X-ray Structure of Isoaspartyl Dipeptidase from E. coli: A Dinuclear Zinc Peptidase Evolved from Amidohydrolases

doi:10.1016/S0022-2836(03)00845-3

J. Mol. Biol. (2003) 332, 243–256

X-ray Structure of Isoaspartyl Dipeptidase from E. coli: A Dinuclear Zinc Peptidase Evolved from Amidohydrolases Daniela Jozic, Jens T. Kaiser, Robert Huber, Wolfram Bode and Klaus Maskos* Max-Planck-Institut fu¨r Biochemie, Abteilung Strukturforschung, Am Klopferspitz 18a, D-82152 Martinsried, Germany

L -aspartyl and L -asparaginyl residues in proteins spontaneously undergo intra-residue rearrangements forming isoaspartyl/b-aspartyl residues linked through their side-chain b-carboxyl group with the following amino acid. In order to avoid accumulation of isoaspartyl dipeptides left over from protein degradation, some bacteria have developed specialized isoaspartyl/b-aspartyl zinc dipeptidases sequentially unrelated to other peptidases, which also poorly degrade a-aspartyl dipeptides. We have expressed and crystallized the 390 amino acid residue isoaspartyl dipeptidase (IadA) from E. coli, and have determined its crystal structure in the absence and presence of the phosphinic inhibitor Asp-C[PO2CH2]LeuOH. This structure reveals an octameric particle of 422 symmetry, with each polypeptide chain organized in a (ab)8 TIM-like barrel catalytic domain attached to a U-shaped b-sandwich domain. At the C termini of the b-strands of the b-barrel, the two catalytic zinc ions are surrounded by four His, a bridging carbamylated Lys and an Asp residue, which seems to act as a proton shuttle. A large b-hairpin loop protruding from the (ab)8 barrel is disordered in the free peptidase, but forms a flap that stoppers the barrel entrance to the active center upon binding of the dipeptide mimic. This isoaspartyl dipeptidase shows strong topological homology with the a-subunit of the binickel-containing ureases, the dinuclear zinc dihydroorotases, hydantoinases and phosphotriesterases, and the mononuclear adenosine and cytosine deaminases, which all are catalyzing hydrolytic reactions at carbon or phosphorous centers. Thus, nature has adapted an existing fold with catalytic tools suitable for hydrolysis of amide bonds to the binding requirements of a peptidase.

q 2003 Elsevier Ltd. All rights reserved. *Corresponding author

Keywords: zinc peptidase; dipeptidase; isoaspartate; inhibitor complex; amidohydrolases

Introduction Proteins undergo several age-dependent modifications such as oxidation, hydrolysis, deamination, racemization, disulfide bond breakage, and ketoamine formation. One of the most prominent ageing reactions in the polypeptide chain is the Present address: J. T. Kaiser, California Institute of Technology, MIC 114-96, Pasadena, CA 91125, USA. Abbreviations used: IadA, isoaspartyl dipeptidase; IaaA, isoAsp aminopeptidase. E-mail address of the corresponding author: [email protected]

succinimide-mediated rearrangement of L -Asp and L -Asn residues to L /D -isoAsp/b-Asp residues, which are amide linked to the following amino acid in the chain through their side-chain b-carboxyl group, possessing an extra methylene group in the polypeptide backbone.1 – 3 The probability of this reaction is higher, when the Asn/Asp residue is located in a flexible segment, or when the carboxyl-sided residue, such as Gly or Ser, mediates enhanced chain flexibility.4 The presence of isoAsp may result in altered biological function as exemplified by the reduced ability of isoAspcalmodulin to exhibit calcium dependent enzyme activation5 and reduced enzyme activity of

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244 modified ribonuclease A6 and Escherichia coli phosphocarrier protein7 (HPr). This modification is also thought to be one of the critical progression factors in Alzheimer’s disease8,9 and may be involved in the onset of autoimmune diseases.10 Structures of isoAsp residues have been reported for several proteins such as bovine pancreatic ribonuclease A,11 hen egg white lysozyme12 and porcine b-trypsin.13 To cope with the accumulation of this modification, nature has evolved two strategies. On the one hand, proteins can be repaired via methyl ester formation catalyzed by the L -isoaspartyl-(D aspartate)-O-methyltransferase,14,15 which recognizes only poorly the L -isoaspartyl residues in dipeptides.16 On the other hand, such damaged proteins are degraded by the cellular protease machinery leading to quite proteolysis-resistant isoAsp-containing b-linked dipeptides,17 which have to be cleaved into the free amino acid residues by a specialized peptidase, the isoaspartyl/ b-aspartyl dipeptidase. Enzymes with isoaspartyl dipeptide cleaving activity were initially isolated from bacteria and mammals.18,19 Experiments with radiolabeled isoaspartyl dipeptides and tripeptides administered to rats indicated that a significant amount of these peptides was metabolized rather than excreted.19 Isoaspartyl dipeptidase was found to be present in rat kidney, liver, brain, lung and muscle. It was partially purified from rat liver and hydrolyzed preferentially isoAsp-Gly dipeptides and also cleaves tripeptides, but has never been characterized at a molecular level.19 More recently, Gary & Clarke20 isolated and overexpressed the 41 kDa isoaspartyl dipeptidase (IadA) from E. coli, which preferentially cleaves isoAsp dipeptides with a hydrophobic carboxyterminal amino acid, but is inactive towards isoAsp dipeptides with Gly or His in second position.20,18 The enzyme formed oligomers of an apparent size of 120 kDa, as determined by gel filtration. L -aAsp-Leu is among the substrates, while tripeptides and g-glutamyl peptides proved to be no substrates for IadA indicating that the enzyme activity does not overlap with g-glutamyltranspeptidase.21 Furthermore, in an attempt to identify peptidases of Salmonella typhimurium which allows growth on the dipeptide Asp-Leu, IadA from S. typhimurium was cloned.22 This dipeptidase shares 82% identity and 90% similarity with the E. coli enzyme and forms oligomeric complexes with molecular weights of about 200 kDa. Besides IadA, Larsen and co-workers22 found a second isoAsp dipeptide-cleaving enzyme, an N-terminal nucleophile protease named isoAsp aminopeptidase (IaaA), which possesses asparaginase activity in addition.23 The presence of such a second isoaspartyl dipeptidase is in agreement with findings of Gary & Clarke that E. coli IadA deletion strains are still able to process isoAsp dipeptides.20 Inhibitor studies confirmed that IadA is a zincdependent enzyme with some sequence similarity

