Structure of the First-Shell Active Site in Metallolactamase: Effect of

Although the crystal structure of the active site of the zinc lactamase from ... fragilis is the initial starting point for the structure optimization...
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J. Phys. Chem. B 2001, 105, 8040-8049

Structure of the First-Shell Active Site in Metallolactamase: Effect of Water Ligands M. Krauss* Center for AdVanced Research in Biotechnology, Maryland Biotechnology Institute, 9600 Gudelsky DriVe, RockVille, Maryland 20850

H. S. R. Gilson VeraChem LLC, 14718 Latakia Place, Gaithersburg, Maryland 20878

N. Gresh Equipe de Pharmacochimie Moleculaire et Cellulaire, UMR 8600 CNRS, U266 INSERM, UniVersite Rene-Descartes, 4, AVenue de l’ObserVatoire, F-75006 Paris, France ReceiVed: June 1, 2001

In this study we have examined the behavior of the first-shell active site of metallolactamases as a function of water both bound directly as a zinc ligand and hydrogen bonded to protein-residue ligands in the active site and as a function of metal-metal distance. The inherent effective interaction energy of the bimetallic metallolactamase active site is relatively flat between the metal cations. This leaves the metal-metal distance susceptible to both perturbations from environmental interactions and protonation of active-site residues. Although the crystal structure of the active site of the zinc lactamase from Bacteroides fragilis is the initial starting point for the structure optimizations, structures very different from the equilibrium crystal structure as well as details of the water hydrogen-bonding pattern in the active site are obtained. Structures with ZnZn distances >4 Å, with each metal cation acting as the center of a separate complex, result when only the crystallographic water that is directly coordinated to the Zn is included in the representation of the active site. When more crystallographic waters are included, the structure remains essentially unchanged from the crystal structure. In addition, a class of structures with even shorter metal-metal distances is calculated with the active site cysteine and the hydroxide ion bound to both zinc cations. This class of conformation is low in energy and includes several hydrogen bonds between the active-site residues and surrounding waters. Protonation of the active site also yields a metal-metal distance either >4 Å or comparable to that in the crystal structure, depending on whether the proton is added to the bridging hydroxide or to the carboxylate of the aspartate 103 ligand. The Zn-O distances of an X-ray structure obtained at a pH of 5.6 agree with those of the active site protonated at the hydroxide. The long-distance structure is the lowest in energy for the Zn-Zn enzyme; however, when zinc is substituted by cadmium, the long-distance structure becomes higher in energy because the cadmium cation does not polarize the ligated water as strongly as zinc. The lowestenergy structure for the Zn-Cd system is predicted to have the zinc bound at the site with three histidine ligands, in agreement with a recent experimental deduction. We suggest that structures that differ from the crystal structure can play a role in the reaction or in the initial stages of metal binding, as indicated by the UV-visible spectrum observed in the cobalt-substituted enzyme. Water that is hydrogen bonded to the active site is also believed to play an important role in the catalytic mechanism; one of the waters may function as the nucleophile.

Introduction Antibiotic resistance of microbial pathogens has emerged as a significant threat in part due to the evolution of a number of metallolactamases with a wide substrate specificity and has led to an interest in the structure and reactivity of these enzymes. Crystal structures of the metallo-β-lactamase from Bacteroides fragilis and Bacillus cereus have yielded different structures and pictures of the metal binding at the active site. We show in this paper that these differences can be explained by considering the pH of the crystal and the effective potential between the metal ions in the active site. * Corresponding author. E-mail: [email protected]. Phone: 301738-6242. Fax: 301-738-6255.

The first structure of a metallolactamase was derived from B. cereus at pH 5.7. This structure has one zinc bound to three histidine and one water or hydroxide ligand.1 The active site of metallolactamase in B. fragilis at pH 7.5 and 9.0, however, was found to contain two zinc cations that are bridged by a hydroxide anion.2,3 This was surprising because the active-site residues in both organisms are identical. Another crystal structure of B. cereus found subsequently at a pH of 4.5 has an active-site structure with the same features as B. fragilis for one of the molecules in the asymmetric unit. However, the other molecule has a large Zn-Zn distance of 4.4 Å4 and appears as two separate Zn sites. Recently, the crystal structure of another metallo-β-lactamase, from Pseudomonas aeruginosa, has been shown to have a bimetallic active site comparable to that of B.

10.1021/jp012099h CCC: $20.00 © 2001 American Chemical Society Published on Web 07/25/2001

First-Shell Active Site in Metallolactamase fragilis.5 The key to the differences between the active sites of the crystal structures in different organisms lies in both the different local protein environments around the first-shell active site for the organisms and the different pH values used in the crystallizations. It is evident that the state of protonation of the active site will have an effect on the structure. This was found in a recent theoretical study of the first-shell active site of zinc lactamase.6 However, the apparently conflicting data on the structures suggest that the local environment could also substantially perturb the first-shell structure. The apparently large perturbation of the structure of the active site of a metalloenzyme is initially surprising since the active site is very ionic. Metal-ligand distances and angles in a large data set of zinc proteins are found to agree well with ideal values deduced from small zinc-complex structures from which we can argue that the protein environment perturbs the active site of metalloenzymes relatively weakly.7 There is also a considerable amount of theoretical calculation for the blue copper proteins showing that in vacuo models of the active site are in reasonable agreement with the experimental structure of the enzyme.8 However, it has been demonstrated that even weak perturbations can have apparently large effects on the conformation of the active site of blue copper proteins because the energy surface can be flat for different conformations of the ligands.9 In this study, we show that the energy surface for the metallolactamase active-site model is relatively flat with variation of the Zn-Zn distance, suggesting that the apparently large perturbations in structure are not accompanied by large energetic changes. This also suggests that structures that are substantially different from the equilibrium crystal structure are not as high in energy as previously believed, so they can play a role in the dynamics and reactivity of the active site. The energy of the active-site structure is calculated as a function of Zn-Zn distance to be almost flat. This energysurface behavior is found both when the net charge of the active site is +1, as is indicated by the structure, and when one of the anionic groups is protonated.6 The long-distance Zn-Zn structure or separated monozinc complexes found in the protonated structure are effectively due to the neutralization of the bridging hydroxide ligand. We show herein that the longdistance structure that is calculated when the active site has a net charge of +1 does not arise in the presence of a hydrogenbonded network of waters that are connected to the bridging hydroxide. There are two components of the environment that can be examined. First, there are the obvious interactions of the firstshell active site with the neighboring protein residues. Second, there are a substantial number of waters revealed in the crystal structure that are either hydrogen bonded directly either to the first-shell ligands or to the zinc itself that also mediate the interaction with the protein through hydrogen bonds. The hydrogen-bonded waters fall into two categories: those involved in through-water bonding with residues in the protein interior and those found to be directed toward the solvent in the substrate-binding region. Insight into the effect of water bonding toward the solvent on the structure of the active site is the goal of this paper. This is achievable since the waters bound in the active site have no hydrogen bonds with polar protein residues other than the residues found in the first shell of the metal active site. We will examine the effect of the protein environment together with that of the waters on substrate binding in a subsequent study. Since the metal-binding residues in the bridging structure are conserved for this enzyme in a majority of organisms with a known sequence,10 we believe that the

