol. (2002) 321, 177–184
COMMUNICATION
Crystal Structure of Human Renal Dipeptidase Involved in b-Lactam Hydrolysis Yasushi Nitanai1*, Yoshinori Satow1, Hideki Adachi2 and Masafumi Tsujimoto2 1 Faculty of Pharmaceutical Sciences, University of Tokyo Hongo, Bunkyo-ku, Tokyo 113 Japan 2
Laboratory of Cellular Biochemistry, RIKEN (The Institute of Physical and Chemical Research) Wako-shi, Saitama 351-0198 Japan
Human renal dipeptidase is a membrane-bound glycoprotein hydrolyzing dipeptides and is involved in hydrolytic metabolism of penem and carbapenem b-lactam antibiotics. The crystal structures of the saccharidetrimmed enzyme are determined as unliganded and inhibitor-liganded forms. They are informative for designing new antibiotics that are not hydrolyzed by this enzyme. The active site in each of the (a/b)8 barrel subunits of the homodimeric molecule is composed of binuclear zinc ions bridged by the Glu125 side-chain located at the bottom of the barrel, and it faces toward the microvillar membrane of a kidney tubule. A dipeptidyl moiety of the therapeutically used cilastatin inhibitor is fully accommodated in the active-site pocket, which is small enough for precise recognition of dipeptide substrates. The barrel and active-site architectures utilizing catalytic metal ions exhibit unexpected similarities to those of the murine adenosine deaminase and the catalytic domain of the bacterial urease. q 2002 Published by Elsevier Science Ltd
*Corresponding author
Keywords: crystal structure; human renal dipeptidase; metabolism of b-lactam antibiotics; cilastatin complex; binuclear metal ions
Human renal dipeptidase (hrDP) (EC 3.4.13.19, membrane dipeptidase and microsomal dipeptidase are synonyms) is a membrane-bound glycoprotein involved in the hydrolysis of dipeptides, and shows activity only toward dipeptides.1,2 In the renal cortex, it is bound to microvillar membranes in the brush-border region of proximal tubules via phosphatidylinositol.3 Although the dipeptidases lack the His-Glu-X-X-His amino acid sequences identified commonly in zinc proteases,4 and has no homology to other metallopeptidase, zinc ions are essential to their hydrolytic functions. Previous mutagenesis studies indicated that Glu125,5 His152,6 His198,6 and His2197 are involved in catalysis. Furthermore, His152 was shown as playing the important role of substrate/ inhibitor binding.6 But, the motif consisting of the
Present address: Y. Nitanai, Laboratory for Structural Biochemistry, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki, Sayo, Hyogo 679-5148, Japan. Abbreviations used: hrDP, human renal dipeptidase; PTE, phosphotriesterase; PEG, polyethylene glycol. E-mail address of the corresponding author: [email protected]
active site and the catalytic mechanism have not been clear. Although general roles in the organism are still unknown, the dipeptidase is well-known as the sole metabolic enzyme for penem and carbapenem b-lactam antibiotics in mammals.8 – 10 It hydrolyzes S(substitute)-L -cysteinyl-glycine adducts such as L -cystinyl-bis-glycine and S-N-ethylmaleimide-L cysteinyl-glycine, and is responsible for conversion of leukotriene D4 to E4.11 These facts suggest that the enzyme plays important roles in the renal metabolism of glutathione, its conjugates, and some drugs. The antibiotics are presently prescribed in clinical practice with the dipeptidyl inhibitor cilastatin.8 The hrDP is a homodimer, each subunit consisting of a 369 amino acid residue peptide (42 kDa), which shows sequence identities of about 75% to mammalian enzymes,12 – 16 and a low value of 23% to the prokaryotic enzyme of Acinetobacter calcoaceticus.17 The subunit has four possible N-glycosylation sites and, consequently, is obtained as a highly glycosylated form of 63 kDa.1 Furthermore, it has a glycosylphosphatidylinositol anchor to microvillar membranes in the brush-border region of the proximal tubules of the renal cortex.1
0022-2836/02/$ - see front matter q 2002 Published by Elsevier Science Ltd
178
Crystal Structure of Human Renal Dipeptidase
Figure 1. (legend opposite)
Crystal Structure of Human Renal Dipeptidase
