Inhibitor Binding by Metallo- â-lactamase IMP-1 from Pseudomonas aeruginosa: QuantumMechanical/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 aquantum mechanical/molecular mechanical (QM/MM) method based on the self-consistent-charge density-functional tight-binding model. The binding patterns of the inhibitors observed in X-ray diffraction experimentsare well reproduced in 600 ps molecular dynamics simulations at room temperature. These inhibitors anchorthemselves in the enzyme active site by direct coordination with the two zinc ions, displacing the hydroxidenucleophile that bridges the two zinc ions. In addition, they also interact with several active-site residues andthose in two mobile loops. The excellent agreement with experimental structural data validates the QM/MMtreatment used in our simulations.

Introduction

The ever increasing level of antibiotic resistance by patho-genic bacteria threatens to jeopardize the treatment of infectiousdiseases, posing a great challenge to public health.1 Resistanceto â-lactam antibiotics, such as penicillin, cephalosphorins, andcarbapenems, is commonly conferred by bacterialâ-lactamases.They are hydrolases that deactivate the antibiotics by thecleavage of the lactam amide (C-N) bond.2 These enzymeshave traditionally been divided into four classes.3,4 Those inclasses A, C, and D rely on an active-site serine in covalentcatalysis,5 while the class B enzymes require one or two zinccofactors, thus called metallo-â-lactamases (MâLs).6 Thecatalytic mechanism of serine-basedâ-lactamases is wellestablished, and several effective inhibitors,7 such as clavulanicacid, are routinely used in clinical settings. However, suchinhibitors have no effect on MâLs because of their completelydifferent catalytic mechanisms. Although various strategies havebeen suggested,8-10 there is still no clinically useful inhibitorof MâLs.

The potential threat of MâLs in the future treatment ofinfectious diseases stems mainly from their broad substrateprofile, which includes mostâ-lactam-based antibiotics and eveninhibitors of serine-basedâ-lactamases.6 Although their preva-lence is still low, recent evidence indicated a rapid proliferationin pathogenic species via plasmid- or integron-mediated mech-anisms.8,11 MâLs have been found worldwide in opportunisticpathogens such asPseudomonas aeruginosaand Stenotroph-omonas maltophilia, and it is only a matter of time before theyspread to major pathogens. Hence, a better understanding ofthe inhibition of MâLs is urgently needed.

MâLs are further classified into three groups.6,12 B1 and B3enzymes contain two zinc ions when fully loaded, but B2enzymes are inhibited by the second metal ion. In this work,we focus on the inhibition of the IMP-1 enzyme fromP.aeruginosa, which belongs to the largest B1 group. This enzyme,which is encoded by theblaIMP gene, has been found to spreadrapidly to other bacterial strains via horizontal gene transfer.11

X-ray structures of several B1 MâLs indicated that they sharea commonRâ/âR asymmetric sandwich fold, despite significantsequence diversity, and the two metal sites are coordinated by

three His residues (Zn1 site) and by Asp, Cys, and His residues(Zn2 site).13-18 The proposed catalytic mechanism for B1 MâLsinvolves a hydroxide nucleophile located between the two zincions, which cleaves the amide bond in the substrate by attackingthe carbonyl carbon in the lactam ring.19,20

Several inhibitors of IMP-1 have been identified. They includethiol compounds,18,21-25 succinic acid,26 thioester deriva-tives,22,27,28tricyclic compounds,29 trifluoromethyl alcohol andketone,30 sulfonyl hydrazone,31 cysteinyl peptide,32 and penicillinderivatives.33 However, their modes of inhibition are not alwaysclear because not all structures have been determined by X-raycrystallographic diffraction. So far, 10 enzyme-inhibitor struc-tures have been published,18,24-26,29 as summarized in Table 1along with the inhibition constants.