J. Phys. Chem. B, Vol. 105, No. 33, 2001 8041 inherent structure is the bimetallic active site and that examination of the structural properties of zinc lactamase should proceed from the bimetallic active-site model. Docking of model substrates into the active site is the basis for much of the speculation on the hydrolysis mechanism. The lactam carboxylate is presumed to bind to a conserved lysine; however, two binding categories can then be distinguished by whether the lactam carboxylate binds to the Zn2 cation directly or through the water (Wat2) that is directly bound to Zn2 in the B. fragilis crystal structure. Concha et al.2 proposed that three different model lactamssampicillin, ceftazidime, and imipenemsall bind the carboxylate through water. They then suggest two possible mechanisms in which either the bridging hydroxide or the apical water bound to Zn2 acts as the initial nucleophile. The apical water would still provide the final proton for transfer to the lactam nitrogen in the mechanism describing the bridging hydroxide as the nucleophile. Although the apical water would make a better nucleophile since it is not bound as strongly to the Zn2, they argued that the weakness in this mechanism is the lack of an appropriate protein residue positioned to assist in the proton transfer. In this paper, the electronic properties of the apical water and its associated network of hydrogen-bonded waters suggest that a polarized water could function as the nucleophile. Metal substitution in the active site offers another tool for probing the effect of water binding on the structure of the active site. A bimetallic active-site structure was found for the CdCd enzyme that is comparable to the zinc structure.11 The preference for Zn and Cd has been studied by the perturbed angular correlation of γ-rays, and the structure around each metal has been deduced.12 Although the Cd-Cd enzyme is reported to be less active than the Zn-Zn enzyme,11 the ZnCd hybrid is found to have a substantially increased activity.13 Data from visible and UV spectroscopy of both the B. cereus and B. fragilis cobalt-substituted enzymes have been studied and found to be the same when the pH was comparable.14-17 The spectroscopic data are used to determine the metal-binding propensity between the two metal sites since the close approach of the cysteine ligand to the cobalt is required for the sulfur ligand-to-metal charge-transfer UV spectrum.18 We predict that an active-site structure with a sulfur anion that is associated with a cysteine ligand coordinated to both Zn anions can form a nonequilibrium but relatively low-energy structure. This is another consequence of the relatively flat energy surface for the inherent bimetallic active site. This could be significant because the role of the cysteine in the binding has been a difficult question to elucidate since the beginning of structural and spectroscopic studies of this class of enzymes,14-16 and continues unanswered to the present date.17,19 Finally, there is the question of whether the bridging ligand is a water or hydroxide anion. Substantial shielding from the bridging ligand is required to keep the metal cations from repelling each other. The calculations will explore whether or not a complete or even partial proton transfer can reduce the effective charge on the hydroxide-bridging ligand sufficiently to effectively dissociate the metal cations. The waters that are hydrogen bonded or ligated to the active-site complex will also play a role in the electronic behavior of the bridging ligand and its effect on the structure. The structure of the first-shell complex is shown here to be stabilized in the bimetallic form by a network of waters, the long-distance structure being possible only if there is a single water included in the calculation. The metal cation lowers the pK of bound water; however, for carbonic anhydrase, the zinc lowers the pK by nearly three units

