doi:10.1016/j.jmb.2011.10.041 J. Mol. Biol. (2012) 415, 205–220

Contents lists available at www.sciencedirect.com

Journal of Molecular Biologyj ourna l homepage: ht tp : / /ees .e lsev ie r.com. jmb

The Evolution of Cefotaximase Activity in theTEM β-Lactamase

Manoj Kumar Singh and Brian N. Dominy⁎Department of Chemistry, Clemson University, Clemson, SC 29634, USA

Received 16 August 2011;received in revised form18 October 2011;accepted 25 October 2011Available online3 November 2011

Edited by D. Case

Keywords:TEM β-lactamase;catalytic activity;enzymatic evolution;MM-GBSA;molecular dynamics

*Corresponding author. E-mail addrdominy@clemson.edu.Abbreviations used: MIC, minimu

concentration; MD, molecular dynamData Bank; MM-GBSA, molecular mBorn surface area.

0022-2836/$ - see front matter © 2011 E

The development of a molecular-level understanding of drug resistancethrough β-lactamase is critical not only in designing newer-generationantibacterial agents but also in providing insight into the evolutionarymechanisms of enzymes in general. In the present study, we have evaluatedthe effect of four drug resistance mutations (A42G, E104K, G238S, andM182T) on the cefotaximase activity of the TEM-1 β-lactamase. Usingcomputational methods, including docking and molecular mechanicscalculations, we have been able to correctly identify the relative order ofcatalytic activities associated with these four single point mutants. Furtheranalyses suggest that the changes in catalytic efficiency for mutant enzymesare correlated to structural changes within the binding site. Based on theenergetic and structural analyses of the wild-type and mutant enzymes,structural rearrangement is suggested as a mechanism of evolution of drugresistance through TEM β-lactamase. The present study not only providesmolecular-level insight into the effect of four drug resistance mutations onthe structure and function of the TEM β-lactamase but also establishes afoundation for a future molecular-level analysis of complete evolutionarytrajectory for this class of enzymes.

© 2011 Elsevier Ltd. All rights reserved.

Introduction

β-Lactam antibiotics are the antibacterial agentsthat inhibit bacterial growth by blocking cell wallsynthesis.1 The first drug in this class was PenicillinG (benzylpenicillin), which was introduced intoclinical practice during the 1940s.2 However, soonafter Penicillin G was brought into regular practiceto treat bacterial infections, bacteria were found todevelop resistance against this drug by secreting anenzyme called β-lactamase.1–3 β-Lactamase is be-lieved to have evolved from Penicillin-bindingproteins,4–7 which are the natural targets of the β-lactam antibiotics. There is another view about the

ess:

m inhibitoryics; PDB, Protein

echanics/generalized

lsevier Ltd. All rights reserve

evolution of serine β-lactamase, which suggests thatthese are ancient enzymes originating over twobillion years ago.8 The β-lactamase catalyticallyhydrolyzes the β-lactam ring and therefore inacti-vates this class of antibiotics. Based on sequencesimilarity, β-lactamase has been subdivided intofour classes.4,9,10 Classes A, C, and D are the active-site serine enzymes, and class B requires a metal(typically Zn2+) for its catalytic activity. The class ATEM β-lactamase is one of the most commonlyfound plasmid-mediated β-lactamases withinGram-negative bacteria,11 and our further discus-sion will be focused on this TEM β-lactamase.The TEM β-lactamase uses the hydroxyl group of

its active-site serine residue (Ser70) as the keyfunctional group for catalyzing the hydrolysisreaction.12 Once the β-lactam antibiotic is accom-modated into the binding pocket of this enzyme,acylation, deacylation, and product release are thethree steps through which hydrolysis takes place.13

Several different mechanisms for this reaction havebeen proposed. According to one of the latest

d.

206 Evolution of Cefotaximase Activity

proposed mechanisms for the hydrolysis ofbenzylpenicillin,12 the key acylation reaction isbelieved to take place in two steps. In the firststep, indirect activation of nucleophile Ser70 takesplace by Glu166. The Glu166 abstracts a proton froma bridging water molecule, and the resultanthydroxyl ion initiates deprotonation of the Ser70side chain. This deprotonated Ser70 attacks thecarbonyl group of the β-lactam ring, thus forming atetrahedral intermediate.12 In the second step ofacylation where the acyl-enzyme complex is formedfrom the tetrahedral intermediate, the leavingthiazolidine nitrogen is protonated by Ser130,which is reprotonated by Glu166 via Lys73.12

These reactions result in the complete breakdownof the lactam bond. In a kinetic study of thehydrolysis reaction of the β-lactamase againstbenzylpenicillin, the rate constants for every stepof the hydrolysis reaction were experimentallydetermined.14 It was found that no single rate-determining step exists for the β-lactamase-cata-lyzed hydrolysis reaction of benzylpenicillin.14 Therate constants for all three steps of hydrolysis(acylation, deacylation, and product release) weredetermined to be approximately the same, and theoverall hydrolysis reaction was found to be diffu-sion controlled. It is therefore concluded that β-lactamase functions as a fully efficient enzyme.4,14

Several new classes of β-lactam antibiotics weredesigned with the aim of synthesizing an antibioticthat cannot be hydrolyzed in order to evade thecatalytic action of β-lactamase. Cefotaxime is one ofthese later-generation drugs (third-generation ceph-alosporin β-lactam antibiotic) that cannot be effi-ciently hydrolyzed by the TEM-1 β-lactamase.11