Crystal Structures of Isoaspartyl Dipeptidase

to bacterial dihydroorotase, defining the M38 family of the MX clan of metalloproteases.18,21 Here we describe the cloning, purification and crystallization of IadA from E. coli. We have solved crystal structures in the presence and in the absence of a transition state analog, AspC[PO2CH2]-LeuOH, which resembles the transition state intermediate formed in the course of the reaction with the substrate aAsp-Leu and explains the preference of IadA for a and b-Asp dipeptides.

Results Overall structure The structure of IadA was solved with the help of multiple wavelengths anomalous diffraction (MAD) data measured at the zinc absorption edge. The anomalous difference Patterson map immediately revealed the presence of two adjacent zinc ions in the active site. In the final structure, the polypeptide chain is fully defined from Met1 up to Glu388, i.e. except for the last two C-terminal residues Thr389 and Ala390. The crystal cell contains two equivalent well demarcated ˚ thick octameric particles of propeller-like 75 A ˚ distance between adjacent vertices. This 120 A propeller is formed by four dimers shaping a central cavity and two pores of exact 4-fold rotational symmetry (Figure 1(a)). The electrostatic surface potential of the octamer is negative, with negatively charged residues being clustered in particular along the central expanding pore (Figure 1(b)). Every 458, each octamer is dissected by local but virtually exact 2-fold rotation axes, conferring each particle a 422 point group symmetry (Figure 1(a)). According to dynamic light scattering data, which yielded an apparent molecular weight of about 330 kDa for the soluble material before crystallization, these octamers are also the predominant IadA particles in solution (data not shown). Each monomer, possessing a sickle-like shape, consists of two adjacent domains, a catalytic domain and a laterally attached b-sandwich domain. The oblate ellipsoidal-shaped catalytic domain formed by residues 55 –354 contains all zinc liganding and catalytic residues together with two coordinated zinc ions. It essentially consists of an elliptically distorted (ab)8 triosephosphate isomerase (TIM)like barrel, where eight twisted parallel b-strands form a closed barrel surrounded by eight a-helices (Figure 2).24,25 The interior of this barrel, mainly filled with bulky hydrophobic side-chains, is confined on the barrel exit (corresponding to the C-terminal ends of the barrel-forming b-strands) by a funnel-shaped pocket lined by the Znbinding and catalytic residues donated from the C-terminal ends of strands s1, s4, s5, s6 and s8 (see Figure 2). On the lower half, this catalytic site depression is bordered by the particularly projecting loops s1-h1 (forming strands sa and sb), s2-h2

Crystal Structures of Isoaspartyl Dipeptidase

245

Figure 1. Stereo view of the quarternary assembly of isoaspartyl dipeptidase (IadA). (a) The octamer can be regarded as a tetramer of dimers with the indicated 422 symmetry. The nomenclature of the monomers as well as the location of the active site zincs (magenta spheres) are given. The monomers in the foreground are colored in light green (A), orange (B), green (C), and blue (D), the monomers in the background are colored in gray. Dimers are formed between molecules A– E, B –F, C –G and D– H, respectively. (b) Surface of isoaspartyl dipeptidase. The electrostatic potential was calculated with GRASP,48 using charges according to Weiner and co-workers.50

and s4-h4. In the dipeptidyl phosphinate complex, this collar-like structure is opposed by the long s8-h8 b-hairpin loop comprising Gly288Ala305 (also denoted as s9 and s10, see below), which acts as a flap, being disordered (open) in the free enzyme but closed in productive enzyme – substrate complexes obstructing access towards the catalytic center (see Figure 4). The opposite barrel side, its entrance, is delimited by the bipartite helix hI/hII and the following

multiple loop segment terminating the polypeptide chain of the catalytic domain. The b-sandwich domain basically consists of a bipartite U-shaped b-sheet to which the N-terminal strands sI– sV and the C-terminal strands sVI – sIX (residues 356– 383) add under formation of the five-stranded and four-stranded partial sheets, respectively (Figure 2). This domain is laterally attached to the TIM barrel entrance via two short linkers formed by Pro62-Gly63 and Thr343-Glu347,