8042 J. Phys. Chem. B, Vol. 105, No. 33, 2001 more than cadmium.20 The electronic behavior of both the bridging ligand and the water bound to the metal are then metal dependent. Methods and Results The initial positions of the waters in the active site are located in the structure in Protein Data Bank file 1znb.21 The crystal structure shows a number of bound waters near or at the active site. Three histidines are bound to Zn1 while the protein ligands bound to Zn2 consist of one cysteine, one histidine, and one aspartate. For the B. fragilis X-ray structure, the active site is bimetallic with a Zn-Zn distance of 3.5 Å. Water hydrogen bonds to the bridging hydroxide, and the aspartate 103 carboxylate oxygen is observed in the active site toward the solvent side. There is also water hydrogen bonded toward the protein side to both aspartate 103 and the sodium cation. Water is ligated directly to the pentacoordinated Zn2 as seen in Figure 1a. As noted in the Introduction, although other zinc lactamase crystal structures are available, a single structure 1znb is used to initiate the entire range of structures reported here. The X-ray structure 1znb was used to initiate theoretical optimizations at the Hartree-Fock (HF) level using the GAMESS set of codes22 or using the polarizable molecular-mechanics procedure, SIBFA (sum of interactions between fragments ab initio computed). SIBFA23 is a many-body classical potential that was calibrated and tested for zinc complexes.24-27 HF optimizations start from SIBFA structures and also from the X-ray structure with the hydrogen atoms added using the MOLDEN code.28 HF constrained optimizations were performed starting from the three structures obtained with SIBFA. Effective core potentials were used with the CEP 4-31G basis set,29,30 which is equivalent to a double-ζ (DZ) basis. Zn-Zn Active Site. The SIBFA optimizations explore the effective potential for the bimetallic Zn-Zn interaction with a bridging hydroxide anion. The SIBFA optimizations were performed at three constrained Zn-Zn distances in the 3.26, 3.48, and 3.78 Å range with the energies provided in Table 1. HF calculations were then performed at these distances. The short-distance structure at 3.26 Å is additionally stabilized by dispersion contributions that become larger as the ligands crowd together. In addition to the bridging hydroxide, the water found to be bound to Zn2 in the crystal structure was retained (Figure 1a) with this formal structure maintained at 3.48 and 3.78 Å. For these distances, the ligated water moves from Zn2 to hydrogen-bond to the bridging hydroxide in HF optimizations (Figure 1b). However, when the Zn-Zn distance is constrained to 3.26 Å, with SIBFA, a structure is optimized with two bridging ligands, the cysteine and the hydroxide, with the water ligated to Zn2 as seen in Figure 1c. This structure is a local minimum at 3.26 Å since another lower-energy structure is obtained by moving the hydrogen bond from the carboxylate of the aspartate to the ligated water, as seen in Figure 1d, from the bridging hydroxide anion. The possibility that the doubly bridged structures may be too strained is investigated by expanding the model to include the CR for all the ligands. Optimizations are initiated with the CR and the Zn-Zn distance (3.20 Å) frozen. By rotating the orientation of the S atom, we obtained two structures, with one resembling a normal bimetallic active-site complex and the other the doubly bridged structure in Figure 1e. These two structures are within 0.5 kcal/mol of each other, the doubly bridged structure being lower in energy. The very different arrangement of the cysteine ligand is seen with the normal, bimetallic site having a Zn1-S distance of 3.84 Å compared to 2.32 Å for

Krauss et al. Zn2-S, while in the doubly bridged structure, the comparable distances are 2.98 and 2.28 Å, respectively. The single-point HF energies at the SIBFA geometries describe a flat effective potential for the Zn-Zn separation, as seen in Table 1 with the energy varying ca. 5 kcal/mol from 3.26 to 3.78 Å and with the minimum moved toward the longer Zn-Zn distance of 3.48 Å. Constrained HF optimizations indicate a more-complex Zn-Zn distance dependence. The equilibrium distance is now 3.61 Å, while the 3.26 Å complex is 12 kcal/mol higher. However, the energy between the 3.48 and 3.26 Å structures is only 3 kcal/mol, showing a flat dependence toward shorter distances. For the Zn-Zn distance of 3.26 Å, a bridging S anion is retained during the optimization. The bridging cysteine coexists with the bridging hydroxide for this class of structures while the bridging-inhibitor thiolate in the structure found for P. aeruginosa5 displaces the hydroxide. The SIBFA structure with the hydrogen bond between the carboxylate and the ligated water (Figure 1d) is calculated to be relatively low in energy, but the HF energy at the SIBFA geometry is much higher. However, the constrained HF optimization at the Zn-Zn distance of 3.26 Å shows this class of conformations with a hydrogen bond to the ligated water to be comparable in energy (3-5 kcal/mol) to those with the widely observed motif of hydrogen bonding between the carboxylate and the bridging hydroxyl31 at short Zn-Zn distances. The hydrogen bond to the ligated water (Figure 1d) is retained with the structure that is 4 kcal/mol lower in energy than the structure with the hydrogen bond to the bridging hydroxide. The different structures in Figure 1 suggest that vibrations along the Zn-Zn direction can alter the ligand hydrogen-bond arrangements as well as the number of bridging ligands. Structures in Figure 1a,b correspond to arrangements of waters and the bridging ligand found in the crystal structure, but structures 1c and 1d are different from the crystal structure. These structures show that a range of structural minima can be created from the crystal structure. These structures expand the range of possible motifs from the X-ray structure and show that an even wider range of minima can be found than previously was reported.6 The new structures may play a role in the structural dynamics, spectroscopy, and reaction path dynamics. The X-ray, SIBFA, and constrained HF structures are then used to initiate unconstrained optimizations of Zn-Zn structures that both relax the original motifs and generate new ones. The standard defaults in GAMESS were used for the optimization. During the course of the optimization that was initiated from structure 1a, the water bends toward the bridging hydroxide, creating a strong H-bond. The water-OH bond is lengthened to about 1 Å, and the Zn-Zn distance is noticeably increased. The proton is subsequently donated to the bridging hydroxide, creating an intermediate H3O2-32,33 that is transformed in the course of the optimization into water ligated to Zn1. The optimized structure (Figure 2a) shows two separate complexes on Zn1 and Zn2 that interact through two ionic hydrogen bonds. This structure is formally identical to structure 6 recently published by Diaz et al.6 The 4.83 Å Zn-Zn distance calculated here is somewhat larger than the 4.49 Å distance reported by Diaz et al.;6 otherwise, the bond distances to the ligands (Table 1) are in good agreement with the largest difference of 0.05 Å, and most are within 0.02 Å. The hydrogen-bond distances between the anionic ligands on Zn2 and the water ligated to Zn1 are all rather short. Both the carboxylate (dO-H ) 1.58 Å) and the hydroxide (dO-H ) 1.50 Å) are hydrogen bonded to a proton from the water. This comparison shows that no essential geometrical difference is expected between the present