In this work, we concentrate on three IMP-1-inhibitorcomplexes whose structures have been solved by X-ray dif-fraction. (The tricyclic compound was not included because itdoes 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 withits two carboxylate groups and interacts with several active-site residues with hydrogen bonds. The interaction patternmimics closely that between a bona fideâ-lactam substrate andthe enzyme. The inhibitor in the second complex (PDB code2DOO)25 is N-[4-({[5-(dimethylamino)-1-naphthyl]sulfonyl}-amino)butyl]-3-sulfanylpropanamide (C4H), which binds bothzinc ions with its thiolate group, thus displacing the hydroxide

TABLE 1: List of X-ray Structures of the IMP-1 EnzymeComplexed with Various Inhibitors

PDBcode inhibitor

IC50

(µM) ref

1DD6 mercaptocarboxylate 0.09 181JJE succinic acid BYS 0.0037 261JJT succinic acid 0.0091HLK

(1KR3)tricyclic compound SB236050 113 29

1VGN 3-oxo-3-[(3-oxopropyl)-sulfanyl]propane-1-thiolate (OPS)

0.080( 0.002a 24

2DOO dansyl-C4S- (C4H) 0.7 25

a kinact (s-1).

9986 J. Phys. Chem. B2007,111,9986-9992

10.1021/jp073864g CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 07/31/2007

nucleophile. Its binding pattern is quite similar to that of themercaptocarboxylate inhibitor studied earlier by Concha et al.18

A unique feature of this inhibitor is its ability as a probe of theIMP-1 enzyme thanks to its dansyl fluorophore. The inhibitor(3-oxo-3-[(3-oxopropyl)sulfanyl]propane-1-thiolate, or OPS) inthe last complex (PDB code 1VGN)24 is similar to C4H in itsbinding pattern, but it also forms a covalent bond with the highlyconserved Lys161. As a result, it inhibits the MâL irreversiblyand represents a new antibiotic strategy.34 The enzyme-inhibitorinteraction patterns are displayed in Figure 1, and they representthe prototypes of mechanism-based inhibition of MâLs.

A major motivation of the current work is to understand thefluctuation of the enzyme-inhibitor complexes at the atomiclevel. Detailed information such as flexibility of ligand bindingcannot be obtained by experiment alone. Computational simula-tions can provide complementary and valuable insights.35

Indeed, our understanding of MâLs has benefited greatly fromcomputer simulations at various levels of sophistication.36-55

Here, we are particularly concerned with the ligand-metalinteractions as well as the electrostatic and “hydrophobic”interactions between the inhibitor and active-site residues. Suchinformation is valuable in designing novel and more efficientinhibitors.

The second goal of our work is to test the validity of aparticular quantum mechanical/molecular mechanical (QM/MM)approach56-58 for characterizing metalloenzymes. The QM/MMapproach is a powerful tool in understanding binding andcatalysis of metal centers in enzymatic systems, mainly becauseof the difficulties of using force fields to describe metal-ligandbonds.59,60 In the current study, we adapted a semiempiricaldensity functional theory for the QM region,61,62 which hasshown much promise in describing zinc enzymes.53-55,58,63Bycomparison with experimental structural data, we can assessits accuracy in describing the metal coordination and hydrogenbond in the enzyme active site.

Computational Methods

Model. The starting geometries of three enzyme-inhibitorcomplexes were adopted from the X-ray structures (PDB codes1JJE,26 1VGN,24 and 2DOO25). We focus on one subunit ofthe dimeric enzyme. In all three models, Zn1 is coordinated byHis77, His79, and His139, while Zn2 is coordinated by His197,one Asp81, and Cys158, as shown in Figure 1. All four Hisresidues were neutral, but the Nδ1 atom was protonated in His77,His139, and His197, while the Nε2 atom of His79 wasprotonated. Both Asp81 and Cys158 were considered to beionized, each carrying a negative charge. In the 1VGN case, a

new residue has to be defined to describe the enzyme-inhibitoradduct on Lys161.

All the heavy atom coordinates were adopted from the crystalstructures without any modification. Hydrogen atoms were thenadded by the HBUILD module of CHARMM.64 The threecomplexes were solvated repeatedly with a preequilibratedTIP3P water65 sphere, which was centered at the Zn1 atom witha radius of 25 Å. Any solvent water molecule that came withina 2.8 Å radius of a non-hydrogen atom in the protein wasdeleted, followed by relaxation of the solvent water sphere withall protein and crystal water fixed. Using the stochastic boundary(SB) protocol,66 the solvated system was divided into thereaction region (r < 22 Å) and buffer region (22 Å< r < 25Å), with atoms outside the 25 Å radius deleted. The energy ofthe entire system was then minimized using both the conjugatedgradient method and the adaptive basis Newton-Raphsonmethod.