In order to obtain diffraction-quality crystals, the human enzyme was expressed in the Pichia pastoris yeast, and its oligosaccharide moieties were trimmed off by treatment with endoglycosidase.18 Here, the structures of the unliganded native and cilastatin-liganded crystals are reported. The hrDP has an elongated ellipsoidal shape of ˚ £ 45 A ˚ £ 45 A ˚ (Figure 1(a) and (b)). The sub90 A units of (a/b)8 barrel architecture (Figure 1(c) and (d)) are related to each other by a non-crystallographic twofold axis. Their barrel axes are twisted about 408 around the long axis of the ellipsoid. The subunits are linked by a disulfide bond between Cys361 side-chains, and interfaced with each other by extensive molecular contacts located mainly at helices a2 and a3. Barrel helices and strands facing the dimer interface are long and in parallel with the barrel axis, and those located away from the interface are short and nearly perpendicular to the axis. Therefore, the barrel is highly distorted; for example, helix a3 proximal to the interface is tilted with respect to distal helix a7 by about 1108. The N-terminal ends of the barrel strands are capped by four helices, aa, a2a, ad, and ae, and the Cterminal ends face the membrane (Figure 1(d)). Ser369, which is modifiable with the glycosylphosphatidylinositol anchor, is located on the surface facing the membrane near the twofold axis. The active site, also located on this surface, is suitable for receiving substrates transported from the membrane. The loop regions downstream of barrel strands b2 and b4 are connected by a disulfide bond between Cys71 and Cys154, and those of b6 and b7 are connected by a disulfide bond between Cys226 and Cys258. Those disulfide bonds correspond precisely to the result of previous mutagenesis studies of cysteine residues.19 It was shown that the Cys361 alone was involved in disulfidelinked dimerization among six cysteine residues, and the others made two disulfide bonds within a subunit. Two of the four possible glycosylation sites, Asn41 and Asn316, show electron densities corresponding to the N-acetylglucosamine structure. These N-glycosylation sites are located on the flanking side of the barrel. Viewed toward the C-terminal ends of the barrel strands, the active site appears as a rectangular ˚ long, 6 A ˚ wide, and 6 A ˚ deep. pocket about 10 A Binuclear zinc ions Zn1 and Zn2 at a distance of ˚ are bridged by the carboxyl oxygen atoms of 3.3 A Glu125 at the bottom of the pocket (Figure 2(a)
179
and (b)). In the native structure, Zn1 is liganded by Glu125 O11, Asp22 Od1, His20 N12, and two water molecules that form a disordered trigonal bipyramid. Zn2 is liganded to Glu125 O12, His198 N12, His219 N12 and two water molecules that form a disordered trigonal bipyramid. In the cilastatin-liganded structure, Zn1 is liganded to those three protein atoms and one water molecule, Wat421, which form a triangular pyramid, and Zn2 is liganded to those three protein atoms, Wat421 and one of the carboxyl oxygen atoms of the cilastatin molecule. These liganded atoms form a square pyramid. Wat421 of the complex structure seems not to correspond to any water molecule in the native structure, but the oxygen atom of the cilastatin molecule seems to correspond to one water molecule in the native structure. A longer sidewall of the pocket is constructed by the carbonyl oxygen atom of Gly291 and the carboxyl side-chains of Asp22 and Asp288 (Figure 2(c)). The opposite sidewall is lined by the basic side-chains of His152, His198, and Arg230. Surfaces of shorter sidewalls are constructed by hydrophobic residues Trp25, Tyr68, and Pro70 on the side near the dimer interface, and by Tyr252, Tyr255, and Pro293 on the other side. The construction of the pocket seems to be reinforced by two adjacent disulfide bonds of Cys71-Cys154 and Cys226-Cys258, and by three proline residues, Pro24, Pro70, and Pro293 (Figure 1(a) and (c)). Although the existence of one zinc ion per one monomer has been reported,20 the structure clearly shows that one monomer needs two zinc ions. Comparing the average B-factor of zinc ions ˚ 2 for the complex) ˚ 2 for the native and 16.2 A (21.1 A with the average B-factor of all protein atoms ˚ 2 for the complex), ˚ 2 for the native and 15.8 A (21.2 A the B-factor of the zinc ions seem to be very reasonable. Then, they should fully occupy their sites and coexist in the structure. Moreover, each zinc ion has its own ligands, just the same as the other binuclear metallo-enzymes (Figures 2(a), (b), and 3(b); and see below). Those are reasons why the active-site structure of binuclear zinc ions is not an artifact. The previous report20 itself suggested that the simple model of the active site as a conventional zinc proteinase was not adequate, because the dipeptidase did not show a linear relationship between the gram atoms of zinc present in the enzyme and the percentage of activity restored.
