9986
J. Phys. Chem. B 2007, 111, 9986-9992
Inhibitor Binding by Metallo-β-lactamase IMP-1 from Pseudomonas aeruginosa: Quantum Mechanical/Molecular Mechanical Simulations Canhui Wang and Hua Guo* Department of Chemistry, UniVersity of New Mexico, Albuquerque, New Mexico 87131 ReceiVed: May 18, 2007; In Final Form: June 15, 2007
The dynamics of the IMP-1 enzyme complexed with three prototypical inhibitors are investigated using a quantum mechanical/molecular mechanical (QM/MM) method based on the self-consistent-charge densityfunctional tight-binding model. The binding patterns of the inhibitors observed in X-ray diffraction experiments are well reproduced in 600 ps molecular dynamics simulations at room temperature. These inhibitors anchor themselves in the enzyme active site by direct coordination with the two zinc ions, displacing the hydroxide nucleophile that bridges the two zinc ions. In addition, they also interact with several active-site residues and those in two mobile loops. The excellent agreement with experimental structural data validates the QM/MM treatment used in our simulations.
Introduction The ever increasing level of antibiotic resistance by pathogenic bacteria threatens to jeopardize the treatment of infectious diseases, posing a great challenge to public health.1 Resistance to β-lactam antibiotics, such as penicillin, cephalosphorins, and carbapenems, is commonly conferred by bacterial β-lactamases. They are hydrolases that deactivate the antibiotics by the cleavage of the lactam amide (C-N) bond.2 These enzymes have traditionally been divided into four classes.3,4 Those in classes A, C, and D rely on an active-site serine in covalent catalysis,5 while the class B enzymes require one or two zinc cofactors, thus called metallo-β-lactamases (MβLs).6 The catalytic mechanism of serine-based β-lactamases is well established, and several effective inhibitors,7 such as clavulanic acid, are routinely used in clinical settings. However, such inhibitors have no effect on MβLs because of their completely different catalytic mechanisms. Although various strategies have been suggested,8-10 there is still no clinically useful inhibitor of MβLs. The potential threat of MβLs in the future treatment of infectious diseases stems mainly from their broad substrate profile, which includes most β-lactam-based antibiotics and even inhibitors of serine-based β-lactamases.6 Although their prevalence is still low, recent evidence indicated a rapid proliferation in pathogenic species via plasmid- or integron-mediated mechanisms.8,11 MβLs have been found worldwide in opportunistic pathogens such as Pseudomonas aeruginosa and Stenotrophomonas maltophilia, and it is only a matter of time before they spread to major pathogens. Hence, a better understanding of the inhibition of MβLs is urgently needed. MβLs are further classified into three groups.6,12 B1 and B3 enzymes contain two zinc ions when fully loaded, but B2 enzymes are inhibited by the second metal ion. In this work, we focus on the inhibition of the IMP-1 enzyme from P. aeruginosa, which belongs to the largest B1 group. This enzyme, which is encoded by the blaIMP gene, has been found to spread rapidly to other bacterial strains via horizontal gene transfer.11 X-ray structures of several B1 MβLs indicated that they share a common Rβ/βR asymmetric sandwich fold, despite significant sequence diversity, and the two metal sites are coordinated by
TABLE 1: List of X-ray Structures of the IMP-1 Enzyme Complexed with Various Inhibitors PDB code 1DD6 1JJE 1JJT 1HLK (1KR3) 1VGN 2DOO a
inhibitor mercaptocarboxylate succinic acid BYS succinic acid tricyclic compound SB236050
IC50 (µM) 0.09 0.0037 0.009 113
3-oxo-3-[(3-oxopropyl)0.080 ( 0.002a sulfanyl]propane-1-thiolate (OPS) dansyl-C4S- (C4H) 0.7
ref 18 26 29 24 25
-1
kinact (s ).