246

Crystal Structures of Isoaspartyl Dipeptidase

Figure 2. Stereo view of the ribbon diagram of the IadA monomer. (a) The a-helices and the b-strands composing the (ab)8-barrel are shown in green and blue, respectively, and two additional helices (hI and hII) on the backside of the barrel are shown in turquoise. The gray balls indicate the beginning and the end of the active-site loop (flap), which is not defined in the native structure. The b-strands in the b-sandwich domain (residues 1 – 61 and 348– 390) are highlighted in light green and orange, respectively. (b) Secondary structure plot. The secondary structure elements of IadA are shown above the amino acid sequence. Secondary structure elements are colored according to (a) except for the TIM-barrel helices, which are highlighted in red. Zinc binding residues are marked with blue background and closed triangles. Asp285, which is involved in zinc binding and catalysis, is marked by an open triangle on top, while Tyr137 is marked by an open triangle below.

247

Crystal Structures of Isoaspartyl Dipeptidase

and is located nearly opposite to the active site. It appears to play a structural role by interacting with the equivalent domain of the dimer partner at the outer end of each blade (Figure 1(a)). In each dimer fragment, two (virtually identical) sickle-like monomers are arranged around a (local) dyad, forming two symmetric essentially hydrophobic contacts via distal parts of both bsandwich and both catalytic domains, respectively. ˚ 2 and is The first, peripheral interface covers 700 A formed through connecting loop residues of the four-stranded sheet of the b-sandwich domain, ˚2 while the second, internal contact covers 680 A and is mediated through side-chains of helix h3 and the s3-h3 loop of the (ab)8 barrel. These dimers are associated around an exact 4-fold rotation axis to form the octamer, leaving a ˚ wide cavity accessible from the outcentral 30 A ˚ wide pores on the front and the side via two 6 A ˚2 back side (Figure 1(a)). The non-symmetric 675 A contacts between the adjacent four “upper” (and accordingly the four “lower”) monomers are made between residues of the s4-h4 loop on one side and helix h4 and the h5-s6 loop on the other side, ˚ 2 contacts between while the symmetric 955 A adjacent upper and lower monomers are mediated through the hairpin strand sb, helix h2 and the C-terminal end. The pores are mainly lined by the s4-h4 loops and helices h4, with the Asp174 sidechains projecting into the pore and creating a strong negative electrostatic potential (see Figure 1(b)). In the octameric particles, the active centers of the individual monomers do not face this central cavity, but are inclined, directed towards the neighboring dimer (blade) and are apparently quite accessible for substrates (in the free state, neglecting the flap).

residues are located at or near the C-terminal ends of the central parallel b-strands s1 (His68 and His70), s4 (Lys162p), s5 (His201), s6 (His230), and s8 (Asp285) and are lining the funnel-shaped depression. In the native structure, all ligands and the two zinc ions are well defined by electron den˚ sity (Figure 3), with both zinc ions placed 3.5 A apart from each other. The more buried zinc, Zn1, is 5-fold coordinated in a distorted trigonal bipyramidal manner, liganded by His68 N12, His70 N12 and water molecule Wat1 arranged in plane with Zn1, and with one of each of the carboxyl oxygens of the Nz-carboxylated Lys162 and of Asp285 as apical ligands. The second more solvent exposed zinc ion (Zn2) is tetrahedrally coordinated by the second oxygen of the carbamylated Lys162 (acting thus as a twisted but bridging ligand), His201 Nd1, His230 N12 and Wat1 (the second bridging ligand) ˚, in a first shell at an average distance of 2.2 A ˚ while Tyr137 Og is more distantly located (3.3 A apart) (see Figure 4). The coordinating His sidechains are further hydrogen bonded through their free N12 or Nd1 function to surrounding enzyme groups or internal ordered water molecules. The electron density of crystals, grown within a few days from freshly purified IadA sample obtained from our overexpressing strain, and immediately frozen and measured, unequivocally indicated the presence of a non-modified Lys162 and of a single zinc ion, coordinated in the middle of all four histidines and Asp285 with an average ˚ (data not shown). Obviously, distance of 3.5 A maturation of IadA requires time and appropriate carbonate supply, and the occupation of the zinc sites is dependent on the presence of such a carbamoyl-lysine. We could not detect any significant activity of IadA prior to this time-dependent activation (data not shown).

The active site of the native enzyme In the active center, four His, one carbamylated Lys and one Asp residue are together involved in binding of the two catalytic zinc ions. These

Binding of the transition state analogue phosphinate inhibitor IadA was successfully co-crystallized with a

Figure 3. Stereo view of the active site of native IadA in density. The zinc-liganding residues (blue), the two zinc ˚ 2Fobs 2 Fcalc electron density is ions (magenta) and the catalytic water/hydroxyl ion (blue) are shown. The final 2.0 A contoured at 1.0s.

