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Crystal Structure of Isoaspartyl Aminopeptidase in Complex with l-Aspartate

2005; Elsevier BV; Volume: 280; Issue: 31 Linguagem: Inglês

10.1074/jbc.m504501200

1083-351X

K. Michalska, Krzysztof Brzeziński, Mariusz Jaskólski,

Protein purification and stability

The crystal structure of Escherichia coli isoaspartyl aminopeptidase/asparaginase (EcAIII), an enzyme belonging to the N-terminal nucleophile (Ntn)-hydrolases family, has been determined at 1.9-Å resolution for a complex obtained by cocrystallization with l-aspartate, which is a product of both enzymatic reactions catalyzed by EcAIII. The enzyme is a dimer of heterodimers, (αβ)2. The (αβ) heterodimer, which arises by autoproteolytic cleavage of the immature protein, exhibits an αββα-sandwich fold, typical for Ntn-hydrolases. The asymmetric unit contains one copy of the EcAIII·Asp complex, with clearly visible l-aspartate ligands, one bound in each of the two active sites of the enzyme. The l-aspartate ligand is located near Thr179, the N-terminal residue of subunit β liberated in the autoproteolytic event. Structural comparisons with the free form of EcAIII reveal that there are no major rearrangements of the active site upon aspartate binding. Although the ligand binding mode is similar to that observed in an l-aspartate complex of the related enzyme human aspartylglucosaminidase, the architecture of the EcAIII active site sheds light on the question of substrate specificity and explains why EcAIII is not able to hydrolyze glycosylated asparagine substrates. The crystal structure of Escherichia coli isoaspartyl aminopeptidase/asparaginase (EcAIII), an enzyme belonging to the N-terminal nucleophile (Ntn)-hydrolases family, has been determined at 1.9-Å resolution for a complex obtained by cocrystallization with l-aspartate, which is a product of both enzymatic reactions catalyzed by EcAIII. The enzyme is a dimer of heterodimers, (αβ)2. The (αβ) heterodimer, which arises by autoproteolytic cleavage of the immature protein, exhibits an αββα-sandwich fold, typical for Ntn-hydrolases. The asymmetric unit contains one copy of the EcAIII·Asp complex, with clearly visible l-aspartate ligands, one bound in each of the two active sites of the enzyme. The l-aspartate ligand is located near Thr179, the N-terminal residue of subunit β liberated in the autoproteolytic event. Structural comparisons with the free form of EcAIII reveal that there are no major rearrangements of the active site upon aspartate binding. Although the ligand binding mode is similar to that observed in an l-aspartate complex of the related enzyme human aspartylglucosaminidase, the architecture of the EcAIII active site sheds light on the question of substrate specificity and explains why EcAIII is not able to hydrolyze glycosylated asparagine substrates. Proteins undergo several age-dependent spontaneous modifications that can limit their useful lifetime. In particular, deamidated, racemized, or isomerized derivatives can be formed in an intramolecular succinimide-mediated rearrangement involving l-asparaginyl or, in a 13–36-fold slower reaction (1.Stephenson R.C. Clarke S. J. Biol. Chem. 1989; 264: 6164-6170Abstract Full Text PDF PubMed Google Scholar), l-aspartyl residues (2.Geiger T. Clarke S. J. Biol. Chem. 1987; 262: 785-794Abstract Full Text PDF PubMed Google Scholar). The major product is an l-isoaspartyl (iAsp) 1The abbreviations used are: iAsp, isoaspartyl residue; AGA, N4-(β-N-acetylglucosaminyl)-l-asparaginase (aspartylglucosaminidase, EC 3.5.1.26); EcAIII, E. coli iaaA gene product; O-MT, l-isoaspartyl(d-aspartyl)-O-methyltransferase (EC 3.4.11.18); Ntn, N-terminal nucleophile; r.m.s.d., root mean square deviation. 1The abbreviations used are: iAsp, isoaspartyl residue; AGA, N4-(β-N-acetylglucosaminyl)-l-asparaginase (aspartylglucosaminidase, EC 3.5.1.26); EcAIII, E. coli iaaA gene product; O-MT, l-isoaspartyl(d-aspartyl)-O-methyltransferase (EC 3.4.11.18); Ntn, N-terminal nucleophile; r.m.s.d., root mean square deviation.