three His residues (Zn1 site) and by Asp, Cys, and His residues (Zn2 site).13-18 The proposed catalytic mechanism for B1 MβLs involves a hydroxide nucleophile located between the two zinc ions, which cleaves the amide bond in the substrate by attacking the carbonyl carbon in the lactam ring.19,20 Several inhibitors of IMP-1 have been identified. They include thiol compounds,18,21-25 succinic acid,26 thioester derivatives,22,27,28 tricyclic compounds,29 trifluoromethyl alcohol and ketone,30 sulfonyl hydrazone,31 cysteinyl peptide,32 and penicillin derivatives.33 However, their modes of inhibition are not always clear because not all structures have been determined by X-ray crystallographic diffraction. So far, 10 enzyme-inhibitor structures have been published,18,24-26,29 as summarized in Table 1 along with the inhibition constants. In this work, we concentrate on three IMP-1-inhibitor complexes whose structures have been solved by X-ray diffraction. (The tricyclic compound was not included because it does not bind tightly with the enzyme.29) The first complex (PDB code 1JJE)26 involves 2-(benzo[1,3]dioxol-5-ylmethyl)3-benzylsuccinic acid (BYS), which binds both zinc ions with its two carboxylate groups and interacts with several activesite residues with hydrogen bonds. The interaction pattern mimics closely that between a bona fide β-lactam substrate and the enzyme. The inhibitor in the second complex (PDB code 2DOO)25 is N-[4-({[5-(dimethylamino)-1-naphthyl]sulfonyl}amino)butyl]-3-sulfanylpropanamide (C4H), which binds both zinc ions with its thiolate group, thus displacing the hydroxide
10.1021/jp073864g CCC: $37.00 © 2007 American Chemical Society Published on Web 07/31/2007
Inhibitor Binding by MβL IMP-1
J. Phys. Chem. B, Vol. 111, No. 33, 2007 9987
Figure 1. Interaction patterns between the IMP-1 enzyme and the three inhibitors.
nucleophile. Its binding pattern is quite similar to that of the mercaptocarboxylate inhibitor studied earlier by Concha et al.18 A unique feature of this inhibitor is its ability as a probe of the IMP-1 enzyme thanks to its dansyl fluorophore. The inhibitor (3-oxo-3-[(3-oxopropyl)sulfanyl]propane-1-thiolate, or OPS) in the last complex (PDB code 1VGN)24 is similar to C4H in its binding pattern, but it also forms a covalent bond with the highly conserved Lys161. As a result, it inhibits the MβL irreversibly and represents a new antibiotic strategy.34 The enzyme-inhibitor interaction patterns are displayed in Figure 1, and they represent the prototypes of mechanism-based inhibition of MβLs. A major motivation of the current work is to understand the fluctuation of the enzyme-inhibitor complexes at the atomic level. Detailed information such as flexibility of ligand binding cannot be obtained by experiment alone. Computational simulations can provide complementary and valuable insights.35 Indeed, our understanding of MβLs has benefited greatly from computer simulations at various levels of sophistication.36-55 Here, we are particularly concerned with the ligand-metal interactions as well as the electrostatic and “hydrophobic” interactions between the inhibitor and active-site residues. Such information is valuable in designing novel and more efficient inhibitors. The second goal of our work is to test the validity of a particular quantum mechanical/molecular mechanical (QM/MM) approach56-58 for characterizing metalloenzymes. The QM/MM approach is a powerful tool in understanding binding and catalysis of metal centers in enzymatic systems, mainly because of the difficulties of using force fields to describe metal-ligand bonds.59,60 In the current study, we adapted a semiempirical density functional theory for the QM region,61,62 which has shown much promise in describing zinc enzymes.53-55,58,63 By comparison with experimental structural data, we can assess its accuracy in describing the metal coordination and hydrogen bond in the enzyme active site. Computational Methods Model. The starting geometries of three enzyme-inhibitor complexes were adopted from the X-ray structures (PDB codes 1JJE,26 1VGN,24 and 2DOO25). We focus on one subunit of the dimeric enzyme. In all three models, Zn1 is coordinated by His77, His79, and His139, while Zn2 is coordinated by His197, one Asp81, and Cys158, as shown in Figure 1. All four His residues were neutral, but the Nδ1 atom was protonated in His77, His139, and His197, while the N2 atom of His79 was protonated. Both Asp81 and Cys158 were considered to be ionized, each carrying a negative charge. In the 1VGN case, a