One of the strategies in designing these newer-generation drugs was to make their side groupsaround the β-lactam ring bulkier so that they cannotfit into the binding pocket of β-lactamase withoutcompromising its ability to bind the Penicillin-binding proteins. It is believed that the bulkiergroups around the β-lactam ring in cefotaximecompared to the benzylpenicillin (Fig. 1) preventits binding to the β-lactamase and therefore prevent

its hydrolysis. However, later, it was found that fivepoint mutations within the TEM-1 β-lactamase geneproduce a variant of β-lactamase that is capable ofhydrolyzing the cefotaxime and therefore causesdrug resistance.15,16 This consists of four missensemutations17 (A42G, E104K, M182T, and G238S) andone 5′ noncoding mutation18 (g4205a). Since thereactive β-lactam ring of cefotaxime is identical withthat of the benzylpenicillin (Fig. 1) and the hydro-lysis reaction catalyzed by β-lactamase is diffusioncontrolled, the cefotaxime binding process can beconsidered the rate-limiting step in the cefotaximaseactivity of β-lactamase, and therefore, it can bethought that, during the evolution of improvedactivity against cefotaxime, β-lactamase increases itsaffinity for the cefotaxime. Since the core structure ofthe β-lactam ring remains unchanged in cefotaximefrom benzylpenicillin, the activation energies for thereaction can be thought to be approximately thesame as in the case of benzylpenicillin. Thishypothesis of substrate binding being the rate-limiting step in the β-lactamase-catalyzed reactionhas already been experimentally verified withcephaloridine19 (another third-generation cephalo-sporin β-lactam antibiotic). In the experimentalstudy, the reaction of mutant β-lactamase wasstudied against benzylpenicillin and cephaloridine,and it was found that the acyl-enzyme structures donot account for the faster hydrolysis of thebenzylpenicillin.19 It was suggested that the relativeactivity toward benzylpenicillin and cephaloridinefor class A β-lactamase must be determined prior toacyl-enzyme formation,19 which is the substratebinding step.Development of drug resistance through the

increased catalytic activity of β-lactamase towardcefotaxime can also be seen as an evolutionary eventwithin a background of an external selectivepressure (i.e., the presence of a drug).2,20 Conse-quently, this process of developing drug resistancein bacteria can be utilized to address questionsrelated to biomolecular evolution. Considering thesefacts, the effect of the genetic background on thephenotypic consequence of a mutation (also termed

Fig. 1. The chemical structures ofbenzylpenicillin and cefotaxime.

207Evolution of Cefotaximase Activity

epistasis) has been experimentally addressed usingTEM β-lactamase and cefotaxime as a modelsystem.21 There are 5!=120 possible mutationaltrajectories (considering all five point mutations inthe gene and assuming that only single mutationstake place at each step) through which the sub-optimal allele (wild-type β-lactamase) can evolve toan optimal allele (five point mutated β-lactamase).In the case of sign epistasis, all mutational trajecto-ries would not be accessible with equalprobability.22 In the experimental study, all possiblemutational trajectories from the suboptimal tooptimal allele were constructed, and their probabil-ity of realization was assessed.21 It was found that102 of the 120 mutational paths were selectivelyinaccessible, and only 18 paths were found toincrease in resistance and fitness at each step,making them accessible through Darwinianselection.21 This experiment has provided concreteevidence of sign epistasis and demonstrated con-straints in biomolecular evolution.22

From the above discussion, it is clear that thedevelopment of a molecular-level understanding ofβ-lactamase evolution has significant implications inthe development of better antibiotics, along withproviding more general insight into enzyme evolu-tionary mechanisms. In the present study, we haveexamined the first stage of mutations (A42G, E104K,M182T, and G238S) in TEM-1 β-lactamase associat-ed with the evolutionary trajectory directed towardcefotaxime resistance.21 The noncoding mutation(g4205a) is not included in our present study as thisdoes not directly affect the chemical structure of theβ-lactamase. As discussed earlier, the substratebinding process is most likely the rate-limitingstep. Therefore, we have estimated the change inbinding affinity for cefotaxime upon these foursingle mutants relative to the wild-type affinity and

compared this to the corresponding experimentallydetermined catalytic activities. We have observedexcellent agreement between change in bindingaffinities of mutant enzymes and the correspondingexperimentally reported minimum inhibitoryconcentrations21 (MICs). The MIC and catalyticactivity have been shown to be linearly correlatedfor bacterial drug resistance.23 We have furtheranalyzed the structural changes upon mutationsand observed that the substantial increase incatalytic activity for the G238S and E104K mutantenzymes is correlated with significant restructuringat the active site, which is consistent with theprevious experimental evidence regarding theG238S mutation.24–26 The present study provides amolecular-level insight into the effect of the fourdrug resistance mutations on the cefotaximaseactivity of β-lactamase, which not only is helpfulin designing new classes of antibiotics but alsoprovides molecular-level insight into the first step ofthe evolutionary trajectory through which bacteriabecome drug resistant. The findings of the presentwork also provide a firm basis for future molecular-level studies of the complete evolutionary trajectoryassociated with drug resistance through TEM β-lactamase and other enzymes of this class.

Results

Docked conformation of cefotaxime

Cefotaxime was docked into the binding pocket ofthe wild-type β-lactamase. The structure with thehighest population, which also has one of the lowestenergies, was selected as the representative of theβ-lactamase-bound structure of cefotaxime (Fig. 2).

Fig. 2. Results of the automateddocking calculation. In this graph,each bar represents the energy andpopulation of a conformationalcluster.

208 Evolution of Cefotaximase Activity

This low-energy solution also has the reactive β-lactam ring of cefotaxime in the correct proximity tothe catalytic residue Ser70. In addition, this confor-mation also makes several contacts (with residuesSer70, Ser130, Ala237, Arg244, Asn132, Lys234, andSer235), which have been recognized as key contactsusing the structurally similar molecules benzylpe-nicillin and cephalosporin in previous studies.27,28 Amajority of these contacts were conserved during amolecular dynamics (MD) simulation. Further de-tails have been provided in Supporting Information.