-containing protein, in which the peptide backbone has been transferred to the side chain forming a β-peptide. Such a serious structural rearrangement usually leads to protein dysfunction. Two mechanisms have been proposed by which organisms may handle the useless proteins and prevent accumulation of the harmful iAsp (3.Mudgett M.B. Lowenson J.D. Clarke S. Plant Physiol. 1997; 115: 1481-1489Crossref PubMed Scopus (55) Google Scholar, 4.Kim E. Lowenson J.D. MacLaren D.C. Clarke S. Proc. Natl. Acad. Sci. 1997; 94: 6132-6137Crossref PubMed Scopus (248) Google Scholar, 5.Larsen R.A. Knox T.M. Miller C.G. J. Bacteriol. 2001; 183: 3089-3097Crossref PubMed Scopus (27) Google Scholar). One well defined mechanism involves the repair of some products of spontaneous damage to intracellular proteins. It is based on the enzyme l-isoaspartyl(d-aspartyl)-O-methyltransferase (O-MT), which is found in organisms ranging from bacteria to mammals and plants and initiates the repair pathway by methylation of l-isoaspartyl residues (and d-aspartyl residues in racemized derivatives) (6.O'Connor C.M. Clarke S. Biochem. Biophys. Res. Commun. 1985; 132: 1144-1150Crossref PubMed Scopus (32) Google Scholar, 7.Lowenson J.D. Clarke S. J. Biol. Chem. 1992; 267: 5985-5995Abstract Full Text PDF PubMed Google Scholar). Despite the quite wide range of recognized substrates, the repair activity of O-MT is limited (8.Lowenson J.D. Clarke S. J. Biol. Chem. 1990; 265: 3106-3110Abstract Full Text PDF PubMed Google Scholar, 9.Lowenson J.D. Clarke S. J. Biol. Chem. 1991; 266: 19396-19406Abstract Full Text PDF PubMed Google Scholar). The damaged proteins that are not identified and repaired by the enzyme are degraded by cellular proteases to free amino acids and to the relatively stable iAsp-containing di- and tripeptides. To prevent accumulation of those harmful isoaspartyl peptides, specialized enzymes with peptidase activity are required (10.Dorer F.E. Haley E.E. Buchan D.L. Arch. Biochem. Biophys. 1968; 127: 490-495Crossref PubMed Scopus (17) Google Scholar). Enzymes with isoaspartyl peptidase activity (EC 3.4.19.5) were initially isolated from mammals (10.Dorer F.E. Haley E.E. Buchan D.L. Arch. Biochem. Biophys. 1968; 127: 490-495Crossref PubMed Scopus (17) Google Scholar) and bacteria (11.Haley E.E. J. Biol. Chem. 1968; 243: 5748-5752Abstract Full Text PDF PubMed Google Scholar). Further studies have revealed the existence of two classes of isoaspartyl peptidases. The first class includes metallopeptidases represented by the Escherichia coli (12.Gary J.D. Clarke S. J. Biol. Chem. 1995; 270: 4076-4087Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and Salmonella enterica (5.Larsen R.A. Knox T.M. Miller C.G. J. Bacteriol. 2001; 183: 3089-3097Crossref PubMed Scopus (27) Google Scholar) proteins, both encoded by the iadA gene. Sequence analysis of the E. coli IadA protein suggested its similarity to bacterial dihydroorotases and imidases, which are involved in the metabolism of pyrimidines (12.Gary J.D. Clarke S. J. Biol. Chem. 1995; 270: 4076-4087Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The crystal structure of E. coli IadA confirmed its structural homology to these enzymes, as well as the presence of zinc cations (13.Thoden J.B. Marti-Arbona R. Raushel F.M. Holden H.M. Biochemistry. 2003; 42: 4874-4882Crossref PubMed Scopus (39) Google Scholar, 14.Jozic D. Kaise J.T. Huber R. Bode W. Maskos K. J. Mol. Biol. 2003; 332: 243-256Crossref PubMed Scopus (31) Google Scholar). The second class of isoaspartyl peptidases is represented by the E. coli iaaA gene product and its S. enterica homolog (5.Larsen R.A. Knox T.M. Miller C.G. J. Bacteriol. 2001; 183: 3089-3097Crossref PubMed Scopus (27) Google Scholar). These enzymes belong to the family of Ntn (N-terminal nucleophile) hydrolases, first classified by Brannigan et al. (15.Brannigan J.A. Dodson G. Duggleby H.J. Moody P.C. Smith J.L. Tomchick D.R. Murzin A.G. Nature. 