new residue has to be defined to describe the enzyme-inhibitor adduct on Lys161. All the heavy atom coordinates were adopted from the crystal structures without any modification. Hydrogen atoms were then added by the HBUILD module of CHARMM.64 The three complexes were solvated repeatedly with a preequilibrated TIP3P water65 sphere, which was centered at the Zn1 atom with a radius of 25 Å. Any solvent water molecule that came within a 2.8 Å radius of a non-hydrogen atom in the protein was deleted, followed by relaxation of the solvent water sphere with all protein and crystal water fixed. Using the stochastic boundary (SB) protocol,66 the solvated system was divided into the reaction region (r < 22 Å) and buffer region (22 Å < r < 25 Å), with atoms outside the 25 Å radius deleted. The energy of the entire system was then minimized using both the conjugated gradient method and the adaptive basis Newton-Raphson method. QM/MM. In the QM/MM approach,67 the enzymatic system is partitioned into a QM region and an MM region. The former is treated with a quantum model for the electrons, while the latter is approximated by a force field. For the dizinc IMP-1 complexes, the QM region includes the two metal ions, their protein ligands, and the entire inhibitor. As a result, the QM model has to be very efficient to handle the large number of electrons. For example, Merz and co-workers have used the semiempirical PM3 method in their QM/MM study of a dizinc MβL (CcrA from Bacteroides fragilis).41 We chose to employ an alternative semiempirical density functional method, namely, self-consistent-charge density-functional tight-binding (SCCDFTB).61 SCC-DFTB is very efficient and reasonably accurate as shown by several recent studies.62 Using a new parametrization of the zinc ion in a biological environment,63 very encouraging results have also been obtained for zinc enzymes.53-55,58 In the current implementation,62 the QM region is approximated by the SCC-DFTB method, while the MM region is approximated by the CHARMM all-atom force field.68 At the boundary, the link atom approach69 was used to saturate the dangling bonds. For the six protein ligands, a link atom was placed between the CR and Cβ atoms in each case. An additional link atom was added in the 1VGN case to cover the covalent bond between the inhibitor and the Lys161 side chain. There are 130, 95, and 115 QM atoms in the 1JJE, 1VGN, and 2DOO complexes, respectively. Molecular Dynamics. In the molecular dynamics (MD) simulations, atoms in the reaction region were governed by Newtonian dynamics on the QM/MM potential. On the other hand, the atoms in the buffer zone were simulated with the
9988 J. Phys. Chem. B, Vol. 111, No. 33, 2007
Wang and Guo TABLE 2: Comparison of Key Geometric Parameters in the 1JJE Complex bond length (Å) or bond angle (deg)
simulation
X-ray (a/b chains)
Zn1-Zn2 Zn1-N2 (His77) Zn1-Nδ1 (His79) Zn1-N2 (His139) Zn2-Oδ2 (Asp81) Zn2-Sγ (Cys158) Zn2-N2 (His197) O3-Zn1 O3-Zn2 O14-Zn2 O4-H-Nδ2 (Asn167) O14-H-Nζ (Lys161) O15-H-Nζ (Lys161) O15-HN (Asn167) N2 (His77)-Zn1-Nδ1 (His79) N2 (His77)-Zn1-N2 (His139) Nδ1 (His79)-Zn1-N2 (His139) N2 (His77)-Zn1-O3 Nδ1 (His79)-Zn1-O3 N2 (His139)-Zn1-O3 Oδ2 (Asp81)-Zn2-Sγ (Cys158) Oδ2 (Asp81)-Zn2-N2 (His197) Sγ (Cys158)-Zn2-N2 (His197) Oδ2 (Asp81)-Zn1-O3 Sγ (Cys158)-Zn1-O3 N2 (His197)-Zn1-O3 Zn1-O3-Zn2
3.67 ( 0.14 1.98 ( 0.05 2.01 ( 0.06 2.00 ( 0.06 2.11 ( 0.07 2.31 ( 0.06 2.06 ( 0.07 2.19 ( 0.09 2.10 ( 0.06 2.76 ( 0.31 2.06 ( 0.35 (3.00 ( 0.26)a 1.93 ( 0.24 (2.86 ( 0.17)a 2.00 ( 0.26 (2.79 ( 0.14)a 1.86 ( 0.14 (2.84 ( 0.13)a 103.9 ( 5.0 105.9 ( 6.1 112.9 ( 5.6 112.7 ( 5.6 111.8 ( 5.6 102.7 ( 5.4 106.9 ( 7.1 88.5 ( 4.4 110.7 ( 6.5 99.7 ( 8.6 111.8 ( 5.6 130.5 ( 7.9 118.0 ( 5.3
3.56/3.60 2.09/2.09 2.06/2.08 1.99/1.98 2.00/2.00 2.28/2.31 2.15/2.15 2.14/2.14 2.00/2.03 2.30/2.32 (2.83/2.86)a (3.36/3.44)a (2.73/2.66)a (2.86/3.04)a 97.7/98.7 105.0/105.4 107.8/109.6 122.5/121.1 113.4/113.5 109.1/107.7 107.0/109.4 89.9/91.3 107.8/110.8 91.3/91.4 110.3/110.1 139.7/135.5 118.7/119.3
a
Distances between heavy atoms (N-O) are given in parentheses.