248

Crystal Structures of Isoaspartyl Dipeptidase

Figure 4. Stereo views of the binding site of IadA shown in standard orientation. (a) The phosphinic inhibitor AspC[PO2CH2]-LeuOH is shown in green (carbon atoms), red (oxygen atoms), dark blue (nitrogen atoms) and purple (phosphorous atoms), while the surrounding IadA residues are colored in blue (carbons). The hydrogen bonding net˚ . (b) Electron density around work and the zinc interactions are shown as dotted lines, with distances depicted in A the bound inhibitor (orange). The inhibitor (green) and the IadA environment (blue) are shown as stick models. The ˚ 2Fobs 2 Fcalc simulated annealing omit map has been contoured at 1.0s. The residues of monomer D which con3.3 A tribute two weak hydrogen bonds to the stabilization of the active-site flap are shown in orange and are marked by asterisks. (c) Electron density around bound asparagine. A sulfate ion is depicted as stick model in yellow. The ˚ 2Fobs 2 Fcalc simulated annealing omit map has been contoured at 1.0s. 2.7 A

249

Crystal Structures of Isoaspartyl Dipeptidase

racemic mixture of the transition state analogue Asp-C[PO2CH2]-LeuOH, where the scissile peptide bond between P1-Asp and P10 -Leu (with P1 and P10 designating the substrate/inhibitor residues N and C-terminal of the scissile bond, and S1 and S10 the opposite peptidase subsites) is replaced by a phosphinic acid group. Due to the method of synthesis, this pseudopeptide contains two asymmetric carbons, giving rise to two pairs of diastereomers.26 The L ,L -stereoisomer was built ˚ electron density of the IadA complex into the 3.3 A due to its occurrence in digests of native proteins and because of the best fit in the electron density (Figure 4(a) and (b)). In this complex, the inhibitor moiety seems to be tightly fixed through its two carboxylate groups with mainly polar groups of the enzyme (Figure 4(a) and (b)), which do not considerably change their conformation and position upon binding except for the flap, that becomes ordered and covers the active site groove. The side-chain carboxylate group of Asp1I (with the suffix I indicating inhibitor residues) is trapped in a narrow cavity, which is left open by the four-glycine stretch Gly73-Gly76, and is hydrogen bonded with the main-chain amide nitrogens of Thr106 and Gly75 and the side-chain of Thr106. The anion hole character of this cavity is underlined by another IadA complex with an S1’ bound Asn molecule, where a sulfate ion replaces this Asp-P1 side-chain carboxyl group (Figure 4(c)). The a-amino group of the dipeptide inhibitor is directed towards the fixed hairpin flap Asn287Ala305, forming hydrogen bonds to Ser289 O and Og (Figure 4(b)). The phosphinate group of the inhibitor “rides” on top of the (imaginary) zinc – zinc line, with the phosphinate oxygens located asymmetrically on either side. Oxygen O2P is placed between both zinc ions but significantly closer to Zn1, and is in addition in hydrogen bond distance to the non-liganding carboxylate oxygen of the catalytic Asp285. Superposition with the native enzyme structure shows that this O2P oxygen is located close to the catalytic water molecule (see Wat1 in Figure 3) in native IadA, indicating that this O2P oxygen corresponds to the catalytic hydroxyl ion after attack of the scissile peptide bond. The phosphinate oxygen O1P, supposed to mimic the carbonyl oxygen of the attacked peptide bond, becomes a fourth binding partner of Zn2. In addition, O1P forms a hydrogen bond to Tyr137 Og, which together with Zn2 represent the electrophiles forming the oxyanion hole for catalysis. The methylene group following the phosphinate group, which mimics the protonated (sp3 hybridized) peptide nitrogen of a bound dipeptide, is hydrogen bonded to the Ser289 carbonyl oxygen, but also positioned in hydrogen ˚ ) to the free Asp285 carboxylbond distance (3.1 A ate oxygen, supposed to function as a proton shuttle during the reaction. The carboxylate group of LeuI2 forms a hydrogen bond network with the two guanidyl groups of Arg233 and Arg169, which both contribute to a

“carboxylate groove”. Both guanidyl groups are further fixed in their proper binding orientation via hydrogen bonds to the side-chain carboxylate groups of Asp204, Asp167 and Glu77, respectively (see Figure 4(b)). The Leu2I side-chain, not well ˚ electron density, extends into defined in the 3.3 A ˚ wide channel, which a mainly hydrophobic 4 –5 A opens to the bulk solvent. Here, small hydrophilic P10 side-chains could interact with Gln290 and larger hydrophobic side-chains could pack between Phe292 and Ile257, while Arg or Lys-P10 side-chains would reach Asp258 and Glu259 at the channel exit (see Figure 4(b)). The flexible b-hairpin flap, especially its N-terminal strand Gly288Phe292 (strand s9), contributes substantially to the formation of the substrate binding sites by covering the bound inhibitor/substrate and orienting the scissile peptide bond. Structural homology with other enzymes Due to its (ab)8 barrel, IadA shows topological similarity with many TIM-like structures.25 Due to the leveling off of the IadA strands s1, s2 and s3 towards the entrance side of the barrel, most of these regular TIM barrel structures deviate considerably from the IadA barrel, however (with about 50% or less of the a-carbon barrel atoms ˚ , using a exhibiting rms deviations of about 1.8 A ˚ cutoff of 2.5 A). A significantly closer topological similarity is found, however, for some pyrimidinases and other (amido)hydrolases such as L and D -hydantoinases, adenosine and cytosine deaminases, dihydroorotases, phosphotriesterase and ureases,27 which catalyze diverse hydrolytic reactions at carbon and phosphorus centers in purine, pyrimidine, cytosine, histidine and urea degradation, but also pyrimidine biosynthesis, detoxification and aminoacylation. IadA exhibits the closest overall structural similarity in the subset of enzymes with binuclear metal centers with the D -hydantoinase from Thermus sp. (PDB code 1GKP)28 and the L -hydantoinase from A. aurescens (PDB code 1GKR)29 containing binuclear zinc centers, and with the a-subunits of the binickel containing ureases from K. aerogenes (PDB code 2KAU)30 and B. paseurii (PDB code 2UBP),31 which all four exhibit, beside the (ab)8 catalytic domain, a somewhat similar b-sandwich domain (see urease in Figure 5(a)). The b-sandwich domains show about 70% of all a-carbon atoms to be topologically equivalent, with rms deviations of about ˚ (hydantoinases) and 1.5 A ˚ (ureases). 1.4 A Further closely related enzymes are the binuclear zinc center-containing structures of the dihydroorotase from E. coli (PDB code 1J79)32 and the phosphotriesterase from Ps. diminuta (PDB code 1PTA),33 which essentially consist of the catalytic domain alone, with about 67 and 69% of the a-car˚ and 1.47 A ˚, bons showing rms deviations of 1.61 A respectively. The similarity to the subset of mononuclear enzymes adenosine34 and cytosine ˚ ). deaminase35 is lower (rms deviations above 1.7 A