1995; 378: 416-419Crossref PubMed Scopus (538) Google Scholar), which, according to a recent study by Elkins et al. (16.Elkins J.M. Kershaw N.J. Schofield C.J. Biochem. J. 2005; 385: 565-573Crossref PubMed Scopus (26) Google Scholar), may actually form a subclass of Ntn-enzymes with more general (not necessarily hydrolytic) activities. Ntn-hydrolases are expressed as enzymatically inactive precursors that become activated upon autoproteolytic cleavage, which generates two subunits, α (N-terminal) and β (C-terminal). The N-terminal residue of subunit β, a threonine, serine, or cysteine, acts as the active nucleophile in the catalytic mechanism. The crystal structures reported for several members of this family, including ornithine acetyltransferase (16.Elkins J.M. Kershaw N.J. Schofield C.J. Biochem. J. 2005; 385: 565-573Crossref PubMed Scopus (26) Google Scholar), human (17.Oinonen C. Tikkanen R. Rouvinen J. Peltonen L. Nat. Struct. Biol. 1995; 2: 1102-1108Crossref PubMed Scopus (156) Google Scholar), and Flavobacterium meningosepticum aspartylglucosaminidases (18.Guo H.C. Xu Q. Buckley D. Guan C. J. Biol. Chem. 1998; 273: 20205-20212Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 19.Xuan J. Tarentino A.L. Grimwood B.G. Plummer Jr., T.H. Cui T. Guan C. Van Roey P. Protein Sci. 1998; 7: 774-781Crossref PubMed Scopus (28) Google Scholar), the proteasome β-subunit (20.Löwe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber R. Science. 1995; 268: 533-539Crossref PubMed Scopus (1363) Google Scholar), glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase (21.Smith J.L. Zaluzec E.J. Wery J.P. Niu L. Switzer R.L. Zalkin H. Satow Y. Science. 1994; 264: 1427-1433Crossref PubMed Scopus (223) Google Scholar), acylases of penicillin G (22.Duggleby H.J. Tolley S.P. Hill C.P. Dodson E.J. Dodson G. Moody P.C.E. Nature. 1995; 373: 264-268Crossref PubMed Scopus (420) Google Scholar) and penicillin V (23.Suresh C.G. Pundle A.V. SivaRaman H. Rao K.N. Brannigan J.A. McVey C.E. Verma C.S. Dauter Z. Dodson E.J. Dodson G.G. Nature Struct. Biol. 1999; 6: 414-416Crossref PubMed Scopus (104) Google Scholar), as well as cephalosporin acylases (24.Kim Y. Yoon K. Khang Y. Turley S. Hol W.G. Structure Fold. Des. 2000; 8: 1059-1068Abstract Full Text Full Text PDF Scopus (98) Google Scholar, 25.Kim J.K. Yang I.S. Rhee S. Dauter Z. Lee Y.S. Park S.S. Kim K.H. Biochemistry. 2003; 42: 4084-4093Crossref PubMed Scopus (45) Google Scholar), have revealed that, although the amino acid sequences of these proteins vary considerably, they all have the same fold. The polypeptide chains of these proteins are organized into a sandwich of two extended parallel β-sheets, flanked on both sides by α-helices. This topology is usually described as the αββα-fold (26.Oinonen C. Rouvinen J. Protein Sci. 2000; 9: 2329-2337Crossref PubMed Scopus (205) Google Scholar), a term that illustrates the spatial organization of the secondary structure elements. Each of the two active sites (at the N-terminal nucleophiles of subunits β) of an Ntn heterotetramer is located between the β-sheets. The iaaA gene product from E. coli was originally classified as a new, plant-type asparaginase, and named EcAIII (27.Blattner F.R. Plunkett G. Bloch C.A. Perna N.T. Burland V. Riley M. ColladoVides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (5935) Google Scholar, 28.Borek D. Podkowinski J. Kisiel A. Jaskolski M. Plant Physiol. 1999; 119: 1568-1569Google Scholar, 29.Borek D. Jaskolski M. Acta Biochim. Polon. 2001; 48: 893-902Crossref PubMed Scopus (77) Google Scholar), because of high sequence homology to enzymes believed to function as plant l-asparaginases. It also shows sequence similarity to aspartylglucosaminidases (AGAs) (30.Borek D. Michalska K. Brzezinski K. Kisiel A. Podkowinski J. Bonthron D.T. Krowarsch D. Otlewski J. Jaskolski M. Eur. J. Biochem. 2004; 271: 3215-3226Crossref PubMed Scopus (52) Google Scholar), which are involved in the breakdown of glycosylated proteins. However, the recently reported kinetic data indicate that the dominant activity of both EcAIII and genuine plant asparaginases is connected with the hydrolysis of isoaspartyl dipeptides, whereas their affinity for l-asparagine is relatively weak, particularly when compared with the affinity of classic bacterial asparaginases (30.Borek D. Michalska K. Brzezinski K. Kisiel A. Podkowinski J. Bonthron D.T. Krowarsch D. Otlewski J. Jaskolski M. Eur. J. Biochem. 2004; 271: 3215-3226Crossref PubMed Scopus (52) Google Scholar). There is also no detectable activity toward N-acetylglucosaminyl-l-asparagine, a typical substrate of aspartylglucosaminidases (5.Larsen R.A. Knox T.M. Miller C.G. J. Bacteriol. 