TABLE 3: Comparison of Key Geometric Parameters in the 1VGN Complex
Figure 2. RMSDs of three enzyme-inhibitor complexes as a function of time.
Langevin dynamics with friction and random forces stemming from the bulk solvent that were not explicitly included in the simulation.66 Nonbonded interactions were cut off at 12 Å, but the electrostatic interactions were dealt with using the extended electrostatics model of the group method,70 which has been shown to provide a balanced treatment of the QM-MM interactions.55 The trajectories were propagated with a time step of 1.0 fs, and the SHAKE algorithm71 was applied to maintain the covalent bonds involving H atoms. All the calculations reported in this work were carried out using the CHARMM suite of simulation codes.64 The temperature was increased to 300 K in 50 ps, and the system was allowed to equilibrate for 200 ps. Data collection was carried out for an additional 400 ps. The coordinate data allow the calculation of B factors according to the following formula:
Bi ) 8π2〈∆ri2〉 )
8π2
N
∑(∆xi2 + ∆yi2 + ∆zi2) N i
bond length (Å) or bond angle (deg)
simulation
X-ray (a/b chains)
Zn1-Zn2 Zn1-N2 (His77) Zn1-Nδ1 (His79) Zn1-N2 (His139) Zn2-Oδ2 (Asp81) Zn2-Sγ (Cys158) Zn2-N2 (His197) S10-Zn1 S10-Zn2 O7-H-Nζ (Lys161) N2 (His77)-Zn1-Nδ1 (His79) N2 (His77)-Zn1-N2 (His139) Nδ1 (His79)-Zn1-N2 (His139) N2 (His77)-Zn1-S10 Nδ1 (His79)-Zn1-S10 N2 (His139)-Zn1-S10 Oδ2 (Asp81)-Zn2-Sγ (Cys158) Oδ2 (Asp81)-Zn2-N2 (His197) Sγ (Cys158)-Zn2-N2 (His197) Oδ2 (Asp81)-Zn1-S10 Sγ (Cys158)-Zn1-S10 N2 (His197)-Zn1-S10 Zn1-S10-Zn2
3.79 ( 0.14 2.01 ( 0.06 2.00 ( 0.06 2.00 ( 0.05 2.06 ( 0.06 2.32 ( 0.06 2.04 ( 0.06 2.36 ( 0.07 2.38 ( 0.07 not stable 104.0 ( 4.7 105.5 ( 4.5 116.8 ( 5.6 113.6 ( 5.5 116.4 ( 5.5 99.6 ( 5.1 112.5 ( 6.6 94.4 ( 4.7 113.4 ( 6.0 106.1 ( 6.0 119.4 ( 5.8 106.8 ( 6.0 106.3 ( 5.5
3.76/3.69 1.98/2.04 1.82/1.92 2.08/2.14 2.23/2.04 2.32/2.18 2.04/2.12 2.19/2.19 2.31/2.44 (3.06/3.27)a 96.8/108.2 110.8/117.6 109.5/113.8 129.7/125.8 117.5/100.8 92.0/89.1 101.2/101.1 99.2/103.5 109.1/115.7 121.9/111.8 107.0/113.0 117.0/110.9 112.7/105.3
a
Distances between heavy atoms (N-O) are given in parentheses.