250

Crystal Structures of Isoaspartyl Dipeptidase

Figure 5. Stereo view of the structural comparison of IadA with urease and dihydroorotase. (a) Superposition of the Ca backbones of IadA (beige), urease (blue, PDB code 2UBP) and dihydroorotase (green, PDB code 1J79). The structural alignment was performed using the program TOP.46 (b) Superposition of the zinc binding residues. The zinc ions of IdaA (beige) are shown with the corresponding residues of urease (blue, PDB code 2UBP) and dihydroorotase (green, PDB code 1J79). The numbers are according to the numbering of IadA.

The sequence identities between these structurally homologous enzymes and IadA based on a topological alignment are relatively low, e.g. only 12 and 7% for the K. aerogenes urease and the E. coli dihydroorotase. Much more similar in structure and sequence are the catalytic centers and their immediate vicinity as exemplified by urease and dihydroorotase in Figure 5(b), but in particular also both hydantoinases. Noteworthy is the presence of phenolic side-chains in the D -hydantoinase and in the dihydroorotase, which might be functionally equivalent to Tyr137 of IadA (see below). In contrast, residues involved in the fixation of the respective substrates differ completely in the different hydrolases. Interestingly, in all of these IadA homologues the active site groove is surrounded by loops, which project away from the (ab)8 barrel and somewhat cover bound substrates, but show completely different conformations. Pre-

sumably, one or the other of these loops is flexible allowing free entry and release to substrates and products. Of the IadA-related enzymes, a flap-like movement of a covering loop has only been shown yet for the exposed helix-turn-helix loop inserted in the s7-h7 loop of the (ab)8 barrel of B. pasteurii urease.31

Discussion When we started this project, it was known that IadA should bear some resemblance with dihydroorotase and ureases, and seemed to be a zinc hydrolase.21 The structure analysis revealed a much closer topological equivalence than expected from the sequence similarity. Thus, IadA exhibits an (ab)8-barrel catalytic domain, as exemplified by dihydroorotase,32 laterally attached to a b-sandwich domain, as known for some of

Crystal Structures of Isoaspartyl Dipeptidase

the structurally related hydrolases, in particular the hydantoinases28,29 and the ureases.30,31 In accordance with these bizinc/binickel hydrolases, IadA possesses in its active-site groove situated at the C-terminal end of the (ab)8 barrel a pair of zinc ions, which are bound and presented by the side-chains of four His residues, of a bridging carbamoyl-lysine, and of an Asp residue. As in the structurally related amidohydrolases, nature might have utilized a nitrogen-carboxylated Lys residue and preferred it over a Glu because of enhanced steric flexibility, potential regulation and enhanced delocalization of the negative charge. On one side, the active-site groove is bordered by a few loops projecting from a few strand-helix loops of the barrel that are opposed by a flap. This flap appears to be flexible in the absence of specifically bound substrates, but closes upon productive binding. In contrast to earlier biochemical data, IadA seems to exist as a homo-octameric particle, ˚ diameter with 422 symmetry and a central 30 A cavity linked to the outside by two small channels only. However, the active-site grooves open towards bulk water, from where substrate molecules have direct access to the active center. The IadA structure was solved for the native enzyme and for a transition state intermediatemimicking complex containing a dipeptidyl phosphinate with an N-terminal L -aAsp and a C-terminal L -Leu residue, i.e. amino acid residues also occurring in dipeptides being hydrolyzed by IadA. Thus, the involvement of distinct residues in substrate binding and cleavage and the interaction geometry of reaction intermediates can be delineated with great confidence. The substrate binding and cleavage mechanism thus should be, similar to that proposed for bizinc exopeptidases (see, e.g. Jozic and co-workers36) and other amidases, as follows (see Figure 4(a)): With the flap open, the dipeptidyl substrate (with residues designated by the suffix S) can, preoriented by opposite electrostatic potentials, enter the active-site groove and bind in such a way that the Asp1S-P1 side-chain carboxylate group and the C-terminal carboxylate become clamped between the anion binding hole and the carboxyl hole. Upon flap closing, induced probably in particular by medium sized hydrophobic P10 -sidechains, Ser289 O will hydrogen bond and fix the scissile peptide bond via its NH group. In this way, water molecule Wat1 (positioned close to O2P of the phosphinate) will be squeezed between the carboxylate group of Asp285 and the scissile peptide carbonyl, acidified mainly through the interaction with Zn1. After abstraction of a proton by the free Asp285 carboxylate oxygen acting as a catalytic base, the resulting Zn1-oriented hydroxyl nucleophile can attack the scissile carbonyl carbon (positioned close to the phosphinate P). The reaction proceeds under formation of and transition through a tetrahedral intermediate, a reaction facilitated by the polarization of the carbonyl group of the scissile peptide bond through Zn2