Figure 1. The overall view of the hrDP. (a) The dimeric structure of the hrDP viewed from the membrane-binding side. Ser369 anchored to the membrane is located on the C-terminal end, and the active sites are located on this side. The a-helices and the b-strands composing (a/b)8 barrels are shown in green and yellow, respectively, and the a-helices capping the barrels are shown in magenta. Zinc ions are drawn as red spheres; cysteine residues forming disulfide bonds are drawn as yellow ball-and-sticks; N-linked N-acetylglucosamine molecules are drawn as pink ball-and-sticks. (b) The dimer viewed from a 908 rotation along the long axis of the dimer. (c) Stereo representation of a monomer subunit. (d) A drawing of the folding of the monomer subunit. The strands or helices are drawn to reflect their relative lengths. The color scheme and labels are consistent with those in (a) to (c). All Figures except Figure 2(a) and (c) were produced with MOLSCRIPT30 and Raster3D.31
180
Crystal Structure of Human Renal Dipeptidase
Figure 2. The hrDP active site with the cilastatin ligand. (a) The Fo 2 Fc electron density maps superimposed on the structural model. The map in white was calculated with phases from a model without the cilastatin and Wat421 molecules, and contoured at 3.0 times the rms values of electron densities. The map in violet was calculated without the zinc ions, and contoured at 12 times the rms values. The cilastatin ligand is shown as a purple stick. The cysteinyl moiety of the ligand is not discernible in the map and is omitted from the drawing. Hydrogen bonds are drawn as broken green lines, and coordinate bonds to zinc ions as broken cyan lines. The Figure was produced with XtalView32 and Raster3D.31 (b) A drawing of the interactions among the cilastatin molecule, the zinc ions and the residues comprising the active site. R1 stands for – (CH2)3SCH2CH(NHþ3 )COO2.The hydrogen bonds are indicated as broken green lines, and the coordinate bonds as broken blue lines. The distances in the subunit A are also shown in the Figure ˚ ). The distance between binuclear Zn ions is 3.3 A ˚ for both the subunits A and B. The distance from Wat421 to the (in A carbonyl carbon atom is shown. The carbon atom of the peptide bond should be attacked by the nucleophile in the case of a substrate. (c) The pocket of the active site viewed from the entrance. The residues constricting the pocket are drawn in CPK model and proximal ones are labeled. Acidic residues are drawn in red; basic in blue; aromatic in purple. The cilastatin molecule is shown in ball-and-sticks. The polar residues are not located uniformly (see the text). (d) The molecular surface is colored by the electrostatic potential calculated by GRASP.33 The view is from the same direction as that of (c). The active site consists of the interface of negative and positive potentials, which is consistent with the non-uniform distribution of the polar residues shown in the (c). The cilastatin molecule is sited on the interface. The Figure was produced with GRASP.33
Crystal Structure of Human Renal Dipeptidase
Figure 3. Comparison of the architectures of the metallo-hydrolases having disordered (a/b)8 barrels. (a) Arrangement of b-strands composing the barrels. Corresponding main-chain atoms of the b-strands are superimposed by least-squares fitting. The strands of subunit A of the hrDP are shown as yellow arrows; urease in sky-blue, phosphotriesterase in orange, and adenosine deaminase in red. The strands labeled b1 through b8 are sequentially arranged from the N-terminal side in each polypeptide. (b) Superimposition of the metal ions and ligands through least-squares fitting. The metal ions and ligand groups of the hrDP subunit A are drawn in yellow, urease in sky-blue, and phosphotriesterase in orange. Carbamoyllysine ligands are represented with Carb-K labels. The fittings in (a) and (b) were calculated independently.