Binding free-energy calculations

The effects of four mutations (A42G, E104K,M182T, and G238S) in TEM-1 β-lactamase on thebinding affinity for cefotaxime were examined. Themolecular mechanics/generalized Born surface area(MM-GBSA) binding affinities were estimated byanalyzing 1000 snapshots, taken during the last10 ns of the production-phase simulation. The MM-GBSA binding affinities to cefotaxime of the wildtype and four mutants are shown in Table 1. Out ofthe four mutants studied, the highest bindingaffinity for cefotaxime was observed for the G238Smutation of TEM-1 β-lactamase. The G238S muta-tion is also the variant that has been shown to exhibitthe maximum MIC (1.410 μg/ml) of cefotaxime.21

As mentioned earlier, the MIC and catalyticefficiency for bacterial resistance are linearlycorrelated.23 Therefore, the G238S mutation contrib-utes the most toward improving the catalyticefficiency of the β-lactamase by increasing itsbinding affinity toward cefotaxime. The E104Kmutation ranks second in increasing the bindingaffinity of the enzyme toward cefotaxime. The MICof cefotaxime for the E104K mutant enzyme wasdetermined to be 0.132 μg/ml,21 which also rankssecond after the G238S mutant enzyme. The M182Tmutation, which has been found to decrease theMICcompared to the TEM-1 β-lactamase, was estimatedto lower the binding affinity of the enzyme towardcefotaxime. The binding affinity of the A42G mutantenzyme, which has an MIC equal to wild type, wasfound to be very close to that of the wild-typestructure. Hence, the binding free-energy data

Table 1. Binding affinity data for TEM-1 β-lactamase and its

ΔGint ΔGCoul ΔGvdW ΔGsol

Wild type 0.39 (0.25) −30.85 (0.32) −32.34 (0.17) 30.40 (0.26) 5G238S 2.47 (0.24) −37.27 (0.31) −46.5 (0.15) 39.61 (0.25) 8E104K 1.59 (0.24) −81.78 (0.49) −39.84 (0.17) 83.82 (0.41) 7A42G 3.48 (0.25) −15.29 (0.31) −42.77 (0.16) 21.43 (0.24) 11M182T 0.55 (0.24) 12.53 (0.38) −41.55 (0.17) 1.93 (0.32) 20

Binding affinities are in kilocalories per mole; ΔGelec=ΔGCoul+ΔGpoin parentheses.

shown in table 1 suggest that cefotaximase activityof TEM-1 β-lactamase evolves by increasing itsbinding affinity (and typically specificity) for cefo-taxime, which is consistent with previous experi-mental studies and proposals.24–26

More insight into the binding mechanism can beobtained by examining the different thermodynam-ic components of the calculated MM-GBSA bindingaffinities. As shown in Table 1, the substantialfavorable change for cefotaxime binding affinity inthe case of the G238S mutant enzyme compared tothe wild-type enzyme is primarily driven by thevan der Waals energy term. This indicates the betteraccommodation of cefotaxime into the bindingpocket of the β-lactamase following the G238Smutation. The E10K mutant enzyme also increasesits binding affinity for cefotaxime compared to thewild type through the van der Waals componentand appears to follow a mechanism similar to thatof the G238S mutant enzyme for increasing itsactivity. In the case of the A42G and M182T mutantenzymes, the favorable van der Waals energy termis either compensated (for A42G) or overcome (forM182T) by unfavorable electrostatic and internalenergy terms.

Structural properties as observed duringMD simulations

Root-mean-square deviation

In order to gain insight into the effect ofmutations on the structural properties of cefotax-ime-bound TEM β-lactamase, we first monitoredthe root-mean-square deviation (RMSD). The aver-age structures of the cefotaxime-bound mutantenzymes from the production run (last 10 ns) werecompared to that of the wild-type average struc-ture. The average structures of the mutant enzymeswere superimposed on the average structure ofwild-type enzyme using the coordinates of themain-chain atoms [N, CA, and C atoms accordingto Protein Data Bank (PDB)29 nomenclature], andthe RMSDs of the overall structure and thestructural units and of the individual residueswere calculated. For convenience, the regionbetween residues 86 and 118 was named AS1

mutants with cefotaxime

ΔGelec ΔGbind ΔΔGvdW ΔΔGelec ΔΔGbind

MIC21

(μg/ml)

.09 (0.15) −32.41 (0.24) 0 0 0 0.088

.65 (0.15) −41.67 (0.24) −14.16 3.56 −9.26 1.410

.84 (0.19) −36.19 (0.27) −7.50 2.75 −3.78 0.132

.77 (0.16) −33.16 (0.24) −10.43 6.68 −0.75 0.088

.26 (0.14) −26.54 (0.23) −9.21 15.17 5.87 0.063

l-sol. The standard errors associated with the averages are given

Table 2. Summary of the RMSDs for different domains ofthe cefotaxime-bound enzymes

G238S E104K A42G M182T

Total 0.84 0.70 0.60 0.63Ω-loop (161–179) 1.27 0.90 0.97 1.08AS1 (86–118) 1.11 1.07 0.86 0.75AS2 (213–229) 1.38 1.01 0.93 1.06AS3 (267–271) 1.72 1.04 0.66 0.50AS 1.26 1.01 0.90 0.90

The RMSDs are given in angstroms; AS=Ω-loop+AS1+AS2+AS3.

209Evolution of Cefotaximase Activity

(active-site region 1). Similarly, the regions fromresidues 213 to 229 and from residues 267 to 271were named AS2 and AS3, respectively.Out of the four mutant structures, the G238S

structure was observed to exhibit the higheststructural deviation from the wild type. Theoverall structural deviation (RMSD=0.84 Å) aswell as the RMSD for the active-site region

(RMSD=1.26 Å) for G238S was found to behighest among the four mutants (Table 2). Thestructural deviation in different parts of theenzyme, as well as in the active-site region of theE104K mutant enzyme, was observed to be thesecond highest after G238S (Table 2). The Ω-loop,which is a relatively flexible part of the enzyme,shows the largest structural deviation upon muta-tion compared to other regions.In order to analyze the structural deviation in

more detail, we have calculated the RMSD forevery residue of the mutant enzyme relative to thewild type. As shown in Fig. 3, three distinctregions in the G238S mutant enzyme (AS1, AS2,and AS3) apart from Ω-loop show significantstructural deviation upon mutation. These fourregions of the enzyme contain most of the residuesthat define the binding pocket for cefotaxime. TheE104K mutant enzyme, which has the secondhighest binding affinity after the G238S mutant,demonstrates a significant structural RMSD in

Fig. 3. The RMSD of the residuesof the cefotaxime-bound mutantenzymes compared to the corre-sponding wild-type structure.