251

and the side-chain hydroxyl group of Tyr137. The transferred proton(s) can be shuttled via the Asp285 carboxylate to the leaving group nitrogen (equivalent to the Leu2I-P10 methylene group in the phosphinate). This favors the break of the C – N amide bond, so that both separated amino acids can leave the substrate binding site. The flap should become flexible again as soon as the peptide bond is broken, thus exhibiting a kind of gating function during catalysis. Tripeptides are probably not bound due to the loss of favorable interactions formed by the C terminus of the dipeptide with the carboxylate hole, in agreement with the lack of activity of IadA against tripeptides.21 The binding geometry of the corresponding bAsp-Leu dipeptide is presumably similar to that of aAsp-Leu (Figure 4(a)), i.e. with the Asp1S-P1 amino group shifted to the other methylene group and directed towards the catalytic center, where it could interact with one of both carboxylate oxygens of the carbamoyl-lysine162. Only such a conformation would allow the tight hydrogen bonds and salt bridge interactions of the dipeptidyl carboxylate groups in the anion hole as well as in the carboxylate groove conferring a precise scissile bond presentation toward the two zinc ions, the squeezed water and the catalytic Asp285 carboxylate proton shuttle. The additional fixation provided by the shifted amino group might contribute to the enhanced hydrolysis activity toward bAsp-Leu compared with aAsp-Leu. As mentioned above, dihydroorotase, which catalyzes the reversible cyclization of carbamoyl L -aspartate to L -dihydroorotate, had originally been identified by sequence search as a structural homologue of IadA,21 what has been confirmed in this work. As shown by the superposition on our IadA-phosphinate complex based on an optimal fit between both proteins (Figure 6), the L -dihydroorotate molecule, tightly fixed via the carboxylate group to the side-chains of Arg20, Asn44 and His254, is bound in the dihydroorotase in such a way that its scissile amide group superimposes with the phosphinate/scissile peptide group of the phosphinate/dipeptide substrate in IadA. The zinc-coordinating residues, the squeezed water molecule and the Asp250 carboxylate shuttle replace the equivalent residues/groups in IadA, so that very similar cleavage intermediates and mechanisms have to be assumed.32 Noteworthy, the Tyr104 phenolic side-chain of dihydroorotase, after a slight rotation, could act, like Tyr137 of IadA, as a second electrophile (Figure 6). An important geometric difference between both enzyme activities is the cis-amide bond in dihydroorotate, compared with the trans-peptide bond in a bound dipeptide substrate of IadA. These different binding geometries are thus not adequately represented by usual comparison schemes stressing the similar geometries of both substrates.20 It might be added that hypoxanthine, soaked into IadA crystals, was trapped in a similar manner as

252

Crystal Structures of Isoaspartyl Dipeptidase

Figure 6. Stereo view of a superposition of the active site of inhibited IadA with the substrate/product filled site of dihydroorotase. The phosphinic inhibitor Asp-C[PO2CH2]-LeuOH and the surrounding IadA residues are shown in beige, while dihydroorotate and dihydroorotase are presented as green stick models. The catalytic water/hydroxyl ion of dihydroorotase is depicted in blue. Structures were superimposed using the active site residues and the program TURBO.51

dihydroorotate in dihydroorotase, likewise exposing its C6 – N1 bond to the catalytic residues (data not shown). In conclusion, for the design of the isoaspartyl dipeptidase, nature has not modified existing peptidases, but has rather adapted the catalytic domain and hydrolase apparatus of the binuclear amidohydrolases to the requirement of specifically binding and cleaving preferentially b-linked AspXaa dipeptides, a nice example for divergent evolution. This molecular evolution resulted in a quite efficient dipeptidase, successfully coping with the problem of degrading bAsp-Xaa dipeptides, which occur by spontaneous protein modification.