All of the critical residues that were reported play an important role in the active site, and it is clear why mutation of these residues causes reduced enzyme activity (Figure 2(a) and (b)). Glu125, His198 and His219 are ligands to the zinc ions and the mutation of these residues should result in removal of the zinc ion(s). His152 is not a ligand to the zinc ion, but it is responsible for the recognition of a substrate or an inhibitor because of making a hydrogen bond, and it would play an important role in the catalytic process (see below). The results of the mutagenesis studies are consistent with the active-site structure, and the active-site structure reveals why mutations lead to reduced activity. Even if cilastatin lacks an N-terminal amino group, it is regarded as a dipeptide analogue that has a dimethylcyclopropyl group and a heptenoic group followed by a cysteinyl moiety as sidechains. The former group is located at the N-terminal end of cilastatin, and the latter group at the C-terminal end. The dipeptidyl moiety of cilastatin is sandwiched between the negatively charged and positively charged sidewalls (Figure 2(c) and (d)). Both ends of the moiety are clamped tightly by the hydrophobic sidewalls. The interactions between cilastatin and hrDP are characterized by the following three observations (Figure 2(a) –(c)). First, the carboxyl terminus of the dipeptidyl moiety has four hydrogen bonds; a pair of hydrogen bonds with the side-chain of Arg230, one with Asp288, and one with Tyr255. Second, the oxygen and nitrogen atoms of the central peptide unit are hydrogen-bonded to His152 N12 and to Gly291 O, respectively. That is the reason why the result of
181
mutation of His152 to Leu lacks the ability of substrate/inhibitor binding.6 Third, the dimethylcyclopropyl group is surrounded by the hydrophobic sidewall of Trp25, Tyr68, and Pro70. The cysteinyl moiety linking to the heptenoic group protrudes into the solvent region, and seems not to be essential to the binding. Wat421 liganded to Zn2 is located immediately above the carbonyl plane ˚ from the of the peptide unit, at a distance of 2.8 A carbonyl carbon atom. Virtually no space is left when the active-site pocket is fully occupied by the dipeptidyl moiety (Figure 2(c)). The bound cilastatin structure explains the substrate specificity of the hrDP with preference for a dipeptide having a hydrophobic bulky group at its N-terminal side.21 When the structures of imipenem and glycyldehydrophenylanine are superimposed on the structure of the cilastatin ligand, their dipeptide backbones are built into the pocket without structural alterations. In the case of the imipenem structure, which was calculated using the semi-empirical molecular orbital method in the Discover/InsightII suite of programs (Biosym, San Diego, California, USA), the backbone atoms are superimposable with ˚ . If a small rms positional difference of 0.25 A the dipeptide substrates are superimposed in the active site, their N-terminal amino groups are placed near the dimethylcyclopropyl carbon atom of the cilastatin, and then the location of the groups will be suitable for interacting with Zn1. According to this structure, Wat421 is possibly activated by Asp288 on the negatively charged sidewall, which is conserved among the mammalian dipeptidase (Figure 2(a) and (b)). The deprotonated Wat421 will be able to function as a nucleophilic reagent and to attack the carbonyl carbon atom of the scissile peptide bond. As a consequence, a tetrahedral adduct is formed. In this intermediate state, the oxyanion of the adduct is stabilized by the positive charge of the conserved His152. This role of His152 in the hydrolysis is comparable to that of the oxyanion holes of serine proteases. The peptide conformation of the bound cilastatin is highly constrained by the dimethylcyclopropyl group, which is in contact with Zn1, Trp25 and Gly291 (Figure 2(a) and (c)). This constrained conformation seems to displace the carbonyl carbon and oxygen atoms of the peptide slightly away from optimum locations in the active-site pocket. It prevents the carbon atom from undergoing nucleophilic attack and hence prevents formation of the tetrahedral adduct. This subtle displacement gives us a clue to understanding how cilastatin works as an inhibitor, not as a substrate. Further analysis of the action of cilastatin as an inhibitor is definitively required (T. P. Smyth, J. G. Wall & Y.N., unpublished results). The murine adenosine deaminase,22 bacterial Klebsiella aerogenes urease,23 and Pseudomonas diminuta phosphotriesterase (PTE)24 are classified as metallo-hydrolases,25 and have a common
182
Crystal Structure of Human Renal Dipeptidase
Table 1. Statistics on the data collections and crystallographic refinements Native
Liganded
30– 2.3 174,691 30,657 92.3 0.093
20– 2.0 185,396 46,290 96.1 0.087
CH3HgCl 9183 0.181 0.62/0.69 1.16/1.73 0.60 (0.59/0.78)
Mersalyl acid 11,830 0.209 0.64/0.70 1.13/1.64
Native
Liganded
C. Crystallographic refinement No. protein atoms No. water molecules No. zinc ions/sugar residues No. cilastatin R Free R (10% data)
5776 213 4/4 – 0.181 0.258
5776 607 4/4 2 0.185 0.254
rms deviations from ideal values ˚) Bond length (A Bond angle (deg.)