QM/MM. In the QM/MM approach,67 the enzymatic systemis partitioned into a QM region and an MM region. The formeris treated with a quantum model for the electrons, while thelatter is approximated by a force field. For the dizinc IMP-1complexes, the QM region includes the two metal ions, theirprotein ligands, and the entire inhibitor. As a result, the QMmodel has to be very efficient to handle the large number ofelectrons. For example, Merz and co-workers have used thesemiempirical PM3 method in their QM/MM study of a dizincMâL (CcrA from Bacteroides fragilis).41 We chose to employan alternative semiempirical density functional method, namely,self-consistent-charge density-functional tight-binding (SCC-DFTB).61 SCC-DFTB is very efficient and reasonably accurateas shown by several recent studies.62 Using a new parametriza-tion of the zinc ion in a biological environment,63 very en-couraging results have also been obtained for zinc enzymes.53-55,58

In the current implementation,62 the QM region is ap-proximated by the SCC-DFTB method, while the MM regionis approximated by the CHARMM all-atom force field.68 Atthe boundary, the link atom approach69 was used to saturatethe dangling bonds. For the six protein ligands, a link atomwas placed between the CR and Câ atoms in each case. Anadditional link atom was added in the 1VGN case to cover thecovalent bond between the inhibitor and the Lys161 side chain.There are 130, 95, and 115 QM atoms in the 1JJE, 1VGN, and2DOO complexes, respectively.

Molecular Dynamics. In the molecular dynamics (MD)simulations, atoms in the reaction region were governed byNewtonian dynamics on the QM/MM potential. On the otherhand, the atoms in the buffer zone were simulated with the

Figure 1. Interaction patterns between the IMP-1 enzyme and the three inhibitors.

Inhibitor Binding by MâL IMP-1 J. Phys. Chem. B, Vol. 111, No. 33, 20079987

Langevin dynamics with friction and random forces stemmingfrom the bulk solvent that were not explicitly included in thesimulation.66 Nonbonded interactions were cut off at 12 Å, butthe electrostatic interactions were dealt with using the extendedelectrostatics model of the group method,70 which has beenshown to provide a balanced treatment of the QM-MMinteractions.55

The trajectories were propagated with a time step of 1.0 fs,and the SHAKE algorithm71 was applied to maintain thecovalent bonds involving H atoms. All the calculations reportedin this work were carried out using the CHARMM suite ofsimulation codes.64 The temperature was increased to 300 K in50 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 ofB factorsaccording to the following formula:

where∆xi, ∆yi, and∆zi are the displacements of atomi at eachtime step from its average positions in thex, y, andzcoordinates.

Results and Discussion

All three systems were found to be quite stable during theentire course of simulation. The root-mean-square deviation(RMSD) of backbone atoms was calculated with regard to theexperimental 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. Theaveraged geometric parameters are listed in Tables 2-4.Snapshots of the active sites of the three complexes are displayedin Figure 3.

As shown in Figures 1 and 3, the succinic acid inhibitor BYSpossesses two carboxylate groups which are both metal bound.One such group mimics the functionally conserved C3/C4

carboxylate group inâ-lactam antibiotic molecules and binds

Figure 2. RMSDs of three enzyme-inhibitor complexes as a functionof time.

Bi ) 8π2⟨∆ri2⟩ )

8π2

N∑

i

N

(∆xi2 + ∆yi

2 + ∆zi2) (1)

TABLE 2: Comparison of Key Geometric Parameters in the1JJE Complex

bond length (Å) orbond angle (deg) simulation

X-ray(a/b chains)