Materials and Methods Cloning procedure The iadA open reading frame from E. coli W3110 was amplified by PCR using primers complementary to the 50 end (50 -CAAGGAGTTATCATGATTGATTATACCG CAGCCGG-30 ) and to the 30 end (50 -CGGGCGGCTG ACAAGCTTTTAAGCCGTTTCAAACGTTCC-30 ) which concomitantly incorporated a BspH I and a Hind III restriction site (underlined), respectively. The purified PCR product was cloned into pTrc99a (Pharmacia) via the restriction sites Nco I and Hind III and the resulting vector was named pIadA. Protein expression and purification Briefly, cultures of E. coli JM109/pIadA were grown at 37 8C, and the production of protein was induced by the addition of 1 mM IPTG at an OD595 ¼ 1.0. Cells were harvested after four hours by centrifugation and were disrupted by sonification. This crude extract was centrifuged and the supernatant was applied to a DE52

column (Whatman). The protein was eluted from the column with a gradient of 0 – 500 mM NaCl in 10 mM Tris-HCl at pH 8.0. Isoaspartyl dipeptidase containing fractions were pooled and ammonium sulfate was added to a final concentration of 1 M before the protein solution was applied onto a Phenyl Sepharose High Performance column (Pharmacia). The proteins were eluted from the column using a linear gradient from 1 M to 0 M ammonium sulfate. Isoaspartyl dipeptidase containing fractions were immediately pooled and concentrated for crystallization or alternatively dialyzed against 10 mM Tris-HCl at pH 8 and further purified on a Sephadex 75 gel filtration column (Pharmacia). Dynamic light scattering Dynamic light scattering experiments were performed using a DynaPro Dynamic Light Scattering Instrument (Protein Solutions, Lakewood, USA) with runs of 25 scans of 15 s each at 295 K. Data were analyzed using the program DYNAMICS v.3.30. IadA in 10 mM Tris-HCl (pH 8) and ,200 mM ammonium sulfate was filtered through 0.1 mm Anotop filters (Whatman, Kent, UK) to remove dust particles and larger aggregates that might interfere with the measurement. Crystallization Crystals were grown at 20 8C by sitting drop vapor diffusion method in droplets composed of one part protein solution (17.5 mg/ml in 10 mM Tris-HC (pH 8) with or without ,200 mM ammonium sulfate) and one part of the crystallization buffers (0.1 M lithium sulfate monohydrate, 0.1 M tri-sodium citrate dihydrate at pH 5.6, 12% w/v PEG 4000 or 20% PEG 3350, 0.2 M ammonium fluoride (pH 6.2)). In order to obtain cocrystals, 4 mM of the phosphinic inhibitor AspC[PO2CH2]LeuOH or 2 mM asparagine was added to the respective crystallization buffers (inhibitor: 20% PEG 3350, 0.2 M ammonium fluoride (pH 6.2); asparagine: 20% PEG 3350, 0.2 M ammonium fluoride (pH 6.2)). For data collections, crystals were transferred into cryobuffer

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Crystal Structures of Isoaspartyl Dipeptidase

(crystallization buffer containing 20% PEG 300) and were flash frozen under a stream of nitrogen at 2180 8C. Due to the method of synthesis, the phosphinic inhibitor used for crystallization experiments contained two asymmetric carbons which gave rise to four different compounds.26 However, omit maps calculated for the inhibitor showed the presence of (L )Asp-C[PO2CH2](L )LeuOH in the active site of IadA. The transition state analogue of Asp-Leu had to be used because (L)isoAspC[PO2CH2](L)-LeuOH was not available. After one week, the crystals reached a final size of 0.5 mm £ 0.3 mm £ 0.25 mm. The crystals are of space group P421 2, contain two molecules in the asymmetric ˚ resolution (see Table 1). unit and diffract up to 2.0 A Crystals of the native enzyme and crystals with the phosphinic inhibitor or asparagine were used for measurements after two to three months, while the crystals containing the apoenzyme were used for data collections after one week. The zinc ion positions in the older crystals were occupied to about 60%. Data collection, structure solution and refinement ˚ were collected from one single MAD data to 2.0 A crystal at three different wavelengths, using synchrotron radiation at the beamline BW6 of DESY (Deutsches Elektronensynchrotron, Hamburg, Germany) with wave˚ ) and f00 lengths at the Zn– K absorption edges f0 (1.2830 A ˚ ˚ (1.2817 A) and a remote wavelength (1.050 A) using a Mar-Research CCD detector (Mar Research, Hamburg, Gemany). The statistics of the MAD data set are summarized in Table 1. At each of the three wavelengths, rotation data were collected in frames of 18 through a contiguous angular range of 908 followed by another

contiguous range of 908 for measuring the Friedel pairs in inverse beam geometry. The diffraction data were processed and scaled by the programs DENZO/SCALEPACK.37 The zinc positions were localized in anomalous difference Patterson maps and were refined with MLPHARE/CCP4.38 After solvent flattening and density ˚ electron density map was modification (DM),39 the 2.0 A clearly interpretable. Model building was performed using the program MAIN.40 The calculations of the electron density maps and the crystallographic refinement were performed using CNS,41 using a simulated annealing procedure. Individual B-factor refinement and three rounds of automatic water building and deletion were performed. A final simulated annealing and positional refinement resulted in an Rfactor of 19.8 and an Rfree of 22.9. All refinements were performed using the maximum-likelihood target with Hendrickson – Lattman coefficients.42 The inhibitor and asparagine complexes were solved by molecular replacement with the program molrep.43 The search model was generated from the coordinates of native isoaspartyl dipeptidase. After rotational search ˚ resolution, two solutions were with data to 3.5 A obtained, accounting for the two molecules in the asymmetric unit and yielding correlation coefficients of 73.3% and 69.7% for the inhibitor and the asparagine containing complexes, respectively, and R-factors of 31.4% and 32.2%, respectively (see Table 2). For all models, strict non-crystallographic symmetry (NCS) restraints were applied, except for the last cycle of refinement. Thereafter, both molecules could be built into the clearly defined electron density. Residues 288– 304 remained undefined in the electron density map of the asparagine containing complex probably due to local disorder.