(1)
where ∆xi, ∆yi, and ∆zi are the displacements of atom i at each time step from its average positions in the x, y, and z coordinates. Results and Discussion All three systems were found to be quite stable during the entire course of simulation. The root-mean-square deviation (RMSD) of backbone atoms was calculated with regard to the experimental coordinates and is shown in Figure 2. The averaged
RMSDs for the 1JJE, 1VGN, and 2DOO complexes are 0.91 ( 0.04, 0.97 ( 0.05, and 0.98 ( 0.07 Å, respectively. The averaged geometric parameters are listed in Tables 2-4. Snapshots of the active sites of the three complexes are displayed in Figure 3. As shown in Figures 1 and 3, the succinic acid inhibitor BYS possesses two carboxylate groups which are both metal bound. One such group mimics the functionally conserved C3/C4 carboxylate group in β-lactam antibiotic molecules and binds
Inhibitor Binding by MβL IMP-1
J. Phys. Chem. B, Vol. 111, No. 33, 2007 9989
TABLE 4: Comparison of Key Geometric Parameters in the 2DOO Complex bond length (Å) or bond angle (deg)
simulation
X-ray (a/b chains)
Zn1-Zn2 Zn1-N2 (His77) Zn1-Nδ1 (His79) Zn1-N2 (His139) Zn2-Oδ2 (Asp81) Zn2-Sγ (Cys158) Zn2-N2 (His197) S25-Zn1 S25-Zn2 O26-H-Nζ (Lys161) N2 (His77)-Zn1-Nδ1 (His79) N2 (His77)-Zn1-N2 (His139) Nδ1 (His79)-Zn1-N2 (His139) N2 (His77)-Zn1-S25 Nδ1 (His79)-Zn1-S25 N2 (His139)-Zn1-S25 Oδ2 (Asp81)-Zn2-Sγ (Cys158) Oδ2 (Asp81)-Zn2-N2 (His197) Sγ (Cys158)-Zn2-N2 (His197) Oδ2 (Asp81)-Zn1-S25 Sγ (Cys158)-Zn1-S25 N2 (His197)-Zn1-S25 Zn1-S25-Zn2
3.79 ( 0.15 2.00 ( 0.05 2.02 ( 0.06 2.02 ( 0.06 2.06 ( 0.06 2.32 ( 0.06 2.05 ( 0.07 2.35 ( 0.07 2.40 ( 0.08 2.22 ( 0.34 (3.10 ( 0.23)b 101.0 ( 4.7 104.8 ( 4.6 107.4 ( 5.5 120.6 ( 6.0 117.7 ( 5.9 103.4 ( 5.3 111.3 ( 6.9 94.3 ( 4.8 109.5 ( 6.6 107.9 ( 6.1 117.1 ( 6.0 113.4 ( 5.9 106.1 ( 5.7
3.70/3.70 2.06/2.11 2.04/2.02 2.09/2.08 2.12/1.94 2.22/2.21 1.96/2.08 2.16 (2.08)/2.35 (2.36)a 2.36 (2.38)/2.30 (2.16)a (3.41/3.45)b 108.4/103.7 107.8/116.2 103.2/107.1 124.5 (122.6)/109.8 (123.0)a 107.0 (103.5)/112.8 (109.9)a 93.5 (98.9)/97.0 (96.2)a 109.9/107.9 100.8/96.9 118.8/113.6 106.4 (101.2)/114.5 (114.2)a 104.4 (108.4)/109.4 (110.1)a 105.9 (105.8)/114.0 (113.5)a 111.9 (109.6)/109.5 (105.4)a
a Numbers in parentheses are from the coordinates of another conformer reported in the PDB. b Distances between heavy atoms (N-O) are given in parentheses.