210 Evolution of Cefotaximase Activity

these four regions (Fig. 3), however, less incomparison to the G238S mutant. For the A42Gand M182T mutants, the structural change is lesssignificant compared to the G238S and E104Kmutant enzymes, other than in the Ω-loop, whichis a very flexible part of the enzyme.

Hydrogen bonds between lactamase andcefotaxime

Hydrogen bonds have a critical role to play instabilizing protein–ligand complexes and thereforecontribute significantly to developing affinity andspecificity for a substrate. The most significanthydrogen bonds between cefotaxime and the enzymevariant structures were examined by analyzing 1000snapshots taken during the 10 ns of the productionsimulations. The enzyme–substrate hydrogen bondswere characterized in terms of the distance betweenheavy atoms, as reported in Table 3.The distances given in Table 3 generally indicate

the restructuring of the ligand in the bindingpocket upon mutations. However, the changes inthe pattern of hydrogen bonding with cefotaximeupon single point mutations were not found to be amajor contributor to change in binding affinity(Table 1). Only two interactions involving thecarbonyl oxygen (O) of the ligand (with residuesAla237 and Ser70) are consistently present withinall mutant and wild-type structures. These in-teractions with the carbonyl oxygen of the ligandhelp to maintain the reactive center of thecefotaxime in the right orientation relative to thecatalytic residues of the enzyme. Since all parts ofthe ligand (β-lactam ring and two side chains) canbe seen to form hydrogen bonds, all three parts ofthe ligand are responsible for binding the substratewithin the active site and contribute toward thespecificity. The negatively charged carboxylategroup of cefotaxime makes a number of H-bondswith residues Ser130, Ser235, and Arg244 in thecarboxylate-group-accommodating pocket andtherefore appears to be a major contributor to the

Table 3. Summary of the average distances between heavy atobetween cefotaxime and enzymes

Hydrogen bond TEM-1 G238S

CEF-O····H-N-Ala237 3.22±0.39 2.92±0.17CEF-O6····H-Nη1-Arg244 3.38±0.47 2.88±0.20CEF-O2····H-Nδ-Asn132 6.16±1.14 3.17±0.31CEF-O5····H-Nζ-Lys234 5.96±0.68 4.31±0.27CEF-O5····H-Oγ-Ser130 6.58±0.60 2.67±0.15CEF-O5····H-Oγ-Ser235 2.65±0.14 3.13±0.31CEF-O6····H-Oγ-Ser235 3.94±0.47 2.78±0.17CEF-O····H-Oγ-Ser70 4.28±0.52 2.94±0.23CEF-N1-H····O_C-Ala237 6.14±0.40 3.41±0.28CEF-N4-H····O_C-Pro167 4.52±1.55 4.66±0.40

All the distances are given in angstroms.

binding affinity (see Discussion). All hydrogenbonding interactions (except CEF-N4-H····O_C-Pro167) reported in the current study have beenfound to be present during an MD simulation ofcephalothin (another cephalosporin β-lactam anti-biotic) bound to TEM-1 β-lactamase.28 The CEF-O5····H-N-Lys234 hydrogen bond is particular tothe E104K mutant and is not very significant inother structures. The residue Ala237, which isinvolved in the formation of the so called “oxya-nion hole,”28 makes two significant interactionswith the ligand. The first interaction is CEF-O····H-N-Ala237, which is very significant in all mutantsas well as in the wild-type structures. The secondinteraction involving Ala237 is CEF-N1H····O_C-Ala237, which is very significant in all mutantstructures but appears to be less significant in thewild-type structure.

Discussion

Mutations can impact the function of a proteinthrough either direct or indirect mechanism. Thedirect means can be among the most obvious andinvolve the gain or loss of important nonbondedinteractions (e.g., hydrogen bonds) with a ligand.Interestingly, none of the four mutant positionsexamined in this study are present within thebinding region of cefotaxime, as observed in thedocking calculations. G238 and E104 are near thecefotaxime binding pocket; however, neither theseresidues nor the mutant residues at thesepositions were found to make any significantcontact with docked structure of cefotaxime. Theresidues A42 and M182 are even further from thebinding pocket. It is therefore not obvious howmutations in any of these positions will affect thebinding affinity toward the substrate. At thesame time, there is no crystal structure availablefor these mutants in the cefotaxime-bound con-formation that could give insight into theirresistance mechanisms.

ms involved in important hydrogen bonding interactions

E104K A42G M182T

3.17±0.45 2.86±0.14 2.93±0.152.85±0.19 3.12±0.33 4.73±0.573.03±0.30 2.91±0.17 3.09±0.314.13±0.34 4.25±0.28 4.83±0.282.71±0.16 2.69±0.15 2.72±0.212.85±0.21 3.01±0.27 5.22±0.442.97±0.22 2.87±0.20 4.61±0.423.47±0.53 3.17±0.21 3.12±0.204.56±1.28 3.37±0.29 3.14±0.265.40±1.31 5.22±0.84 5.10±0.76

211Evolution of Cefotaximase Activity

Since wild-type β-lactamase cannot properlyaccommodate cefotaxime in its binding pocket,structural changes within the active site uponmutation have been proposed as a possible mech-anism for developing resistance.24–26 The G238Smutation causes a maximum increase in catalyticefficiency compared to the other three single pointmutations, and therefore, it has been the center ofattention. Considering the physiological implica-tions of the G238S mutation, an experimental studywas performed to correctly identify the role of Ser.24