Table 1. Data collection and refinement statistics for native isoaspartyl dipeptidase Data set

Edge

Peak

Remote

˚) Wavelength (A 1.2830 1.2817 1.0500 ˚) Resolution (A 19.8–2.0 19.8–2.0 19.8–2.0 Redundancy 3.80 3.47 1.41 Unique reflections 64,424 60,181 60,055 Completeness overall (%) 94.2 94.6 90.2 Outermost shell (%) (2.03–2.00) 94.2 92.4 87.8 I/s 22.7 23.8 14.9 5.5 6.2 4.3 Rsyma (%) Space group P4212 ˚ ˚ ˚ Cell dimensions a ¼ 116.64 A b ¼ 116.64 A c ¼ 137.12 A Volume fraction of protein (%) 56.4 ˚ 3/Da) 2.84 Vm (A Total number of residues 742 Total non-H-atoms 5500 Number of water molecules 540 Number of zinc atoms 4 Temperature factors ˚ 2) 25.8 Protein (A ˚ 2) 36.6 Solvent (A ˚ 2) 28.3 Metal (A ˚) Resolution range of reflections used (A 20.0–2.0 19.76 Rfactor (%)b 22.85 Free Rfactor (%)b Stereochemical ideality ˚) Bond (A 0.005 Angle 1.438 P P P a Rsym ¼ P hkl i lIhkl;i 2 kIl Phkl l= lkIlhkl l: b Rfactor ¼ lFobs 2 Fcalc l= Fobs ; where Fobs is the observed and Fcalc is the calculated structure factor amplitude. The Rfree was calculated for 10% of the reflections excluded from the refinement.

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Crystal Structures of Isoaspartyl Dipeptidase

Table 2. Data collection and refinement statistics for isoaspartyl dipeptidase in complex with asparagine or the transition state inhibitor AspC[PO2CH2]LeuOH Data set

IadA-Asparagine

IadA-AspC[PO2CH2]LeuOH

˚) Resolution (A 20.0–2.7 26.0–3.3 Unique reflections 24,987 14,699 Completeness overall (%) 93.5 96.8 Outermost shell (%) 92.1 (2.8–2.7) 94.4 (3.42–3.30) a 11.8 15.2 Rsym (%) P4212 Space group P4212 Cell constants a 117.48 119.14 b 117.48 119.14 c 137.96 138.15 Total number of residues 746 776 Total non-H-atoms 5564 5729 Number of water molecules 90 42 Number of zinc atoms 4 4 Temperature factors ˚ 2) 40.2 39.4 Protein (A ˚ 2) 29.6 40.1 Solvent (A 2 ˚ ) 50.7 47.3 Metal (A ˚ 2) 59.2 42.6 Ligand (A 20.86/26.56 24.41/28.58 Rfactor/Rfreeb Sterochemical ideality Bond 0.006 0.011 ˚) Angle (A 1.52 1.58 P P P a Rsym ¼ P hkl i lIhkl;i 2 kIl Phkl l= lkIlhkl l: b Rfactor ¼ lFobs 2 Fcalc l= Fobs ; where Fobs is the observed and Fcalc is the calculated structure factor amplitude. The Rfree was calculated for 5% of the reflections excluded from the refinement.

Edman degradation yielded no indications for a cleavage site in this area. IadA contains three cis-proline residues found at positions 20, 140 and 260, which are located at least ˚ away from the active site. According to Ramachan15 A dran plots calculated with the program PROCHECK,44 all main-chain angles of the final models except those of Ala208 fall into the allowed and generously allowed regions. However, the electron density corresponding to Ala208 is well-ordered and the disallowed angles may be explained by its position at the end of a type III turn. Buried surface areas were calculated with the help of the program AREAIMOL, using a probe radius of ˚ .45 Searches for structural neighbors were per1.4 A formed using the program TOP.46 Figures were drawn with MOLSCRIPT,47 GRASP48 and Raster3D.49 Protein Data Bank accession codes The structures reported here have been deposited in the RCSB Protein Data Bank. The references are 1PO9 for native IadA, 1POJ for the inhibitor complex and 1POK for the complex with asparagine.

Acknowledgements We thank G. Bourenkow (DESY, Hamburg) for his help with measuring MAD data of the native enzyme, and V. Dive (Centre d’Etudes Saclay, France) for the phosphinic inhibitor. Additionally, we thank the SFB469, the Fonds der Chemischen Industrie and the EU project SPINE for financial support.

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Edited by I. Wilson (Received 25 April 2003; received in revised form 19 June 2003; accepted 27 June 2003) Note added in proof: After this manuscript was submitted, a paper describing the X-ray structure of IadA from E. coli and a product complex with aspartate was published. (Thoden, J. B., Marti-Arbona, R., Raushel, F. M. & Holden, H. M. (2003). Biochemistry, 42, 4878 –4882.) The binding site for the aspartate residue and the closure of the active site by the active site loop in the presence of aspartate are in accordance with our findings.