with Zn2. The other carboxylate displaces the zinc-bridging hydroxide and binds with both zinc ions with one of its oxygen atoms. In particular, the Zn1-Zn2 distance is 3.67 ( 0.14 Å, which can be compared with the experimental values of 3.56 and 3.60 Å found in the two subunits.26 This distance is also similar to the typical values found in several B1 enzymes where a hydroxide bridges the two ions.19 Interestingly, the slightly tighter binding of the bridging oxygen (O3) with Zn1 was also reproduced in our simulation. The larger O3-Zn2 distance can probably be attributed to the pentacoordination of Zn2. Table 1 also shows that the coordination bonds of the two metal ions are well reproduced by the simulation, as evidenced by the agreement of the bond lengths and bond angles with experimental data. These ligand-metal bonds are generally quite rigid, with only small fluctuations. The only exception is the O14-Zn2 bond, which fluctuates substantially and has a larger bond length (2.76 ( 0.31 Å) than the experimental value (2.30/ 2.32 Å). There are several hydrogen bonds between the BYS inhibitor and active-site residues. Both oxygen atoms of the Zn2-binding carboxylate are hydrogen bonded with the side chain of the highly conserved Lys161, as evidenced by the O15‚‚‚H and O14‚‚‚H distances of 2.00 ( 0.26 and 1.93 ( 0.24 Å. These hydrogen bonds are similar to that between a bona fide substrate and the same Lys residue.41,72 The inhibitor O15 atom is also hydrogen bonded with the backbone NH of Asn167 with an O‚‚‚H distance of 1.86 ( 0.14 Å. Finally, the NH2 side chain of Asn167 binds with O4 with a weak hydrogen bond with an O‚‚‚H distance of 2.06 ( 0.35 Å. In other MβLs, it has been suggested that a very similar interaction pattern exists between the enzyme and a substrate, and the Asn167 side chain (or its equivalent) provides an oxyanion hole during catalytic cleavage of the lactam ring by the zinc-bridging hydroxide nucleophile.14,73 In addition to the hydrogen bond network, the side chain of Trp28 also provides quadrupolar interaction with the benzo[1,3]dioxole moiety in BYS, as shown in Figure 3. This hydrophobic pocket was first noted in the IMP-1 complex with a mercaptocarboxylate inhibitor.18 These extensive interactions
with the enzyme are consistent with the fact that the BYS inhibitor has the smallest IC50 among all known inhibitors of IMP-1. Both the OPS and C4H inhibitors anchor at the enzyme active site with thiolate coordination bonds with both zinc ions. As shown in Figures 1 and 3, the thiolate is in the bridge position, displacing the hydroxide nucleophile, thus deactivating the enzyme. The same binding mode was also observed earlier in the IMP-1 complexed with a mercaptocarboxylate inhibitor.18 In both complexes, the Zn-Zn distances are almost identical (3.79 ( 0.14 and 3.79 ( 0.15 Å), which can be compared with the experimental values ranging from 3.69 to 3.76 Å. This distance is slightly larger than the case with succinic acid, in which the bridging atom is oxygen. The overall agreement with experimental bond lengths and bond angles is quite good, as shown in Tables 3 and 4. In the simulations, the distances between the sulfur atom and the two zinc ions are almost the same, while the X-ray structures seem to suggest a slightly shorter S-Zn1 distance. It is interesting to compare our QM/MM results with earlier MD simulations of the IMP-1 enzyme complexed with a mercaptocarboxylate inhibitor.49 It was shown that the X-ray structure of the complex18 could not be maintained in the simulation if a force field based on a nonbonded approach was used. Reasonable reproduction of the experimentally observed structure was only possible when the zinc ions were simulated by a cationic dummy atom approach in which the tetrahedral coordination of the Zn(II) was maintained by four such dummy atoms. Even then, the calculated Zn-Zn distance was about 0.2 Å longer than the X-ray structure, and the ligand-metal bond lengths were found to be significantly shorter. Similar magnitudes of errors have also been observed in several force field based MD simulations of other dinuclear MβLs.39,42,44 The OPS substrate is immobilized in 1VGN by a covalent bond with the side chain of Lys161, and its interactions with other active-site residues are not strong. The X-ray structure of 1VGN suggests that there might be a hydrogen bond between O7 and the NH2 side chain of Asn167, but this interaction is
9990 J. Phys. Chem. B, Vol. 111, No. 33, 2007
Figure 3. Snapshots of the active site in three enzyme-inhibitor complexes.
quite weak as evidenced by the O7-N distance of 3.06/3.27 Å.24 Indeed, this hydrogen bond was unstable in our simulation, and water molecules were found intermittently between the two groups. The two carbonyl groups in the OPS inhibitor were found in our simulation to be surrounded by solvent water molecules. The C4H inhibitor in 2DOO forms a hydrogen bond with the Lys161 with its sulfonyl group. However, the strength of this hydrogen bond is weak, as evidenced by the relatively long H-O distance and large fluctuation (2.22 ( 0.34 Å), presumably due to competition by other hydrogen bonds formed between the sulfonyl group and a number of solvent water molecules. In addition, the naphthyl ring of the substrate is found to interact
Wang and Guo
Figure 4. Space-filling rendition of the binding patterns in three complexes. Key residues (Trp28 and Asn167) in the two loops are shown to interact with the inhibitors.