0.007 1.40
0.007 1.46
A. Diffraction data collection ˚) Spacing (A No. measured reflections No. unique reflections Completeness (%) Rmerge(I ) B. Isomorphous phasing statistics Heavy-atom derivatives ˚) No. reflections (up to 3.0 A RF versus native data RCullisa Phasing powera Overall figure-of-meritsb
a b
K2PtCl4 11,611 0.160 0.63/0.72 0.98/1.62
The values are for the acentric and centric reflections. The values in the parentheses are for the acentric and centric reflections.
distorted (a/b)8 barrel architecture. When the main-chain atoms of the barrel b-strands of the hrDP are superimposed on the corresponding atoms of these structures, the b-strands show an unexpected and remarkable superposition (Figure ˚ from the 3(a)), with an rms difference of 1.29 A ˚ adenosine deaminase; 1.56 A from the urease, and ˚ from the PTE. The urease and the PTE have 1.77 A binuclear metal ions, each bridged by the carboxyl group of carbamoyllysine, Ni ions in the urease and Zn ions in the PTE, in a spatial arrangement very similar to that of the hrDP (Figure 3(b)). The residues liganding to these binuclear ions are also superimposable in a common sequential order of the liganded groups. These metallo-hydrolases with the (a/b)8 barrel architecture possibly have a common ancestral origin or have been evolved to adopt binuclear catalysis.
Experimental details The recombinant hrDP was expressed as a 369 residue polypeptide in the yeast P. pastoris, and its oligosaccharide moieties were trimmed off by treatment with the endoglycosidase Hf.18 The saccharide-trimmed hrDP retained full activity and its monomeric subunit showed a molecular mass of 42 kDa. Crystallization conditions were as described.18 Briefly, droplets containing 10 mg/ml of protein and 7.5% (w/v) PEG 8000 (pH 5.6) were equilibrated against reservoir solutions of 15%
PEG 8000 (pH 5.6). Box-shaped crystals were obtained after a few days. The macro-seeding technique was adopted for obtaining lager crystals. The diffraction data from unliganded native crystals were recorded at 277 K on Fuji Imaging Plates using Cu Ka X-rays, and were integrated into intensities using the DENZO and SCALEPACK program suite.26 The cilastatin-liganded crystals were prepared by soaking the unliganded crystals in a solution of 30% (w/w) PEG 8000, 15% (v/v) glycerol, 2 mM ZnCl2, and 25 mM cilastatin at pH 7.6, and their diffraction intensities were collected at 120 K. The structure was determined by multiple isomorphous replacement phasing from three kinds of derivative crystals, combined with the solventflattening and non-crystallographic symmetry (NCS) molecular averaging proceeded under the CCP4 suite.27 In the CH3HgCl2 derivative, four heavy-atom sites were found near His90 and Cys93 of both subunits. In the mersalyl acid derivative, there were six sites, near Asn32, His90 and Cys93 of both subunits. In the K2PtCl4 derivative, four of five sites were found near Met30 and Met95 of both subunits, and the last one seemed to be on the NCS axis of the dimer, near both Cys361. Structural models were constructed and refined using the TRUBO-FRODO28 and X-PLOR suite.29 To avoid the phase bias, omit maps and difference Fourier maps were used to build and check the models. The initial models were constructed
Crystal Structure of Human Renal Dipeptidase
without any N-acetylglucosamine molecules, because there was no information about which possible N-glycosylation sites (Asn41, Asn263, Asn316 and Asn342) are linked to the sugar moiety. In the course of the refinement, the extra electron densities extending from the side-chains of Asn41 and Asn316 of both subunits appeared, then the sugars were constructed in the models. The other sites did not show any clear extra density. Finally, omit refine maps were calculated to check all of the models. A Ramachandran plot of the native structure shows that 86.4% of the residues belong to the most favored regions, 12.8% to the additionally allowed regions, 0.8% to the generously allowed regions, 0% to the disallowed regions; in the complex structure, the values were 86.6%, 12.6%, 0.8% and 0%, respectively. Statistics on the data collection and crystallographic refinements are given in Table 1.
Protein Data Bank accession codes The coordinate set have been deposited with the RCSB Protein Data Bank, with the accession code of 1ITQ for the unliganded crystal and 1ITU for the liganded crystal.
Acknowledgments We specially thank Timothy P. Smyth on comments and suggestions on the manuscript. This study was supported by the JSPS-RFTF program 96L00505 grant to Y.S.
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Edited by R. Huber (Received 11 March 2002; received in revised form 19 June 2002; accepted 19 June 2002)