Zn1-Zn2 3.67( 0.14 3.56/3.60Zn1-Nε2 (His77) 1.98( 0.05 2.09/2.09Zn1-Nδ1 (His79) 2.01( 0.06 2.06/2.08Zn1-Nε2 (His139) 2.00( 0.06 1.99/1.98Zn2-Oδ2 (Asp81) 2.11( 0.07 2.00/2.00Zn2-Sγ (Cys158) 2.31( 0.06 2.28/2.31Zn2-Nε2 (His197) 2.06( 0.07 2.15/2.15O3-Zn1 2.19( 0.09 2.14/2.14O3-Zn2 2.10( 0.06 2.00/2.03O14-Zn2 2.76( 0.31 2.30/2.32O4-H-Nδ2 (Asn167) 2.06( 0.35 (3.00( 0.26)a (2.83/2.86)a

O14-H-Nú (Lys161) 1.93( 0.24 (2.86( 0.17)a (3.36/3.44)a

O15-H-Nú (Lys161) 2.00( 0.26 (2.79( 0.14)a (2.73/2.66)a

O15-HN (Asn167) 1.86( 0.14 (2.84( 0.13)a (2.86/3.04)a

Nε2 (His77)-Zn1-Nδ1 (His79) 103.9( 5.0 97.7/98.7Nε2 (His77)-Zn1-Nε2 (His139) 105.9( 6.1 105.0/105.4Nδ1 (His79)-Zn1-Nε2 (His139) 112.9( 5.6 107.8/109.6Nε2 (His77)-Zn1-O3 112.7( 5.6 122.5/121.1Nδ1 (His79)-Zn1-O3 111.8( 5.6 113.4/113.5Nε2 (His139)-Zn1-O3 102.7( 5.4 109.1/107.7Oδ2 (Asp81)-Zn2-Sγ (Cys158) 106.9( 7.1 107.0/109.4Oδ2 (Asp81)-Zn2-Nε2 (His197) 88.5( 4.4 89.9/91.3Sγ (Cys158)-Zn2-Nε2 (His197) 110.7( 6.5 107.8/110.8Oδ2 (Asp81)-Zn1-O3 99.7( 8.6 91.3/91.4Sγ (Cys158)-Zn1-O3 111.8( 5.6 110.3/110.1Nε2 (His197)-Zn1-O3 130.5( 7.9 139.7/135.5Zn1-O3-Zn2 118.0( 5.3 118.7/119.3

a Distances between heavy atoms (N-O) are given in parentheses.

TABLE 3: Comparison of Key Geometric Parameters in the1VGN Complex

bond length (Å) orbond angle (deg) simulation

X-ray(a/b chains)

Zn1-Zn2 3.79( 0.14 3.76/3.69Zn1-Nε2 (His77) 2.01( 0.06 1.98/2.04Zn1-Nδ1 (His79) 2.00( 0.06 1.82/1.92Zn1-Nε2 (His139) 2.00( 0.05 2.08/2.14Zn2-Oδ2 (Asp81) 2.06( 0.06 2.23/2.04Zn2-Sγ (Cys158) 2.32( 0.06 2.32/2.18Zn2-Nε2 (His197) 2.04( 0.06 2.04/2.12S10-Zn1 2.36( 0.07 2.19/2.19S10-Zn2 2.38( 0.07 2.31/2.44O7-H-Nú (Lys161) not stable (3.06/3.27)a

Nε2 (His77)-Zn1-Nδ1 (His79) 104.0( 4.7 96.8/108.2Nε2 (His77)-Zn1-Nε2 (His139) 105.5( 4.5 110.8/117.6Nδ1 (His79)-Zn1-Nε2 (His139) 116.8( 5.6 109.5/113.8Nε2 (His77)-Zn1-S10 113.6( 5.5 129.7/125.8Nδ1 (His79)-Zn1-S10 116.4( 5.5 117.5/100.8Nε2 (His139)-Zn1-S10 99.6( 5.1 92.0/89.1Oδ2 (Asp81)-Zn2-Sγ (Cys158) 112.5( 6.6 101.2/101.1Oδ2 (Asp81)-Zn2-Nε2 (His197) 94.4( 4.7 99.2/103.5Sγ (Cys158)-Zn2-Nε2 (His197) 113.4( 6.0 109.1/115.7Oδ2 (Asp81)-Zn1-S10 106.1( 6.0 121.9/111.8Sγ (Cys158)-Zn1-S10 119.4( 5.8 107.0/113.0Nε2 (His197)-Zn1-S10 106.8( 6.0 117.0/110.9Zn1-S10-Zn2 106.3( 5.5 112.7/105.3

a Distances between heavy atoms (N-O) are given in parentheses.