with the side chain of Trp28 via quadrupolar forces. These interactions can be seen in Figure 3, but they are weak compared with the coordination of its thiolate with the two zinc ions, as evidenced by a large fluctuation between the centers of the two rings (5.18 ( 0.33 Å). To gauge the strength of the inhibitor-enzyme interaction, it is perhaps instructive to compare the B factors of two key active-site residues, namely, Lys161 and Asn167, in the three complexes. As shown in Table 5, the fluctuation of these residues varies substantially. In the 1JJE complex, both residues are involved in hydrogen bonds with the inhibitor. As a result, both the backbone and side chain are relatively stable. In the
Inhibitor Binding by MβL IMP-1
J. Phys. Chem. B, Vol. 111, No. 33, 2007 9991 Conclusions
Figure 5. Comparison of CR B factors of the three enzyme-inhibitor complexes.
TABLE 5: Calculated B Factors for Two Key Active-Site Residues B factors (Å2) residue
atom
1JJE
1VGN
2DOO
Lys161
CR Nζ CR Nδ2
12.26 15.58 25.44 39.66
11.79 24.53 158.12 508.10
11.26 17.89 49.01 157.39
Asn167
1VGN complex, on the other hand, the lack of strong hydrogen bonds with the inhibitor leads to large B factors for Asn167. Similarly, the B factors for Asn167 in the 2DOO complex are also quite large, because of the absence of hydrogen bonds. On the other hand, the hydrogen bond with the sulfonyl group significantly stabilizes the Lys161 side chain. In addition to the direct metal coordination, the inhibitors are also partially held in the active site by two loop structures, as shown in Figure 4. The minor loop consisting of residues 166-170 and its effect on binding have been discussed above in the context of the key residue Asn167. The major loop comprised of the 25-29 residues is essentially a β-sheet flap, and it interacts with the inhibitor mainly through Trp28. The latter loop has been shown experimentally to be an important determinant in substrate/inhibitor binding of several MβLs. Specifically, NMR relaxation studies observed significant rigidification of this loop in the presence of a tight-binding inhibitor.74,75 In addition, mutation of Trp28 in IMP-1 was found to be detrimental to substrate binding.76 Its role in clamping down the substrate is also supported by computational studies.42,43 To further understand the role of these loops in the inhibitor binding by IMP-1, we have computed the B factors of the backbone CR atoms for all residues in the three complexes. As shown in Figure 5, the fluctuation of the residues in the two loops is very pronounced. The 25-29 loop of the 1VGN complex is the most mobile among the three enzyme-inhibitor complexes, judging from the corresponding B factors in Figure 5. This can be readily understood because this small inhibitor is covalently linked to Lys161 and has little interaction with the residues in this loop, as shown in Figure 4. On the other hand, the BYS inhibitor in the 1JJE complex, which is also shown in Figure 4, is much bulkier, and the interactions with both loops are important binding determinants. This is reflected by the relatively small B factors for the residues in the two loops. The 2DOO complex is an intermediate case, as shown in Figure 4, and the fluctuation is thus reflected by the corresponding B factors. These observations provide further supporting evidence for the functionality of these mobile loops in ligand binding, which has to be taken into consideration in designing new and enzyme-specific inhibitors.