This study investigated the impact of numeroussubstitutions at the G238 position on the catalyticactivity of the lactamase. Based on a systematicanalysis of the activity data, the hydroxyl group ofSer was not found to make any direct contributiontoward the binding affinity for cefotaxime throughhydrogen bonding, and therefore, restructuringwithin the active site was suggested as the possiblereason for the increase in the catalytic activity of β-

Fig. 4. The active-site region of the averaged structures of twild type; red, mutants. Ligand structures belong to mutant e

lactamase upon G238S mutation.24 However, theexperiments performed did not address the detailednature of the structural changes that take place as aresult of this important mutation critical for resis-tance to extended spectrum antibiotics.As shown in Figs. 3 and 4, active-site restructuring

is evident in the G238S mutant enzyme, which isconsistent with the observations made in priorexperimental studies.24–26 The most significantrestructuring was found to take place in the Ω-loop. As shown in Fig. 4, the Ω-loop in the G238Smutant enzyme opens up, providing more space forthe ligand to fit into the binding pocket. Significantstructural changes in other parts of the active site(AS1, AS2, and AS3) were also observed apart fromstructural changes in the Ω-loop (Figs. 3 and 4),which aid in the ability of the enzyme to bind tighterto cefotaxime. Also consistent with prior experimen-tal observations, no hydrogen bonding interactionwith the hydroxyl group of the Ser238 residue to any

he mutant enzymes superimposed over wild type. Green,nzymes.

Fig. 5. The superimposition of the average ligandstructures from the G238S mutant enzyme simulation(blue) and the M182T mutant enzyme simulation (green).The ligandbound in theG238Smutant enzyme's pocket canbe seen as adopting an extended conformation comparedto the ligand bound to the M182T mutant enzyme.

212 Evolution of Cefotaximase Activity

part of cefotaxime was observed during the MDsimulation. Therefore, the entire increase in bindingaffinity is coming from the active-site restructuringcaused by the G238S mutation, which is in agree-ment with previous experimental observations.24

Structural changes in the binding pocket of theE104K mutant enzyme are also very significant. Inthe case of the E104K mutation, the most significantstructural changes were observed in the AS1, AS2,and AS3 regions (Figs. 3 and 4), along withsignificant structural changes in the Ω-loop. Asmentioned earlier, these structural changes in theactive-site region appear to help in increasing thebinding affinity of the E104Kmutant enzyme towardcefotaxime. Structural changes in the active-siteregions are less significant for the A42G and M182Tmutants, which do not demonstrate an increase incatalytic efficiency (Figs. 3 and 4). The correlationbetween structural changeswithin the active site andincreases in catalytic activity for cefotaxime indicatethat the restructuring of active site is a generalmechanism for increasing the resistance againstcefotaxime within TEM β-lactamase.As noted above, while the conformational

changes observed upon mutation of A42 andM182 are less dramatic than changes occurringupon the mutation of G238 or E104, some confor-mational changes did occur. The, albeit weaker,sensitivity of the active site toward these moredistant positions might be indicated through ananalysis of correlated motions obtained using theANM (Anisotropic Network Model) web server.30

An analysis of the superposition of the first 10dynamic modes associated with the elastic networkbased on the 1BTL crystal structure demonstratescorrelation between the motion of A42 and M182and the motion of loops within the active site.Specifically, the dynamics of A42 appear positivelycorrelated to the dynamics of the loop labeled asAS3 and negatively correlated to the dynamics ofthe Ω-loop. The dynamics of M182 are positivelycorrelated to the movements of the Ω-loop.The relative cefotaximase activities of β-lactamase

mutants are communicated not only through theactive-site conformations but also through theaccessible conformations of the cefotaxime substrate.The C2 atom of the dihydrothiazine ring of cefotax-ime can flip between up and down conformationswith respect to the plane formed by the C, S, and C3atoms of the ring, and therefore, the cefotaxime canadopt two distinct conformations based on theorientation of the C2 atom. These conformations ofcefotaxime are termed as the C2-up and C2-downconformations. The majority of crystal structures ofthird-generation cephalosporin β-lactam antibioticsbound to β-lactamase have been reported to exhibitthe C2-down conformation.28 In a previous theoret-ical study, it has been suggested that the C2-downconformation in cephalosporin is energetically

favored.28 It has also been suggested that thepreference between the C2-up and C2-down confor-mations is mainly governed by the conformationalpreferences of the antibiotic side groups.28,31

In the present study, we have further rationalizedthe structural implications of the C2-down confor-mation. We have observed that, in G238S mutantenzyme, which has the highest activity againstcefotaxime, the cefotaxime adopts the C2-downconformation 98.6% of the time during a 10-nsproduction run. For the M182T mutant enzyme,which has the lowest activity among the mutantenzymes, the C2-up conformation was populatedduring 100% of the production simulation. Asshown in Fig. 5, in the case of the C2-downconformation, the two side chains of cefotaximeadopt an extended conformation relative to the C2-up conformation. This can be considered indicativeof the volume difference between the active sites ofthese two mutant enzymes. The G238S mutantenzyme is providing an extended binding pocketcompared to the M182T mutated enzyme, helping tobetter accommodate the cefotaxime. The popula-tions of the C2-down conformation for E104K,A42G, and wild-type structures were found to be44.3%, 6.2%, and 17.4%, respectively.As mentioned earlier regarding MM-GBSA, apart

from being computationally efficient and reliable inpredicting relative binding affinities, the MM-GBSAbinding affinity can be efficiently decomposed intoper-residue contributions, which can provide fur-ther insight into binding mechanism. In the presentstudy, we have decomposed the total bindingaffinity as well as the van derWaals and electrostaticcomponents of the binding affinity into per-residuecontributions.For the wild-type structure, the major contribution

to the binding affinity comes from the residue R244,which accommodates the carboxylic group attachedto the dihydrothiazine ring of cefotaxime. R244contributes toward the binding affinity primarilythrough the electrostatic component (Fig. 6). S235 is

Fig. 6. The per-residue decompo-sition of theMM-GBSA binding freeenergy of the wild-type enzyme forcefotaxime. The first graph repre-sents the per-residue decompositionof the total binding free energy. Thesecond graph represents the per-residue decomposition of the elec-trostatic component (Columbic andpolar solvation) of binding freeenergy, and the third graph repre-sents the per-residue decompositionof the van der Waals component ofthe binding free energy. The per-residue contribution from the li-gand residue is not represented inthe above graphs.