9988 J. Phys. Chem. B, Vol. 111, No. 33, 2007 Wang and Guo

with Zn2. The other carboxylate displaces the zinc-bridginghydroxide and binds with both zinc ions with one of its oxygenatoms. In particular, the Zn1-Zn2 distance is 3.67( 0.14 Å,which can be compared with the experimental values of 3.56and 3.60 Å found in the two subunits.26 This distance is alsosimilar to the typical values found in several B1 enzymes wherea hydroxide bridges the two ions.19 Interestingly, the slightlytighter binding of the bridging oxygen (O3) with Zn1 was alsoreproduced in our simulation. The larger O3-Zn2 distance canprobably be attributed to the pentacoordination of Zn2.

Table 1 also shows that the coordination bonds of the twometal ions are well reproduced by the simulation, as evidencedby the agreement of the bond lengths and bond angles withexperimental data. These ligand-metal bonds are generally quiterigid, with only small fluctuations. The only exception is theO14-Zn2 bond, which fluctuates substantially and has a largerbond length (2.76( 0.31 Å) than the experimental value (2.30/2.32 Å).

There are several hydrogen bonds between the BYS inhibitorand active-site residues. Both oxygen atoms of the Zn2-bindingcarboxylate are hydrogen bonded with the side chain of thehighly conserved Lys161, as evidenced by the O15‚‚‚H andO14‚‚‚H distances of 2.00( 0.26 and 1.93( 0.24 Å. Thesehydrogen bonds are similar to that between a bona fide substrateand the same Lys residue.41,72 The inhibitor O15 atom is alsohydrogen bonded with the backbone NH of Asn167 with anO‚‚‚H distance of 1.86( 0.14 Å. Finally, the NH2 side chainof Asn167 binds with O4 with a weak hydrogen bond with anO‚‚‚H distance of 2.06( 0.35 Å. In other MâLs, it has beensuggested that a very similar interaction pattern exists betweenthe enzyme and a substrate, and the Asn167 side chain (or itsequivalent) provides an oxyanion hole during catalytic cleavageof the lactam ring by the zinc-bridging hydroxide nucleo-phile.14,73 In addition to the hydrogen bond network, the sidechain of Trp28 also provides quadrupolar interaction with thebenzo[1,3]dioxole moiety in BYS, as shown in Figure 3. Thishydrophobic pocket was first noted in the IMP-1 complex witha mercaptocarboxylate inhibitor.18 These extensive interactions

with the enzyme are consistent with the fact that the BYSinhibitor has the smallest IC50 among all known inhibitors ofIMP-1.

Both the OPS and C4H inhibitors anchor at the enzyme activesite with thiolate coordination bonds with both zinc ions. Asshown in Figures 1 and 3, the thiolate is in the bridge position,displacing the hydroxide nucleophile, thus deactivating theenzyme. The same binding mode was also observed earlier inthe 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 withthe experimental values ranging from 3.69 to 3.76 Å. Thisdistance is slightly larger than the case with succinic acid, inwhich the bridging atom is oxygen. The overall agreement withexperimental bond lengths and bond angles is quite good, asshown in Tables 3 and 4. In the simulations, the distancesbetween the sulfur atom and the two zinc ions are almost thesame, while the X-ray structures seem to suggest a slightlyshorter S-Zn1 distance.