We in this work investigated the dynamics of the IMP-1 enzyme complexes with three inhibitors that deactivate the MβL by displacing the hydroxide nucleophile bridging the zinc ions. The experimental structures of the complexes are well reproduced by MD trajectories based on a QM/MM approach. Particularly encouraging is the excellent characterization of the metal-ligand interactions in the active site, which provides further evidence in support of the validity of the semiempirical SCC-DFTB in treating the metal centers in enzymes. The most important anchoring interaction of the inhibitors with the enzyme is probably the direct metal binding. For OPS, a covalent bond with Lys161 provides an additional anchoring point for the inhibitor. However, the succinic acid BYS was found to be stabilized by an elaborate hydrogen bond network and the hydrophobic interaction with Trp28 via a quadrupolar force. Hydrophobic interactions and hydrogen bonds contribute too in the case of C4H. All these binding determinants are reasonably reproduced by the QM/MM simulations. Acknowledgment. This work was supported by the National Institutes of Health (Grant R03AI068672). Some of the calculations were carried out at the National Centers for Supercomputing Applications (NCSA). References and Notes (1) Neu, H. C. Science 1992, 257, 1064. (2) Knowles, J. R. Acc. Chem. Res. 1985, 18, 97. (3) Ambler, R. P. Phil. Trans. R. Soc. London 1980, B289, 321. (4) Bush, K.; Jacoby, G. A.; Medeiros, A. A. Antimicrob. Agents Chemother. 1995, 39, 1211. (5) Fisher, J. F.; Meroueh, S. O.; Mobashery, S. Chem. ReV. 2005, 105, 395. (6) Bush, K. Clin. Infect. Dis. 1998, 27, S48. (7) Payne, D. J.; Du, W.; Bateson, J. H. Expert Opin. InVest. Drugs 2000, 9, 247. (8) Walsh, T. R.; Toleman, M. A.; Poirel, L.; Nordmann, P. Clin. Microbiol. ReV. 2005, 18, 306. (9) Toney, J. H.; Moloughney, J. G. Curr. Opin. InVest. Drugs 2004, 5, 823. (10) Spencer, J.; Walsh, T. R. Angew. Chem., Int. Ed. 2006, 45, 1022. (11) Livermore, D. M.; Woodford, N. Curr. Opin. Microbiol. 2000, 3, 489. (12) Galleni, M.; Lamotte-Brasseur, J.; Rossolini, G. M.; Spencer, J.; Dideberg, O.; Frere, J.-M. Antimicrob. Agents Chemother. 2001, 45, 660. (13) Carfi, A.; Pares, S.; Duee, E.; Galleni, M.; Duez, C.; Frere, J.-M.; Dideberg, O. EMBO J. 1995, 14, 4914. (14) Concha, N. O.; Rasmussen, B. A.; Bush, K.; Herzberg, O. Structure 1996, 4, 823. (15) Carfi, A.; Duee, E.; Galleni, M.; Frere, J.-M.; Dideberg, O. Acta Crystallogr. 1998, D54, 313. (16) Carfi, A.; Duee, E.; Paul-Soto, R.; Galleni, M.; Frere, J.-M.; Dideberg, O. Acta Crystallogr. 1998, D54, 47. (17) Fabiane, S. M.; Sohi, M. K.; Wan, T.; Payne, D. J.; Bateson, J. H.; Mitchell, T.; Sutton, B. J. Biochemistry 1998, 37, 12404. (18) 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. (19) Wang, Z.; Fast, W.; Valentine, A. M.; Benkovic, S. J. Curr. Opin. Chem. Biol. 1999, 3, 614. (20) Crowder, M. W.; Spencer, J.; Vila, A. J. Acc. Chem. Res. 2006, 45, 13650. (21) Goto, M.; Takahashi, T.; Yamashita, F.; Koreeda, A.; Mori, H.; Ohta, M.; Arakawa, Y. Biol. Pharm. Bull. 1997, 20, 1136. (22) Greenlee, M. L.; Laub, J. B.; Balkovec, J. M.; Hammond, M. L.; Hammond, G. G.; Pompliano, D. L.; Epstein-Toney, J. H. Bioorg. Med. Chem. Lett. 1999, 9, 2549. (23) Mollard, C.; Moali, C.; Papamicael, C.; Damblon, C.; Vessilier, S.; Amicosante, G.; Schofield, C. J.; Galleni, M.; Frere, J.-M.; Roberts, G. C. K. J. Biol. Chem. 2001, 276, 45015. (24) Kurosaki, H.; Yamaguchi, Y.; Higashi, T.; Soga, K.; Matsueda, S.; Yumoto, H.; Misumi, S.; Yamagata, Y.; Arakawa, Y.; Goto, M. Angew. Chem., Int. Ed. 2005, 44, 3861. (25) Kurosaki, H.; Yamaguchi, H.; Yasuzawa, H.; Jin, W.; Yamagata, Y.; Arakawa, Y. ChemMedChem 2006, 1, 969.
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