2001; 183: 3089-3097Crossref PubMed Scopus (27) Google Scholar, 30.Borek D. Michalska K. Brzezinski K. Kisiel A. Podkowinski J. Bonthron D.T. Krowarsch D. Otlewski J. Jaskolski M. Eur. J. Biochem. 2004; 271: 3215-3226Crossref PubMed Scopus (52) Google Scholar). In addition, it has been shown that the homologous enzyme from S. enterica is not a rigorous dipeptidase, because it is also capable of hydrolyzing tripeptides with an N-terminal isoaspartyl residue (5.Larsen R.A. Knox T.M. Miller C.G. J. Bacteriol. 2001; 183: 3089-3097Crossref PubMed Scopus (27) Google Scholar). Among dipeptides, iAsp-Leu is recognized with the highest affinity. The structure of free EcAIII has been determined and deposited in the Protein Data Bank (PDB) independently by two groups. Borek et al. (31.Borek, D. (2001) Structural and Biochemical Studies of Asparaginases, Ph.D. Thesis, A. Mickiewicz University, PoznanGoogle Scholar) determined the structure of EcAIII at low and high calcium concentration (PDB accession codes 1K2X and 1JN9, respectively), and later Prahl et al. (32.Prahl A. Pazgier M. Hejazi M. Lockau W. Lubkowski J. Acta Cryst. D. 2004; 60: 1173-1176Crossref PubMed Scopus (16) Google Scholar) reported a structure that is essentially identical to the former (1T3M). Here, we describe the crystal structure of EcAIII in complex with l-aspartate (PDB accession code 1SEO), which is a product of the two reactions catalyzed by the enzyme (Fig. 1), as well as a mimic of the substrate of the l-asparaginase reaction. We report the ligand binding mode and investigate the conformational changes in the active site and in the overall architecture of the protein that occur upon ligand binding. Finally, the structure of the EcAIII·Asp complex provides insights into the catalytic mechanism and substrate specificity of the enzyme. Crystallization—EcAIII was produced and purified as previously described (30.Borek D. Michalska K. Brzezinski K. Kisiel A. Podkowinski J. Bonthron D.T. Krowarsch D. Otlewski J. Jaskolski M. Eur. J. Biochem. 2004; 271: 3215-3226Crossref PubMed Scopus (52) Google Scholar). Single crystals of a complex between EcAIII and l-aspartate were obtained using a modification of the protocol developed for the free enzyme (33.Borek D. Jaskolski M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 1505-1507Crossref PubMed Scopus (19) Google Scholar). The crystals were grown by the hanging drop, vapor diffusion method at room temperature. The precipitant solution contained 17% polyethylene glycol 4000, 13% polyethylene glycol 400, 80 mm CaCl2, 100 mm Tris/HCl, pH 8.5, and 100 mm sodium l-aspartate. The molar excess of l-aspartate was about 200-fold. The protein solution at a concentration of 15 mg/ml was mixed with equal volumes of the precipitant solution (2 plus 2 μl). The crystals reached their maximum size of 0.2 × 0.2 × 0.2 mm in 12 days. They belong to the orthorhombic system, space group P212121. Data Collection and Processing—X-ray diffraction data extending to 1.9-Å resolution were collected for a single crystal flash-frozen at 100 K in a gas nitrogen stream using the mother liquor as cryoprotectant. Synchrotron radiation generated at the MAX-lab (Lund, Sweden) beam-line I711 was used. The data were processed and scaled with DENZO and SCALEPACK from the HKL suite (34.Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar). Table I lists the data collection and processing statistics.Table IData collection and processing statisticsRadiation sourceLund, MAX-lab, I711Wavelength (Å)1.095Temperature of measurements (K)100Space groupP212121Cell dimensions (Å)a = 49.9, b = 77.3, c = 147.5Mosaicity (°)0.66Resolution range (Å)20.0–1.9 (1.97–1.90)aValues in parentheses correspond to the last resolution shellOscillation step (°)0.75No. of images160Reflections collected154,645Unique reflections43,625 (4,085)Completeness (%)95.8 (91.3)Redundancy3.5 (3.0)〈I〉/〈σI〉15.0 (2.2)RintbRint = ΣhΣj|Ihj – 〈Ih〉|/ΣhΣjIh j, where Ihj is the intensity of observation j of reflection h0.064 (0.256)a Values in parentheses correspond to the last resolution shellb Rint = ΣhΣj|Ihj – 〈Ih〉|/ΣhΣjIh j, where Ihj is the intensity of observation j of reflection h Open table in a new tab Structure Determination and Refinement—The structure of the EcAIII·Asp complex was solved by molecular replacement using the program MolRep (35.Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4098) Google Scholar). The search model, consisting of the complete (αβ)2 heterotetramer, was generated from the coordinates of the free enzyme deposited in the PDB under the accession code 1K2X (31.Borek, D. (2001) Structural and Biochemical Studies of Asparaginases, Ph.D. Thesis, A. Mickiewicz University, PoznanGoogle Scholar). The molecular replacement approach gave a clear solution characterized by an R factor of 36% and a correlation coefficient of 67%. The presence of the ligand molecules in the two active sites was clearly visible in the preliminary Fo - Fc electron density maps generated with phases calculated from the protein model only. Residues 1 and 162–178 from chain A (subunit α), 1 and 158–178 from chain C (subunit α), and 314–321 from chains B and D (subunits β) were not visible in the electron density maps even at the conclusion of the refinement. Cysteine 63 in both subunits α (chains A and C) carries a clear covalent modification at the Sγ atom, which according to the appearance of the electron density maps has been modeled as a β-mercaptoethanol residue. As in the free enzyme, the α subunits coordinate a structurally important sodium cation. At the initial stage of the refinement, the CNS program (36.Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunsteve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar) was used. The final refinement was carried out in Ref-mac5 (37.Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13712) Google Scholar) using the maximum-likelihood target. TLS parameters (38.Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1639) Google Scholar) defined for each of the polypeptide chains were also optimized in the refinement. 270 water molecules were included in the final model. The final R and Rfree factors converged at, respectively, 16.4% and 18.9%, and the model is characterized by very good geometry (r.m.s.d. from ideal bond lengths 0.014 Å, from ideal bond angles 1.39°). Manual modeling was done using the Xfit program from the XtalView package (39.McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2016) Google Scholar). The refinement statistics are given in Table II.Table IIRefinement statisticsResolution (Å)20.0–1.9No. of reflections in working set42,200No. of reflections in test set1372RaR = Σh||Fo| – |Fc||/Σh||Fo| for all reflections, where Fo and Fc are observed and calculated structure factors, respectively. Rfree is calculated analogously for the test reflections, randomly selected, and excluded from the refinement/Rfree (%)16.4/18.9No. of atoms (protein/ligand/solvent/Na+/Ca2+/Cl-)4267/18/270/6/1/1r.m.s.d. from idealityBond lengths (Å)0.014Bond angles (°)1.391Average B factor (Å2)14.7Residues in the Ramachandran plot (%)Most favored regions92.4Allowed regions7.6a R = Σh||Fo| – |Fc||/Σh||Fo| for all reflections, where Fo and Fc are observed and calculated structure factors, respectively. Rfree is calculated analogously for the test reflections, randomly selected, and excluded from the refinement Open table in a new tab Protein Data Bank Accession Code—The atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank with the accession code 1SEO. Structure Solution and Crystal Packing—EcAIII in complex with l-aspartate crystallizes in the orthorhombic space group P212121 with unit cell dimensions a = 49.9, b = 77.3, c = 147.5 Å. The asymmetric unit contains the (αβ)2 assembly, i.e. a dimer of heterodimers (Fig. 2a), corresponding to a Matthews volume of 2.3 Å3/Da and solvent content of 46.4% (41.Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7894) Google Scholar). Because one of the polymorphs of the free enzyme (1K2X) crystallizes in the same space group with nearly identical cell parameters (a = 50.3, b = 77.6, c = 148.2 Å) and the same cell contents (31.Borek, D. (2001) Structural and Biochemical Studies of Asparaginases, Ph.D. Thesis, A. Mickiewicz University, PoznanGoogle Scholar), it seemed natural that an electron density map calculated with the "complex" amplitudes and "free" phases should provide the structure solution. However, such a map was not interpretable. For that reason the structure was solved by the molecular replacement method with the free enzyme as the probe. The solution revealed that, despite apparent crystallographic similarities, these two crystals have quite different molecular packing. Description of the Structure—The molecular structure is created from four polypeptide chains, which have arisen from the breakdown, into subunits α and β, of two original precursors, 321 residues each. The biological assembly is, therefore, a heterotetramer, or a dimer of heterodimers, (αβ)2. The maturation process consists of the proteolytic splitting of the Gly178-Thr179 peptide and of additional degradation of all the termini, except of the N terminus of subunit β with the catalytic Thr179. The removal of the Met1 residue at the N terminus of subunit α is carried out by an E. coli methionyl aminopeptidase (42.Hirel P.H. Schmitter J.M. Dessen P. Fayat G. Blanquet S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8247-8251Crossref PubMed Scopus (660) Google Scholar). The C termini of both subunits are degraded in a slower process, which after prolonged incubation produces the following products: Gly2–Ala161 (subunit α, 160 residues) and Thr179–Gly315 (subunit β, 137 residues) (30.Borek D. Michalska K. Brzezinski K. Kisiel A. Podkowinski J. Bonthron D.T. Krowarsch D. Otlewski J. Jaskolski M. Eur. J. Biochem. 2004; 271: 3215-3226Crossref PubMed Scopus (52) Google Scholar). Although the maturation process leaves the two disjointed chain ends spatially separated, the α and β subunits originating from the same precursor are intertwined into a single protein core, particularly with regard to the extended β-sheet, which is created from strands contributed by both subunits. In particular, the N-terminal β-chains of both subunits are placed as adjacent antiparallel strands within the β-sheet structure (Fig. 2). In the final model built into electron density maps, chain A corresponds to the full mature subunit α, residues 2–161, whereas four residues are missing from the C terminus of chain C (2–157), presumably as a result of disorder. Both β subunits, chains B and D, are composed of residues 179–313, i.e. they have two residues missing at their C termini. In both α subunits, the Sγ atom of Cys63 is chemically modified by a covalent S–S attachment of β-mercaptoethanol (used in the protein preparation protocol), resulting in a modified residue annotated as Cme63. The modification does not seem to have any structural or functional consequences. The topology of the EcAIII (αβ) heterodimer is similar to that of other Ntn-hydrolases, with a typical αββα-layer structure (26.Oinonen C. Rouvinen J. Protein Sci. 2000; 9: 2329-2337Crossref PubMed Scopus (205) Google Scholar) (Fig. 2). The protein core is composed of two open β-sheets, positioned to face each other, with their strands following the same general direction. The smaller one consists of four antiparallel β-strands belonging to subunit β. The larger β-sheet is composed of eight β-strands contributed by both subunits and an additional (ninth) strand (S3β′) contributed by the other heterodimer. The overall antiparallel character of this β-sheet is violated by strand S4α, which is parallel to S3α. The layer of helices that pack on the outside of the nine-stranded β-sheet consists of five α-helices and of two 310 helices. The four-stranded β-sheet is flanked by four α-helices forming a broad crossover loop between the antiparallel strands S5β and S6β. Although EcAIII has a general fold similar to aspartylglucosaminidases, structural alignments reveal several important differences visible especially in the organization of the secondary structure elements of subunit α (Fig. 2c). In particular, the N-terminal elements S1α and H2α of EcAIII are much longer, placing the loop connecting them ∼17 Å away from the corresponding AGA loop. Metal Coordination—The loop between H3α and S2α comprises 19 residues and has a complicated conformation supported by a metal cation with a coordination geometry that is close to octahedral. The