First-Shell Active Site in Metallolactamase

J. Phys. Chem. B, Vol. 105, No. 33, 2001 8043

Figure 1. (a) Zn-Zn first-shell active site, formally equivalent to the X-ray structure from 1znb. (b) Zn-Zn first-shell active site with W1 hydrogen bonded to the bridging hydroxide. (c) Zn-Zn first-shell active site with two bridging ligands, hydroxide and methyl sulfide, and the carboxylate hydrogen bonded to hydroxide. (d) Zn-Zn first-shell active site with two bridging ligands and water hydrogen bonded to carboxylate. (e) Zn-Zn first-shell active site with two bridging ligands and water hydrogen bonded to hydroxide.

results and that of Diaz et al. even though a DZ basis set is used with the ECP. In other metalloenzyme studies, it was shown that comparable structures are obtained with DZ and DZd basis sets.34 On the other hand, one of the constrained HF structures (Figure 1b), the B. fragilis crystal structure,2 and structure 5 of Diaz et al.6 all show that a water can hydrogen-bond to the bridging hydroxide without increasing the Zn-Zn distance. The

crystal structure has water that is bound to the bridging hydroxide as well as one water ligated to Zn2. An oxygen is positioned 2.8 Å from the bridging oxygen in the appropriate orientation for a hydrogen bond that could be used to initiate the HF optimization. A constrained HF optimization at 3.78 Å, starting from the SIBFA structure with the water originally ligated to Zn2, transfers the water to a hydrogen bond to the bridging hydroxide. Starting an unconstrained optimization from

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TABLE 1: Binding and Total Energy of Active Sites as a Function of Zn-Zn Distance Zn-Zn 3.78 Å

3.48 Å

3.61 Å

3.26 Å

3.26 Å*

SIBFA (kcal/mol) ∆E ∆E + Edisp SIBFA geometry constrained optimizationf

1167.0 1269.6

1172.1 1278.0

1177.3 1286.3

1189.9 1302.6

722.85910a

HF (total -E, a.u.) 722.86477a

722.85551c

722.84660d

722.91563b

722.91089a

722.90042e

722.90607d

722.92545

a Figure 1a with water ligated to Zn2. b Figure 1b with water hydrogen bonded to the bridging ligand. c Figure 1c with the bridging S and hydroxyl; water is ligated to Zn2. d Figure 1d with the bridging S and hydroxyl; aspartate 103 is hydrogen bonded to the ligated water (3.26 Å*). e Figure 1e with bridging S and hydroxyl; water is hydrogen bonded to hydroxide. f There are two minima for unconstrained optimizations with a single water in the active site in addition to that in the bridging ligand; long Zn-Zn of 4.83 Å (Figure 2a), -722.93098, and short Zn-Zn of 3.61 Å, -722.92545 (Figure 2b); unconstrained optimization for structure 1d lowers the energy to -722.90658 a.u.

structure 1b, we find that the Zn-Zn distance is optimized to 3.61 Å with water hydrogen bonded to the bridging hydroxide of 1b. This structure (1b) is formally the same as structure 5 observed in ref 6. One of the points of this paper is to illustrate that a range of apparently diverse but energetically close structures can be generated from a single X-ray structure through a series of both constrained and unconstrained optimizations. Classical many-body potentials such as SIBFA provide a relatively inexpensive way of first examining this complicated energy surface; however, the molecular-mechanics optimization cannot transfer protons, so structures such as 2a will only result from an ab initio optimization. Starting with one water bound to Zn2 as in Figure 1a, we find that a structure (2a) is optimized with a Zn-Zn distance >4 Å. However, the crystal structure unambiguously shows that there are a number of waters hydrogen bonded to the first-shell ligands. The relative instability in the Zn-Zn distance found in optimizing a motif with a single-water ligand disappears when two or more waters are included in the model. Several of the possible arrangements of clustered waters are shown in Figure 2b-e, with the interatomic distances described in Table 2. One water was added to the original motif using the coordinates of ligated water after the addition of hydrogen atoms to the crystal structure. Now, the HF optimization does not tend toward the large Zn-Zn structure as the hydrogen bond to the added water prevents the formation of such a hydrogen bond to the bridging hydroxide and stabilizes the bimetallic complex. Structures are presented for two and three additional waters in Figure 2b-e. This list certainly does not exhaust possible structures that can be dynamically accessed in solution or additional minima for the HF surface. As an example, we show a structure in Figure 2f in which the water displaces the bond from Zn2 to the carboxylate OD1 and two strong ionic hydrogen bonds keep the acetate bound to the bridged complex with both Zn cations now with a tetrahedral coordination. Protonated Active Site. The next class of structures for the Zn-Zn enzyme is obtained by adding a proton to the first-shell model. Protonation can occur at the bridging hydroxide, the carboxylate oxygen, and even the cysteine sulfur. Here, we will examine the consequences of protonating the bridging hydroxide and the carboxylate. Starting from structure 1a with water ligated to Zn2, we find that the lowest-energy optimized structure is obtained by protonating either the bridging hydroxide or the carboxylate. This structure (3a) depicts a long Zn-Zn distance (5.39 Å) and is shown in Figure 3a with the bond distances described in Table 2. This structure differs from the longdistance protonated structure, 6H, reported by Diaz et al.6 since the ligand on Zn1 is a hydroxide rather than a water. The present