It is interesting to compare our QM/MM results with earlierMD simulations of the IMP-1 enzyme complexed with amercaptocarboxylate inhibitor.49 It was shown that the X-raystructure of the complex18 could not be maintained in thesimulation if a force field based on a nonbonded approach wasused. Reasonable reproduction of the experimentally observedstructure was only possible when the zinc ions were simulatedby a cationic dummy atom approach in which the tetrahedralcoordination of the Zn(II) was maintained by four such dummyatoms. Even then, the calculated Zn-Zn distance was about0.2 Å longer than the X-ray structure, and the ligand-metalbond lengths were found to be significantly shorter. Similarmagnitudes of errors have also been observed in several forcefield based MD simulations of other dinuclear MâLs.39,42,44

The OPS substrate is immobilized in 1VGN by a covalentbond with the side chain of Lys161, and its interactions withother active-site residues are not strong. The X-ray structure of1VGN suggests that there might be a hydrogen bond betweenO7 and the NH2 side chain of Asn167, but this interaction is

TABLE 4: Comparison of Key Geometric Parameters in the 2DOO Complex

bond length (Å) orbond angle (deg) simulation

X-ray(a/b chains)

Zn1-Zn2 3.79( 0.15 3.70/3.70Zn1-Nε2 (His77) 2.00( 0.05 2.06/2.11Zn1-Nδ1 (His79) 2.02( 0.06 2.04/2.02Zn1-Nε2 (His139) 2.02( 0.06 2.09/2.08Zn2-Oδ2 (Asp81) 2.06( 0.06 2.12/1.94Zn2-Sγ (Cys158) 2.32( 0.06 2.22/2.21Zn2-Nε2 (His197) 2.05( 0.07 1.96/2.08S25-Zn1 2.35( 0.07 2.16 (2.08)/2.35 (2.36)a

S25-Zn2 2.40( 0.08 2.36 (2.38)/2.30 (2.16)a

O26-H-Nú (Lys161) 2.22( 0.34 (3.10( 0.23)b (3.41/3.45)b

Nε2 (His77)-Zn1-Nδ1 (His79) 101.0( 4.7 108.4/103.7Nε2 (His77)-Zn1-Nε2 (His139) 104.8( 4.6 107.8/116.2Nδ1 (His79)-Zn1-Nε2 (His139) 107.4( 5.5 103.2/107.1Nε2 (His77)-Zn1-S25 120.6( 6.0 124.5 (122.6)/109.8 (123.0)a

Nδ1 (His79)-Zn1-S25 117.7( 5.9 107.0 (103.5)/112.8 (109.9)a

Nε2 (His139)-Zn1-S25 103.4( 5.3 93.5 (98.9)/97.0 (96.2)a

Oδ2 (Asp81)-Zn2-Sγ (Cys158) 111.3( 6.9 109.9/107.9Oδ2 (Asp81)-Zn2-Nε2 (His197) 94.3( 4.8 100.8/96.9Sγ (Cys158)-Zn2-Nε2 (His197) 109.5( 6.6 118.8/113.6Oδ2 (Asp81)-Zn1-S25 107.9( 6.1 106.4 (101.2)/114.5 (114.2)a

Sγ (Cys158)-Zn1-S25 117.1( 6.0 104.4 (108.4)/109.4 (110.1)a

Nε2 (His197)-Zn1-S25 113.4( 5.9 105.9 (105.8)/114.0 (113.5)a

Zn1-S25-Zn2 106.1( 5.7 111.9 (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 givenin parentheses.

Inhibitor Binding by MâL IMP-1 J. Phys. Chem. B, Vol. 111, No. 33, 20079989

quite weak as evidenced by the O7-N distance of 3.06/3.27Å.24 Indeed, this hydrogen bond was unstable in our simulation,and water molecules were found intermittently between the twogroups. The two carbonyl groups in the OPS inhibitor werefound in our simulation to be surrounded by solvent watermolecules.

The C4H inhibitor in 2DOO forms a hydrogen bond withthe Lys161 with its sulfonyl group. However, the strength ofthis hydrogen bond is weak, as evidenced by the relatively longH-O distance and large fluctuation (2.22( 0.34 Å), presumablydue to competition by other hydrogen bonds formed betweenthe sulfonyl group and a number of solvent water molecules.In addition, the naphthyl ring of the substrate is found to interact

with the side chain of Trp28 via quadrupolar forces. Theseinteractions can be seen in Figure 3, but they are weak comparedwith the coordination of its thiolate with the two zinc ions, asevidenced by a large fluctuation between the centers of the tworings (5.18( 0.33 Å).