metal is chelated exclusively by main-chain C=O groups provided by residues Leu60, Glu61, Cme63, Phe66, Ala68, and Ile70 in this loop (Fig. 3a). By analogy to the structure of the free enzyme (31.Borek, D. (2001) Structural and Biochemical Studies of Asparaginases, Ph.D. Thesis, A. Mickiewicz University, PoznanGoogle Scholar), the metal cation has been identified as Na+. This interpretation has been confirmed by (i) unacceptable B factors refined for species with significantly different numbers of electrons and by (ii) the coordination geometry, in particular when interpreted by the bond valence method (43.Muller P. Kopke S. Sheldrick G.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 32-37Crossref PubMed Scopus (72) Google Scholar). The coordination spheres of both Na+ centers are very similar, with the Na+...O distances ranging from 2.2 to 2.7 Å, and the O...Na+...O angles distributed within the intervals 81–107 and 159–173°. Quaternary Structure of EcAIII—In analogy to aspartylglucosaminidases, EcAIII forms a dimer of heterodimers (αβ)2. The interface between the two (αβ) heterodimers involves a number of hydrogen bonds and hydrophobic contacts. The main interactions are found between helices H2β and between strand S4α from one (αβ) unit and strand S3β′ from the other. This antiparallel interaction (which occurs twice in the heterotetramer) extends the eight-stranded β-sheet formed within one (αβ) heterodimer into a nine-stranded structure, within which three out of the four polypeptide chains of the (αβ)2 heterotetramer meet. Additional hydrogen bonds responsible for oligomerization are formed between helix H1β and two loops from the complementary (αβ) unit. The first loop connects helix H4α with strand S4α, whereas the second loop is a hairpin connecting strands S2α and S3α. Similar regions determine the quaternary structure of AGAs, but the fragment classified here as S3β (44.Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (11986) Google Scholar) was interpreted as a loop in the aspartylglucosaminidase fold (17.Oinonen C. Tikkanen R. Rouvinen J. Peltonen L. Nat. Struct. Biol. 1995; 2: 1102-1108Crossref PubMed Scopus (156) Google Scholar, 18.Guo H.C. Xu Q. Buckley D. Guan C. J. Biol. Chem. 1998; 273: 20205-20212Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The S3β element belongs to the most conserved region of the amino acid sequences of the compared enzymes (Gly206–Pro212 in EcAIII numbering). The main difference between the interfaces found in the present isoaspartyl peptidase and in aspartylglucosaminidases is the presence in EcAIII of a pair of interacting Tyr251 residues at the C termini of the H2β helices, which bring these helices closer together. The tetrameric structure of EcAIII is found both in crystal and in solution (30.Borek D. Michalska K. Brzezinski K. Kisiel A. Podkowinski J. Bonthron D.T. Krowarsch D. Otlewski J. Jaskolski M. Eur. J. Biochem. 2004; 271: 3215-3226Crossref PubMed Scopus (52) Google Scholar). This is also true for human AGA (17.Oinonen C. Tikkanen R. Rouvinen J. Peltonen L. Nat. Struct. Biol. 1995; 2: 1102-1108Crossref PubMed Scopus (156) Google Scholar), whereas for the bacterial enzyme no (αβ)2 tetramers have been observed by size exclusion chromatography (19.Xuan J. Tarentino A.L. Grimwood B.G. Plummer Jr., T.H. Cui T. Guan C. Van Roey P. Protein Sci. 1998; 7: 774-781Crossref PubMed Scopus (28) Google Scholar). The lower stability of F. meningosepticum AGA tetramers cannot be attributed to the absence of a random coil domain at the C terminus of subunit α (19.Xuan J. Tarentino A.L. Grimwood B.G. Plummer Jr., T.H. Cui T. Guan C. Van Roey P. Protein Sci. 1998; 7: 774-781Crossref PubMed Scopus (28) Google Scholar), because no such domain is present in EcAIII. The non-crystallographic symmetry of the molecule corresponds to a nearly ideal 2-fold rotation (179.7°), as in the free enzyme, but the r.m.s.d. between the superposed Cα atoms is relatively high (0.45 Å). The source of this discrepancy lies in some small but significant deviations between the α-subunits (0.50 Å), whereas the β-subunits are practically identical (0.28 Å). The differences between the α-subunits seem to be due to packing intera