result agrees with the short Zn1-O distance found by Fabiane et al.4 for the crystal structure of B. cereus obtained at a pH of 4.5. The Zn-Zn distance for the two independent molecules in the crystal is relatively long and is presumed to result from a protonated active site. The hydroxide O bound to Zn1 is hydrogen bonded to the water that is bound to Zn2 with a distance of 1.77 Å. The proton of the hydroxide hydrogen bonds to S with a distance of 2.40 Å. Finally, the proton bound to OD2 of the carboxylate has a short, presumably strong, hydrogen bond to the hydroxide O with a distance of 1.45 Å. The optimization was repeated for this structure with a polarized basis set with d functions on all heavy atoms and gave essentially the same results. For example, the Zn-Zn distance decreases slightly to 5.26 Å from the 5.39 Å value obtained with the DZ basis. The small quantitative changes found for enzyme model structures when using a polarized basis set were noted earlier.34 If water is hydrogen bonded to the bridging hydroxide when the proton is added to the carboxylate, the optimization results in structure 3b with a shorter Zn-Zn distance of 3.72 Å, while the total energy is only 3 kcal/mol higher than that for structure 3a. Other protonated structures are possible, including the structure with the cysteine bridge found by Diaz et al.6 Cd in the Active Site. Optimization of some comparable ZnCd, Cd-Zn, and Cd-Cd models was performed with first-shell model structures from 2b-e. Not all the possible structures have been explored since the qualitative features of the hydrogenbonded water are illustrated by the results of the Zn-Zn models. The initial structure is the comparable Zn-Zn structure theoretically obtained with one exception. In one Cd-Cd case, we again start from the crystal structure of B. fragilis, with only one water ligated to Cd2. This motif was examined to see if the Cd-Cd enzyme would also optimize to a Cd-Cd bond distance >4 Å. This optimization did not occur. Although water does not stay ligated to Cd2 during the optimization but moves to hydrogenbond to the bridging hydroxide, the final structure is formally identical to one that starts from the Zn-Zn structure of 1b. Water is not sufficiently activated by the Cd cation, with the OH bond distance never exceeding 1 Å in the course of the optimization as it does with Zn. The proton on the ligated water is attracted to the O of the bridging hydroxide; but since the proton is not sufficiently activated by the Cd cation, the H3O2moiety is not formed. The hydrogen-bond attraction is sufficient to transfer the water from binding to Cd2 to the hydroxide. A structure with a large Cd-Cd separation would be obtained if we start the optimization from the comparable long-distance Zn-Zn structure 3a. However, any Cd-Cd structure comparable

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Figure 2. (a) Zn-Zn first-shell active site with metal cations separated by a long distance and with water bound to the Zn1. (b) Zn-Zn first-shell active site with water1 ligated to Zn2 and water2 hydrogen bonded to both water1 and the hydroxide. (c) Zn-Zn first-shell active site with water1 ligated to Zn2 and water2 both hydrogen bonded to water1 and oriented away from the hydroxide. (d) Zn-Zn first-shell active site with water1 ligated to Zn2; hydrogen bond-cluster links water1 to water2 to water3 to carboxylate to hydroxide. (e) Zn-Zn first-shell active site with water1 ligated to Zn2; hydrogen-bond cluster links water1 to water2 to hydroxide, and water2 hydrogen-bonds to water3. (f) Zn-Zn first-shell active site with water1 ligated to Zn2; carboxylate is displaced from Zn2 and hydrogen-bonds to water1 and the hydroxide.

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TABLE 2: Bond Distances (Å) for the Zn-Zn Active Siteb structure

expa

2a

2b

M1-HN M1-HNδ M1-HN M1-OB M2-HN M2-S M2-DO M2-OB M2-W1O M1-M2

2.1/2.2 2.0/2.1 2.0/2.0 1.9/1.9 2.1/2.2 2.3/2.4 2.3/2.1 2.1/2.2 2.3/2.2 3.5

2.04 2.05 2.04 1.94 2.07 2.36 2.01 3.38 1.97 4.83

2.05 2.07 2.05 1.95 2.07 2.33 1.99 2.04

2c

2.05 2.09 2.05 1.91 2.20 2.41 2.06 2.07 2.22 3.61 3.71

2d

2e

2f

3a

3b

2.06 2.09 2.09 1.91 2.16 2.39 2.03 2.15 2.23 3.75

2.05 2.09 2.05 1.91 2.20 2.42 2.07 2.06 2.17 3.70

2.05 2.11 2.05 1.93 2.20 2.39 2.06 2.18 2.12 3.76

2.07 2.07 2.04 1.97 2.01 2.31 2.04

2.07 2.06 2.06 1.98 2.04 2.30 2.10 2.09

2.10 5.39 3.72

a Experimental first shell formally resembles the structure found in Figure 2c. Protonated first shell is represented by structures 3a and 3b. b Total HF energy for structures 2a (-722.93098), 2b (-722.92546), 2c (-739.76156), 2d (-739.74960), 2e (-756.60312), and 2f (-756.59854).

to 3a would be at a higher energy than the Cd-Cd version of structure 1b. For two waters, the Cd-Cd conformations comparable to the Zn-Zn structures in Figure 2b,c are obtained. This structure has water ligated to Cd2 since the additional water stabilizes a local minimum and prevents the transfer of the ligated water to a hydrogen bond on the hydroxide. The Cd-ligand bond distances in Table 3 are in agreement with experiment11 with the exception of the value for the bridging hydroxide for one of the molecules in the asymmetric unit. We suggest that either the hydrogen-bonded waters are not sufficiently localized in the crystal structure, making the refinement of the waters and thus the hydroxide difficult, or there is another ligand besides the hydroxide. The final optimizations are for Zn-Cd and Cd-Zn based on the Zn-Zn structures 1b and 2b. For the Cd-Zn structure, water is initially bound to Zn2 but moves to hydrogen-bond the bridging hydroxide during the optimization (Figure 1b). The details of these structures are also summarized in Table 3. The lowest-energy structures for each class are those for the tetracoordinated Zn and the pentacoordinated Cd. As additional waters are added, the energy difference between the tautomers moderately increases. Thus, structures 5a (Cd1Zn2, Figure 1b) and 5c (Cd1Zn2, Figure 1b) are separated by only 3 kcal/mol, and 5b (Zn1Cd2, Figure 2c) and 5d (Zn1Cd2, Figure 2e) are separated by 5 kcal/mol. Discussion The number and binding arrangement of the waters have a substantial effect on the Zn-Zn distance for the first-shell models. The single water initially bound to the Zn2 cation is activated to attack the bridging hydroxide, forming an effective H3O2- complex. The delocalization of the charges in this complex leaves the oxygen between the metal cations with a charge that is insufficient to screen the cations. The unscreened divalent cations repel against the ligands that are reorganizing, leading to longer-distance stable structures. This behavior was noted earlier for metal complexes and suggested as a motif for oligozinc enzymes.32,33 However, this long-distance active-site structure is an artifact of the simple model with only one ligated water. Additional waters, hydrogen bonded to the ligated water, stabilize the bimetallic active site. Additional binding to the protein environment would only reinforce the stabilization of the short Zn-Zn distance. The structure of the Cd-Cd first-shell active site provides additional insight since the water that is bound to Cd2 is not as activated by the larger and less-positive cation. Creating an