213Evolution of Cefotaximase Activity

the other residue that significantly contributes(∼2 kcal/mol) toward the binding affinity throughthe electrostatic component. The residues S70, Y105,S130, N132, V216, G326, and A237 contributethrough van der Waals interactions (Fig. 6).We further analyzed the differential binding

affinities [ΔΔG(mutant)=ΔG(mutant)−ΔG(wild-type)] of mutant enzymes for cefotaxime comparedto the wild-type enzyme. The differential increasein binding affinity of the G238S mutant enzymemainly originates from the residues Y105, N170,K234, A237 and S238 (Fig. 7). Of these residues,Y105, N170, A237 and S238 contribute toward thedifferential stabilization through van der Waalsinteractions (Fig. 8). The residue K234 increases thedifferential binding affinity through the electrostat-ic term (Fig. S1 in Supporting Information). In thecase of the E104K mutant enzyme, residues K104,Y105, N170, and A237 contribute toward thedifferential stabilization through van der Waalsinteractions (Fig. 8). In the E104K mutant enzyme,K234 contributes significantly through electrostaticinteractions (Fig. S1 in Supporting Information).

The R244 position in E104K mutant is lessdifferentially destabilizing (3.64 kcal/mol) com-pared to the G238S mutant enzyme (4.08 kcal/mol). The R244 position in the A42G mutantenzyme contributes more toward differential de-stabilization (5.11 kcal/mol) compared to theE104K and G238S mutant enzymes, but lesscompared to the M182T mutant enzyme where itis highest with a value of 7.4 kcal/mol (Fig. 7).R244 also contributes to differential stabilizationthrough van der Waals interactions in all themutant enzymes (Fig. 8). In the case of the A42Gmutant enzyme, the contributions to differentialbinding affinity though van der Waals interactionscome from residues P167, N170, A237, and R244(Fig. 8). The residues N132 and K234 contributethrough electrostatic interactions in the A42Gmutant enzyme (Fig. S1 in Supporting Informa-tion). The residues of the M182T mutant enzymemake several differential stabilizing and destabiliz-ing contributions to binding affinity. The differen-tially stabilizing contributions from M69, S70, N132,P167, N170, K234, and A237 are overwhelmed by

Fig. 7. Per-residue decomposi-tion of the differential binding freeenergy of the mutant enzymes forcefotaxime, relative to the wild-typebinding free energy. The per-resi-due contribution of the ligandresidue is not represented in theabove graphs.

214 Evolution of Cefotaximase Activity

differential destabilizing contributions from S130,V216, S235, and R244.In order to catalytically hydrolyze cefotaxime, the

β-lactamase evolves by expanding its preexistingability to catalytically hydrolyze benzylpenicillin.Since the mutants G238S and E104K retain someactivity against benzylpenicillin while gaining ac-tivity against cefotaxime,32 the first step of evolutioncan be considered a process of increasing itsfunctional promiscuity. According to one proposedmechanism underlying enhanced substrate promis-cuity during biomolecular evolution,33,34 the proteinbecomes more flexible after mutating and can shiftbetween alternate conformations without any sig-nificant free-energy change. On a potential energydiagram (Fig. 9), we can think of two potentialenergy wells each associated with alternate struc-tures. Well A corresponds to the native conforma-tion, and B corresponds to an alternate conformationthat is required for a new function. Based on anevolutionary mechanism involving enhanced

flexibility,33,34 both conformations A and B becomealmost equal in energy with a very low energybarrier between them (case I in Fig. 9), and therefore,there is no significant energetic penalty associatedwith the conformational change. However, usually,only one type of β-lactam antibiotic is used in thetreatment of a bacterial infection, and therefore,bacteria typically gain no advantage in evolving forpromiscuity. Therefore, the observed promiscuitymay just be a side effect. In this case, there is apossibility that conformation B becomes more stablerelative to conformation A, as suggested in previousstudies.33–36 This can be represented as cases II andIII in Fig. 9 (G23S, E104K, and M182T mutationshave been shown to increase the fold stability of β-lactamase37,38). This will result in a pre-adoptedconformation of the enzyme that can preferentiallybind to the new ligand.In order to address these possibilities, we have

analyzed the structural properties of the unboundwild-type and mutant enzymes during MD

Fig. 8. Per-residue decomposi-tion of the differential van derWaals component of binding freeenergy of the mutant enzymes forcefotaxime, compared to the wild-type affinity. The per-residue con-tribution of the ligand residue is notrepresented in the above graphs.

215Evolution of Cefotaximase Activity

simulations. More details about the analyses ofunbound enzymes can be found in SupportingInformation. In brief, we did not observe anyconsiderable change in flexibility (as quantified bythe root-mean-squared fluctuation of α carbonatoms) within the binding region of the unboundmutant enzymes. However, in the G238S mutant,we have observed a considerable change in thestructure of the active-site region during the courseof the production simulation. Similarly for E104K,

Fig. 9. Coarse-grained depiction of a potential energy profevolution through a mechanism involving increased flexibilityinvolving the differential stabilization of an alternate conform

we observed a significant structural change in AS1region of the active site. Since the timescale overwhich the conformational transition between statesA and B takes place is longer than a few nanosec-onds, our observations based on a nanosecondtimescale MD simulations are clearly suggestiverather than conclusive evidence regarding themechanism of evolution-driven structural change.However, the suggestion is that mutations cause achange in the equilibrium conformation of the

ile of two conformations of an enzyme. Case I depicts the. Cases II and III depict the evolution through a mechanismation (B).