To gauge the strength of the inhibitor-enzyme interaction,it is perhaps instructive to compare theB factors of two keyactive-site residues, namely, Lys161 and Asn167, in the threecomplexes. As shown in Table 5, the fluctuation of theseresidues varies substantially. In the 1JJE complex, both residuesare involved in hydrogen bonds with the inhibitor. As a result,both the backbone and side chain are relatively stable. In the

Figure 3. Snapshots of the active site in three enzyme-inhibitorcomplexes.

Figure 4. Space-filling rendition of the binding patterns in threecomplexes. Key residues (Trp28 and Asn167) in the two loops areshown to interact with the inhibitors.

9990 J. Phys. Chem. B, Vol. 111, No. 33, 2007 Wang and Guo

1VGN complex, on the other hand, the lack of strong hydrogenbonds with the inhibitor leads to largeB factors for Asn167.Similarly, theB factors for Asn167 in the 2DOO complex arealso quite large, because of the absence of hydrogen bonds. Onthe other hand, the hydrogen bond with the sulfonyl groupsignificantly stabilizes the Lys161 side chain.

In addition to the direct metal coordination, the inhibitorsare also partially held in the active site by two loop structures,as shown in Figure 4. The minor loop consisting of residues166-170 and its effect on binding have been discussed abovein the context of the key residue Asn167. The major loopcomprised of the 25-29 residues is essentially aâ-sheet flap,and it interacts with the inhibitor mainly through Trp28. Thelatter loop has been shown experimentally to be an importantdeterminant in substrate/inhibitor binding of several MâLs.Specifically, NMR relaxation studies observed significant ri-gidification of this loop in the presence of a tight-bindinginhibitor.74,75In addition, mutation of Trp28 in IMP-1 was foundto be detrimental to substrate binding.76 Its role in clampingdown the substrate is also supported by computational stud-ies.42,43

To further understand the role of these loops in the inhibitorbinding by IMP-1, we have computed theB factors of thebackbone CR atoms for all residues in the three complexes. Asshown in Figure 5, the fluctuation of the residues in the twoloops is very pronounced. The 25-29 loop of the 1VGNcomplex is the most mobile among the three enzyme-inhibitorcomplexes, judging from the correspondingB factors in Figure5. This can be readily understood because this small inhibitoris covalently linked to Lys161 and has little interaction withthe residues in this loop, as shown in Figure 4. On the otherhand, the BYS inhibitor in the 1JJE complex, which is alsoshown in Figure 4, is much bulkier, and the interactions withboth loops are important binding determinants. This is reflectedby the relatively smallB factors for the residues in the two loops.The 2DOO complex is an intermediate case, as shown in Figure4, and the fluctuation is thus reflected by the correspondingBfactors. These observations provide further supporting evidencefor the functionality of these mobile loops in ligand binding,which has to be taken into consideration in designing new andenzyme-specific inhibitors.

Conclusions

We in this work investigated the dynamics of the IMP-1enzyme complexes with three inhibitors that deactivate the MâLby displacing the hydroxide nucleophile bridging the zinc ions.The experimental structures of the complexes are well repro-duced by MD trajectories based on a QM/MM approach.Particularly encouraging is the excellent characterization of themetal-ligand interactions in the active site, which providesfurther evidence in support of the validity of the semiempiricalSCC-DFTB in treating the metal centers in enzymes.

The most important anchoring interaction of the inhibitorswith the enzyme is probably the direct metal binding. For OPS,a covalent bond with Lys161 provides an additional anchoringpoint for the inhibitor. However, the succinic acid BYS wasfound to be stabilized by an elaborate hydrogen bond networkand the hydrophobic interaction with Trp28 via a quadrupolarforce. Hydrophobic interactions and hydrogen bonds contributetoo in the case of C4H. All these binding determinants arereasonably reproduced by the QM/MM simulations.

Acknowledgment. This work was supported by the NationalInstitutes of Health (Grant R03AI068672). Some of the calcula-tions were carried out at the National Centers for Supercom-puting Applications (NCSA).

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Figure 5. Comparison of CR B factors of the three enzyme-inhibitorcomplexes.

TABLE 5: Calculated B Factors for Two Key Active-SiteResidues

B factors (Å2)

residue atom 1JJE 1VGN 2DOO

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