H3O2- complex is more difficult, as is polarizing the hydrogenbonded waters that could be the basis for the lower activity of the Cd-Cd enzyme. Two Cd-Cd molecules are found in the asymmetric unit of the crystal structure with Cd-Cd distances of 3.7 and 3.8 Å.11 The calculated Cd-Cd distance is in reasonable agreement within the experimental accuracy of the structure. The comparative behavior of the Zn-Cd with the Cd-Zn with one water in the first shell started with the formal structure 1b was to initiate the two bimetallic active site structures. The Zn1-Cd2 structure is lower in energy than the Cd1-Zn2 structure, which results from both the binding of the Cd site to the cysteine ligand and the increased propensity of Cd over that of Zn to become pentacoordinated. The in vacuo energy difference of g5 kcal/mol is sufficient that only one structure is observed experimentally.12 The calculation of the structures constrained at a range of Zn-Zn distances provides one route to generate classes of structures that differ from the X-ray structure, although all the initial optimizations start from the X-ray structure. Optimization with Zn-Zn constrained to 3.26 Å results in a fundamental change in structure with the cysteine sulfur bridging the two zinc cations. There is little evidence of strain in this structure since both singly and doubly bridged structures have essentially the same energy when optimized with the CR frozen. The existence of this structure would explain the contradictory spectral data in the cobalt-substituted enzyme.17 In this study, the charge-transfer band that results from the interaction with the cobalt and the cysteine appears at very low cobalt concentrations when cobalt is reconstituted in the apolactamase enzyme from B. cereus. However, it is believed that the cobalt binds to the Zn1 site, not the Zn2 site. No charge-transfer band would be visible if this were the case.18 If the metal-binding site is as flexible as these calculations indicate, the cysteine residue would be able to interact with the cobalt bound in the Zn1 site, resulting in a charge-transfer band even at concentrations in which there is only one metal that is bound. Protonation of the first-shell structure for Zn-Zn can also lead to a long-distance structure. A protonated active site is certainly occurring in the crystal structure obtained with a pH of 4.5.4 However, the present calculations predict that the protonated form can lead to both long (3a) and short (3b) ZnZn structures. The long-distance structure (3a) obtained here differs from an earlier prediction for such a structure6 by depicting a hydroxide ligand rather than a water bound to Zn1. This is more in agreement with the hydroxide associated with Zn1 acting as the single-zinc nucleophile. Two structures, one at a long and the other at a shorter distance, but both at distances longer than that observed for B. fragilis, 2 are observed in the asymmetric unit for the crystal structure formed at acidic conditions. The experimental distances for the Zn1-O and Zn2-O bonds suggest that both of these sites are protonated.4 The predicted long Zn-Zn distance is larger than the observed values, suggesting that the constraints of the protein environment prevent the creation of the larger predicted separation. The observed difference between the Zn-Zn distance in the two independent molecules in the crystal structure also suggests that the proton is binding to two different sites. This enzyme can be active as a single- or double-zinc enzyme, depending on the organism. The relatively flat effective potential, the strong dependence on the water binding, and the possibility that protonation of the active site leads to a separation into two metal complexes are probable contributory factors. The protein environment in B. cereus acts on this inherent lack of

First-Shell Active Site in Metallolactamase

J. Phys. Chem. B, Vol. 105, No. 33, 2001 8047

Figure 3. (a) Protonated Zn-Zn first-shell active site with separated metal-cation complexes; hydroxide anion bound to Zn1 and Zn2 with carboxylate protonated. (b) Protonated Zn-Zn first-shell active site with a short Zn-Zn distance; water is hydrogen bonded to hydroxide, and the proton is bound to carboxylate.

TABLE 3: Bond Distances (Å) for the Cd-Cd, Zn-Cd, and Cd-Zn Active Sitesb Cd-Cd

Zn-Cd

Cd-Zn

structure

expa

4a

4b

5a

5b

5c

5d

M1-HN M1-HNδ M1-HN M1-OB M2-HN M2-S M2-DO M2-OB M2-W1O M1-M2

2.1/2.3 2.1/2.2 2.1/2.1 1.9/2.1 2.5/2.3 2.5/2.5 2.3/2.2 2.4/2.7 2.2/2.2 3.8/3.7

2.31 2.27 2.27 2.16 2.30 2.52 2.22 2.27 2.49 3.94

2.28 2.31 2.27 2.13 2.33 2.57 2.26 2.34 2.44 3.98

2.07 2.05 2.05 1.94 2.30 2.51 2.22 2.28

2.06 2.06 2.08 1.91 2.36 2.56 2.25 2.37

2.27 2.32 2.27 2.18 2.06 2.34 1.99 2.03

2.33 2.28 2.27 2.16 2.07 2.35 1.99 2.01

3.84

3.92

3.67

3.65

a

Experimental first shell formally resembles the structure found in Figure 4b. b Total HF energy for structures 4a (-604.97131), 4b (-621.80189), 5a (-663.95144), 5b (-680.78469), 5c (-663.94634), and 5d (-680.77682).