216 Evolution of Cefotaximase Activity

unbound state that is more accommodating tocefotaxime binding. In the case of ligand-boundstructures, these conformational changes are accel-erated and more easily observable in the MDsimulations due to the presence of the ligand inthe binding pocket of enzyme. Whether the evolu-tionary mechanism toward enhanced substratepromiscuity involves enhanced flexibility or a shiftin the equilibrium conformation of the unboundenzyme, it is suggested that the enhanced affinitytoward cefotaxime is accomplished by minimizingthe protein reorganization energy upon binding.

Conclusions

In the present work, we have been able tosuccessfully distinguish the beneficial mutations(G238S and E104K) from non-beneficial mutations(A42G and M182T) during the first step of β-lactamase evolution toward cefotaximase activity.We have further detailed the structural rearrange-ment that occurs within the active site of the enzymeupon mutation, driving the acquisition of drugresistance. Binding free-energy decomposition de-scribes these structural changes in terms of localinfluences on cefotaxime binding affinity leading toimproved cefotaximase activity. The mechanismdescribed here and linked to single amino acidmutants of the TEM-1 β-lactamase may also providesignificant insight into the effect of combinations ofthese mutations. The energetic calculations incombination with the structural analyses presentedin the current study can be helpful in the develop-ment of new-generation antibiotics.

Methods

To assess the effect of four mutations (G238S, E104K,A42G, and M182T) on the binding affinity of TEM β-lactamase for cefotaxime, we simulated modeled mutantenzymes as well as the wild-type enzyme bound tocefotaxime. As there is no crystal structure at present forcefotaxime bound to the TEM β-lactamase, cefotaximewas first docked into the binding pocket of the wild-typeenzyme structure. In the next step of our study, structuresof mutant enzymes bound to cefotaxime were generatedby modeling the mutations in the wild-type complexstructure. MD simulations and MM-PBSA free-energycalculations were performed in subsequent steps. Thedetails of the methodology used in the present study aregiven below.

Docking of cefotaxime into the binding pocket

Cefotaxime was docked into the crystal structure of theTEM-1 (wild type) β-lactamase (PDB ID: 1BTL39) usingAutoDock4.40 The amino acid sequence of this crystalstructure is consistent with the definition of the wild-type

enzyme used in a corresponding experimental study.21

This structure has also been used in several previoustheoretical studies involving benzylpenicillin andcephalosporin.27,28 Further, Autodock has been success-fully used to determine the enzyme-bound structures ofbenzylpenicillin and cephalosporin, which are in closestructural resemblance with cefotaxime.27,28 The C2-down conformer of dihydrothiazine ring of cefotaximewas used for docking as this conformer has beensuggested to be more stable and abundant in thestructurally similar molecule cephalosporin in the en-zyme-bound state.28 This conformation was held fixedduring the docking.Prior to the docking calculations, the active-site region

was defined as in previous studies.27,28 In order to coverthe entire region of the binding site, we used a sufficientlylarge grid around the catalytic site of the enzyme withinAutoDock4. The conformational searches were performedusing a genetic algorithm, and a maximum number ofenergy evaluations per genetic algorithm run was set to25,000,000. The rest of the parameters was set to thedefault values within AutoDock4.

Ligand parameterization

The parameterization of cefotaxime was performed onits docked conformation (without the receptor). Thecefotaxime was assigned force field parameters from thegeneral Amber force field41 for small molecules, using theANTECHAMBER package.42 The partial atomic chargeson the cefotaxime were derived using R.E.D. tools.43 Thecefotaxime was first minimized in Gaussian0344 using theHF/6-31G⁎ basis set. In the next step, partial atomiccharges were derived using a two-stage restrictedelectrostatic potential45 fitting protocol available withinthe R.E.D. tools.

Modeling the mutations

The mutations in the TEM-1 β-lactamase enzyme weregenerated using the tools within MODELLER, as devel-oped by Feyfant et al.46 This method uses a combination ofa knowledge-based potential and a simulated annealingprotocol to build side-chain conformations of mutantresidues. The details of this protocol can be found in theoriginal publication.46 Since none of the mutant positionswere found to make any significant contact with cefotax-ime in the wild-type modeled complex, the presence of theligand in the binding pocket was not a requirement for theaccurate modeling of side-chain conformations of mutantamino acids. Therefore, the mutations were built withinthe original crystal structure of TEM-1 β-lactamase (PDBID: 1BTL).

System preparation and the MD simulation protocol

In order to accurately model the Michaelis complex, weplaced a water molecule between residues Ser70 andGlu166 of the enzyme models based on structuralinformation from previous studies.12,28 This water mole-cule has a key role to play during catalysis;12 however, thiswater has not been shown to make any significant

217Evolution of Cefotaximase Activity

contribution toward ligand specificity.27,28 All enzyme–ligand complexes were solvated in an octahedral simula-tion box of TIP3P water molecules, using tleap withinAmber 10.47,48 The Amber ff99SB49 force field was used todescribe the protein part of the enzyme–cefotaximecomplex. The minimum distance between any of theatoms of the solvated complex and the box boundary wasmaintained to at least 10 Å. The systems (all enzyme–ligand complexes as well as unbound enzymes) wereneutralized by the addition of potassium ions.During all energy minimization and MD simulations,

the electrostatic interactions between atoms were treatedusing a particle mesh Ewald method,50 with a B-splineorder of 4 and direct space cutoff of 10 Å. The Lennard-Jones interactions were computed within a cutoff of 10 Å.All the bonds involving the hydrogen were constrainedusing SHAKE51 in Amber 10.In the next step, minimizations of the systems were

carried out using the SANDER module of the Amber 10package. In the first part of the minimization, the soluteatoms were held harmonically restrained (with a veryhigh force constant of 500 kcal/mol/Å2), and the systemswere minimized for 1000 steps using a steepest descentalgorithm and 1000 steps using a conjugate gradientalgorithm. In the next part of the minimization, the forceconstant for the positional restraint on the solute atomswas reduced to 50 kcal/mol/Å2, and the systems werefurther minimized for 1000 steps using the steepestdescent algorithm.Once the systems were relaxed, the gradual heating of

the systems was performed within the SANDER module.The systems were gradually heated from 0 K to 300 Kduring 100 ps of simulation, with a 1-fs time step forintegration. The solute atoms were restrained with a forceconstant of 5 kcal/mol/Å2, in order to avoid anysignificant structural deformation during the heatingprocess. After heating, the density equilibration simula-tion was performed. The MD simulations of the systemswere performed in a constant pressure and temperature(NPT) ensemble for 500 ps, with a pressure relaxation timeof 1.0 ps and 2-fs time step for integration. The soluteatoms were restrained with a relatively weak forceconstant of 2 kcal/mol/Å2 during the NPT simulation.The temperature was maintained to an average value of300 K using Langevin dynamics, with collision frequencyof 2 ps− 1. After achieving a density equilibrationcorresponding to 1 atm pressure, all the restraints on thesystems were removed, and further simulations wereperformed in a constant volume ensemble (NVT) withinthe PMEMDmodule of Amber 10, using a 2-fs time step ofintegration. A total of 30 ns of equilibration and 10 ns ofproduction simulations were performed for each free-energy estimation and structural analysis.

MM-GBSA binding affinity calculation

The binding affinities of cefotaxime to the wild typeas well as mutant enzymes were estimated using anMM-GBSA protocol.52–54 The MM-GBSA protocol notonly is computationally efficient in comparison to thetraditionally used MM-PBSA and other free-energyestimation techniques but also has been shown to bein excellent qualitative agreement with MM-PBSA andexperiment.55

According to the MM-GBSA protocol, the free energy ofbinding can be estimated using the following formulation.

GMM−GBSA¼GMMþGsol − TS

DGbind¼GMM−GBSAcomplex − GMM−GBSA

receptor − GMM−GBSAligand

DGbind¼DGMMþDGsol − TDS

Where the ΔGbind is the binding free energy, ΔGMM isthe molecular mechanics free-energy contribution to thebinding free energy (which is contribution from the gas-phase energies of isolated molecules), ΔGsol is thesolvation contribution to the binding free energy, and−TΔS is the contribution from the conformationalentropy change. In previous studies, it has beensuggested that the conformational entropy changeupon single point mutation is negligible and can safelybe ignored,56,57 and therefore, the conformational entro-py change upon mutation has not been included in thepresent study. The Ω-loop (residues 161–179) of theligand-free β-lactamase exhibits long timescalemotions,58 which cannot be properly sampled on thenanosecond timescale simulation. Therefore, in order toavoid the error caused by the insufficient sampling ofthe Ω-loop, we estimated the MM-GBSA energies ofcomplex and receptor from same trajectory. In order toaccount for the reorganization energy of cefotaximeupon binding, a trajectory from separate simulation wasused for the ligand MM-GBSA free-energy calculation.In a previous study, it has been shown that includingthe ligand reorganization energy improves the bindingaffinity prediction.59

The molecular mechanics contribution to the bindingaffinity (ΔGMM) can be written as the contribution fromColumbic (ΔGelec) interactions, van der Waals (ΔGvdW)interactions, and internal energy change (ΔGinte). Theinternal energy term here includes bond, angle, anddihedral energies of a molecule.

DGMM¼DGelecþDGvdWþDGinte

The Columbic, van der Waals, and internal energycontributions to the binding affinity were estimatedusing exactly the same parameters that have been usedfor energy minimization and MD simulations. Whilecalculating the interaction energies, we used no cutofffor nonbonded interactions.The solvation free-energy contribution to the binding

affinity can be written as sum of polar (ΔGsolpol) and

nonpolar (ΔGsolnon-pol) contributions.

DGsolv¼DGsolþDGnon�polsol

The polar contribution to the solvation free energy wasestimated using generalized Born method in Amber 10.The nonpolar contribution to the solvation free energywas calculated using solvent-accessible surface areamethod as

DGsol¼g × SASA

The value of 0.0072 kcal/mol/Å2 was used for surfacetension coefficient γ. The solvent-accessible surface area

218 Evolution of Cefotaximase Activity

was determined with the MOLSURF method, as imple-mented in Amber 10.

Per-residue decomposition

The free energy estimated using an MM-GBSA methodcan be efficiently decomposed into its per-residue con-tributors, which can provide further insight into a bindingmechanism. The per-residue decomposition of the MM-GBSA binding affinity has been successfully used inaddressing several questions, including the prediction ofsome drug-resistant mutations in human immunodefi-ciency virus type 1 protease.60

Thedetails of the per-residuedecomposition ofMM-GBSAbinding free energy in AMBER can be found elsewhere.61 Inbrief, the contribution of jth residue to the binding free energyfrom ith snapshot of species x can be written as:

Gxði; jÞ = Gxgasði; jÞ + Gx

solvationði; jÞ −TSxði; jÞThe contribution of residue j electrostatic (Columbic+

polar solvation) free energy can be obtained by:

Gxelec i; jð Þ = −

12

Xiaj

Xk

1 −exp −kð Þ

ew

� �qkql

fGBkl rklð Þ

+12

Xiaj

Xk p l

qkqlrkl

where the first term designates the electrostatic contribu-tion to the solvation free energy calculated using general-ized Born. The second term stands for the Columbicinteraction energy. ɛw is the dielectric constant, qk and ql arethe partial charges on the atoms k and l, and rkl is thedistance between respective atoms. The internal energies(bond, angle, and dihedral energies) were decomposedbased on atomic contributions from the residues.

Acknowledgement

This work was supported by an NSF CAREERaward (MCB 0953783).

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2011.10.041

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