stability toward larger metal-metal distances or two independent metal cation complexes to create the long-distance form of the active site. The Zn2 or less strongly bound cysteine site is closer to a cationic residue in the second shell of the active site and more accessible to solvent that would facilitate the removal of the metal cation. The very different binding constants for the different metal sites in B. cereus have been attributed to the nearby presence of another positively charged arginine residue.3 The importance of the binding of the waters and their effect on structure and reactivity in the enzyme active site have also been emphasized recently36 while binding to the protein environment. Understanding the detailed behavior requires the presence of the protein environment that can be created with the effective fragment potential (EFP).37 This will be reported later. However, we can speculate that the guanidinium cation of the arginine is so close to the cysteine (4 Å) and aspartate (3 Å) that it competes with Zn2 as the Zn1-Zn2 distance increases, especially if more water can bind to this complex. The Zn2 complex is bonded to the Zn1 complex in structure 3a by hydrogen bonds that will be weakened by the access to water. The binding of the aspartate

and the cysteine is shared in part by both metal cations in the bimetallic active site. This is seen clearly when the cysteine bridges both cations for metal-metal distances shorter than 3.3 Å. The comparable cation in B. fragilis is a Na+ located at >6 Å away from Zn2 and shielded by through-bond water. The HF equilibrium minimum has a Zn-Zn distance of 3.61 Å, about 0.1 Å larger than that of the experiment,2 with the SIBFA data suggesting that the effective potential for the ZnZn separation is flat toward shorter distances. This unusual flatness would be accentuated by the inclusion of dispersion interactions. MP2 optimization of the dispersion effect is not tractable at this time for such a large model system. We can only speculate that vibrational distortions can access the shorter Zn-Zn distances where the cysteine sulfur begins to bridge both Zn1 and Zn2. This provides a means of generating the ligandto-metal transitions in the partially substituted cobalt enzyme without having to ensure that the cobalt is replacing zinc at the M2 site. This situation could be complicated by the differential effects of the protein environment in the enzyme from different organisms on the effective Co-M (M ) Zn or Co) potential. The implications of these results for the mechanism will be considered while realizing that the model must be expanded to include the protein environment for any complete treatment. Docking of penicillin to the active site2 suggested that the carboxylate of the substrate binds to a conserved lysine while the carbonyl on the lactam ring is hydrogen bonded to a conserved asparagine. Formation of the salt bridge with the lysine is likely to be the ordering interaction since the energetics of this bond exceed that of any other hydrogen bond possible. The nucleophile in this case was suggested to be the ligated water,2 although the bridging hydroxide is usually suggested.38,39 However, the crystal structure and the present calculations suggest that there will a tight cluster of second-shell waters hindering the approach of the substrate to the active site. The strong binding of the water cluster is indicated by the short distance of the hydrogen bonds (1.65 Å) calculated for the two waters bonded to the ligated water. In both cases, these waters

8048 J. Phys. Chem. B, Vol. 105, No. 33, 2001 SCHEME 1a

a Water that is hydrogen bonded between the first-shell active site and the substrate is suggested as the nucleophile. Hydrogen bonds from the lactam carboxylate to the conserved lysine and the nucleophile are indicated by a solid arrow while the attack of the OH- and the temporary transfer of the H+ are indicated by the dashed arrow.

are substantially polarized with the hydrogen-bonded proton bond distance stretched by 0.03 Å relative to the O-H bond that is not directly hydrogen bonded. This allows a nucleophilic attack involving the polarized waters in which the aspartate hydrogen bond is maintained or rearranged toward the polarized water. A source for the proton is also maintained. The proton can be abstracted by the carboxylate and used for the proton transfer to the N atom of the lactam, with the H-bond rearrangement contributing to the rate-limiting barrier.10 Parking of the proton or competition of the binding of the proton to protein residues bonded to the lactam nitrogen can account for the late protonation step.39 Scheme 1 describes this process with the water initially hydrogen bonded between the carboxylate of the lactam substrate and either Zn2 or the bridging hydroxide. The involvement of the aspartate in the mechanism has been emphasized in two recent papers.40,41 Answering the question of whether or not this route for generating the nucleophile is competitive with the direct action of the bridging hydroxide requires the introduction of the protein environment with the EFP. Preliminary results in an MD and SIBFA simulation, with ampicillin bound to the B. fragilis active site, find that the lysine-carboxylate salt bridge binds through water to the zinc cation42 and leaves substantial room for a network of waters. In parallel, more detailed comparisons between SIBFA and ab initio computations are being undertaken, including energydecomposition analyses with the Restricted Variational Space Approximation,43 an extension of the basis sets with polarization functions, and the inclusion of a correlation by the DFT approach. Conclusion Five significant points summarize the conclusions of this study. (1) The interaction energy between the metal cations is calculated to be relatively weak. The inherent energetics of the supermolecule complex do not differ substantially between a complex with a metal-metal distance near the crystal-structure value and a complex with a much larger distance (>4 Å). This suggests that modest interaction energies from the protein environment can have disproportionate effects on the activesite structure. (2) Relatively low-energy, unusual structures can be created from optimizations that start from the crystal structure. As an example, we cite the doubly bridged structure obtained when the Zn-Zn distance is constrained to be slightly shorter than that of the crystal structure. (3) The “neutral” long-

Krauss et al. range structure is very dependent on the number of waters that are included in the active-site complex. More waters stabilize the bridging hydroxide and prevent the effective protonation of this ligand that leads to the long Zn-Zn distance. This can be considered an instance in which the local environment, i.e., the waters in the active site, has effected the structure in a substantial fashion. Adding more protein environment will further stabilize the short-distance structure. (4) The protonated structure agrees with the Zn-O distances found for the structures in ref 4. This suggests that the X-ray structures formed at low pH are protonated at the active site. An accurate description of the site will require the protein environment. (5) Polarized waters are calculated to form a tightly hydrogen-bonded network. The short H-bond distance and the long, calculated OH-donor bonds suggest that one of these waters can act as the nucleophile. Scheme 1 was suggested to represent the attack of the OH on the lactam carbonyl with the proton directed toward a number of possible temporary binding sites, with the carboxylate of the aspartate considered to be the most likely candidate. References and Notes (1) Carfi, A.; Pares, S.; Duee, E.; Galleni, M.; Duez, C.; Frere, J.-M.; Dideberg, O. EMBO J. 1995, 14, 4914-4921. (2) Concha, N. O.; Rasmussen, B. A.; Bush, K.; Herzberg, O. Structure 1996, 4, 823-836. (3) Carfi, A. D. E.; Paul-Soto, R.; Galleni, M.; Frere, J. M.; Dideberg, O. Acta Crystallogr., Sect. D 1998, 54, 45-57. (4) Fabiane, S. M.; Sohi, M. K.; Wan, T.; Payne, D. J.; Bateson, J. H.; Mitchell, T.; Sutton, B. J. Biochemistry 1998, 37, 12404-12411. (5) Concha, N. O.; Janson, C. A.; Rowling, P.; Pearson, S.; Cheever, C. A.; Clarke, B. P.; Lewis, C.; Galleni, M.; Frere, J. M.; Payne, D. J.; Bateson, J. H.; Abdel-Meguid, S. S. Biochemistry 2000, 39, 4288-4298. (6) Diaz, N.; Suarez, D.; Merz, K. M., Jr. J. Am. Chem. Soc. 2000, 122, 4197-4208. (7) Alberts, I. L.; Nadassy, K.; Wodak, S. J. Protein Sci. 1998, 7, 17001716. (8) Pierloot, K. D. K. J.; Ryde, U.; Olsson, M. H. M.; Roos, B. O. J. Am. Chem. Soc. 1998, 120, 13156-13166. (9) DeKerpel, J. O. A.; Ryde, U. Proteins: Struct., Funct., Genet. 1999, 36, 157-174. (10) Wang, Z. F. W.; Valentine, A. M.; Benkovic, S. J. Curr. Opin. Chem. Biol. 1999, 3, 614-622. (11) Concha, N. O.; Rasmussen, B. A.; Bush, K.; Herzberg, O. Protein Sci. 1997, 6, 2671-2676. (12) Paul-Soto, R.; Zeppezauer, M.; Adolph, H. W.; Galleni, M.; Frere, J. M.; Carfi, A.; Dideberg, O.; Wouters, J.; Hemmingsen, L.; Bauer, R. Biochemistry 1999, 38, 16500-16506. (13) Paul-Soto, R. H.-V. M.; Galleni, M.; Bauer, R.; Zeppezauer, M.; Frere, J. M.; Adolph, H. W. FEBS Lett. 1998, 438, 137-140. (14) Baldwin, G. S.; Galdes, A.; Hill, H. A.; Waley, S. G.; Abraham. E. P. J. Inorg. Biochem. 1980, 13, 189-204. (15) Bicknell, R. S. A.; Waley, S. G.; Auld, D. S. Biochemistry 1986, 25, 7208-7215. (16) Crowder, M. W.; Wang, Z.; Franklin, S. L.; Zovinka, E. P.; Benkovic, S. J. Biochemistry 1996, 35, 12126-12132. (17) Orellano, E. G.; Girardini, J. E.; Cricco, J. A.; Ceccarelli, E. A.; Vila, A. J. Biochemistry 1998, 37, 10173-10180. (18) Gilson, H. S. R.; Krauss, M. J. Am. Chem. Soc. 1999, 121, 69846989. (19) Paul-Soto, R.; Bauer, R.; Frere, J. M.; Galleni, M.; Meyer-Kalucke, W.; Nolting, H.; Rossolini, G. M.; DeSeny, D.; Hernandez-Vallardes, M.; Zeppezauer, M.; Adolph, H. W. J. Biol. Chem. 1999, 274, 13242-13249. (20) Garmer, D. R.; Krauss, M. J. Am. Chem. Soc. 1992, 114, 64876493. (21) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235-242. (22) Schmidt, M. W.; Baldridge, K. K.; Boatz, J. A.; Elbert, S. T.; Gordon, M. S.; Jensen, J. H.; Koseki, S.; Matsunaga, N.; Nguyen, K. A.; Su, S. J.; Windus, T. L.; Dupuis, M.; Montgomery, J. A. J. Comput. Chem. 1993, 4, 1347-1363. (23) Gresh, N. J. Chim. Phys. 1997, 94, 1365-1416. (24) Gresh, N. J. Comput. Chem. 1995, 16, 856-882. (25) Garmer, D. R.; Gresh, N.; Roques, B. P. Proteins 1998, 31, 4260. (26) Rogalewicz, F.; Ohanessian, G.; Gresh, N. J. Comput. Chem. 2000, 21, 963-973.

First-Shell Active Site in Metallolactamase (27) Tiraboschi, G.; Gresh, N.; Giessner-Prettre, C.; Pedersen, L. G.; Deerfield, D. W. J. Comput. Chem. 2000, 21, 1011-1039. (28) Schaftenaar, G.; Noordik, J. H. J. Comput.-Aided Mol. Des. 14, 123-134. (29) Stevens, W. J.; Basch, H.; Krauss, M. J. Chem. Phys. 1984, 81, 6026-6033. (30) Stevens, W. J.; Krauss, M.; Basch, H.; Jasien, P. G. Can. J. Chem. 1992, 70, 612-630. (31) Wilcox, D. E. Chem. ReV. 1996, 96, 2435-2458. (32) Ruf, M. W. K.; Vahrenkamp, H. J. Am. Chem. Soc. 1996, 118, 9288-9294. (33) Vahrenkamp, H. Acc. Chem. Res. 1999, 32, 589-596. (34) Krauss, M. Int. J. Quantum Chem. 2000, 76, 331-340. (35) Krauss, M. J. Chem. Inf. Comput. Sci. 2001, 41, 8-17. (36) Dunitz, B. D.; Beachy, M. D.; Cao, Y.; Whittington, D. A.; Lippard,

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