Structural Basis of APH(3')-IIIa-Mediated Resistance to N1-Substituted Aminoglycoside Antibiotics

Butirosin is unique among the naturally occurring aminoglycosides,having a substituted amino group at position 1 (N1) of the 2-deoxystreptaminering with an (S)-4-amino-2-hydroxybutyrate (AHB) group. Whilebacterial resistance to aminoglycosides can be ascribed chieflyto drug inactivation by plasmid-encoded aminoglycoside-modifyingenzymes, the presence of an AHB group protects the aminoglycosidefrom binding to many resistance enzymes, and hence, the antibioticretains its bactericidal properties. Consequently, several semisyntheticN1-substituted aminoglycosides, such as amikacin, isepamicin,and netilmicin, were developed. Unfortunately, butirosin, amikacin,and isepamicin are not resistant to inactivation by 3'-aminoglycosideO-phosphotransferase type IIIa [APH(3')-IIIa]. We report herethe crystal structure of APH(3')-IIIa in complex with an ATPanalog, AMPPNP [adenosine 5'-(β, ${gamma}$ -imido)triphosphate], andbutirosin A to 2.4-Å resolution. The structure shows thatbutirosin A binds to the enzyme in a manner analogous to other4,5-disubstituted aminoglycosides, and the flexible antibiotic-bindingloop is key to the accommodation of structurally diverse substrates.Based on the crystal structure, we have also constructed a modelof APH(3')-IIIa in complex with amikacin, a commonly used semisyntheticN1-substituted 4,6-disubstituted aminoglycoside. Together, theseresults suggest a strategy to further derivatize the AHB groupin order to generate new aminoglycoside derivatives that canelude inactivation by resistance enzymes while maintaining theirability to bind to the ribosomal A site.

INTRODUCTION

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INTRODUCTION

Aminoglycosides encompass a vast group of molecules with importantantibiotic activities. They are often prescribed for the treatmentof serious nosocomial infections and, in some cases, protozoaninfections (6). Moreover, their potential as antiviral agentshas also been under intense investigation in recent years (33,47, 50). The high efficacy of aminoglycosides as antibacterialagents is in part due to their bactericidal capability. Mostaminoglycosides kill the bacteria by targeting the A site ofthe 16S rRNA (40). Upon binding of the drug, key nucleotidesin the decoding region of the ribosome undergo conformationalchanges that promote the interactions between mRNA and near-cognateor noncognate tRNA, leading to errors in protein translation(45). Unfortunately, the extensive use of aminoglycosides hasundermined their effectiveness, due to the emergence of resistancein pathogens. Resistance to aminoglycosides can predominantlybe attributed to covalent modification of the drug catalyzedby plasmid-encoded aminoglycoside-modifying enzymes, especiallythe O-phosphotransferases, which generally give high levelsof resistance (16). Covalent modification diminishes the antibiotics'ability to bind to their targets, and therefore, they can nolonger exert their bactericidal effects (34).

Butirosins are unique among the naturally occurring aminoglycosidesdue to the (S)-4-amino-2-hydroxybutyrate (AHB) side chain moietysubstituted at the amino group at position 1 (N1) of the 2-deoxystreptaminering. While most aminoglycosides routinely lose their potencydue to inactivation by an aminoglycoside-modifying enzyme, butirosinis able to elude many inactivating enzymes, thus retaining itsbactericidal capabilities (51). This observation prompted thedevelopment of semisynthetic N1-substituted aminoglycoside antibioticssuch as amikacin and arbekacin (kanamycin A and dibekacin derivatedat N1 by AHB, respectively) (23, 26, 30, 31), isepamicin (gentamicinB substituted with 4-amino-2-hydroxypropionyl at N1) (41), andnetilmicin (sisomicin with an ethyl group introduced at N1)(54). These compounds have been shown to be clinically usefulagainst some aminoglycoside-resistant strains, such as methicillin-resistantStaphylococcus aureus (29). It is thought that the AHB and otheracyl side chains at the 1-amino position hinder binding to theaminoglycoside-modifying enzymes without affecting binding tothe ribosome A site. Recently, the capability of amikacin bindingto the ribosome was illustrated by Kondo et al. in the crystalstructure of an RNA fragment containing the A site in complexwith amikacin (28).

Nonetheless, many of these N1-substituted aminoglycosides arenot immune to inactivation by all aminoglycoside kinases. Forexample, butirosin, amikacin, and isepamicin can be inactivatedby 3'-aminoglycoside O-phosphotransferase type IIIa [APH(3')-IIIa]via the addition of a phosphate group. APH(3')-IIIa is foundin many gram-positive bacteria, such as enterococci and staphylococci,and is perhaps the most-studied aminoglycoside resistance factor,as it has an unusually broad substrate profile (37).

Considerable knowledge of the accommodation of the structurallydiverse substrates by APH(3')-IIIa has been gained in recentyears. Crystal structures of APH(3')-IIIa in the apo, nucleotide-bound,and ternary complexes, with both the nucleotide and the aminoglycosideantibiotic, have been determined (9, 18, 24). The ternary complexesconfirmed the importance of electrostatic interactions in substratebinding and revealed that discrete pockets and a flexible bindingloop are used to accommodate aminoglycosides of different shapesand sizes (18). To date, the conformations of several aminoglycosides,including those that are substituted at N1 such as amikacin,isepamicin, and butirosin, bound to APH(3')-IIIa, have beenstudied by nuclear magnetic resonance (NMR) spectroscopy (12-14,48). These studies suggest that different aminoglycosides mayassume markedly different conformations and that there is littlesimilarity between the binding properties of 4,5- and 4,6-disubstitutedaminoglycosides. Moreover, a comparison of the aminoglycosideconformations observed in the ternary crystal structures ofAPH(3')-IIIa and those obtained from NMR experiments shows limitedresemblance. Most importantly, none of the NMR studies haveindicated how the substitution at N1 is accommodated by thekinase. Here, we report the crystal structure of APH(3')-IIIain complex with AMPPNP [adenosine 5'-(β, ${gamma}$ -imido)triphosphate;a nonhydrolyzable ATP analog] and butirosin A, as well as acomputationally deduced model for APH(3')-IIIa bound with amikacin,a prototypical semisynthetic N1-substituted aminoglycoside.These results improve our understanding of the effectivenessof APH(3')-IIIa as a resistant enzyme and provide informationfor the development of new derivatives.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Crystallization and data collection. APH(3')-IIIa was obtained based on the procedure described byMcKay et al. (37). Butirosin A-bound APH(3')-IIIa was crystallizedfrom a solution containing 15 mg/ml of protein, 2.5 mM of AMPPNP(Sigma-Aldrich, St. Louis, MO), 1.5 mM of butirosin A (Sigma-Aldrich,St. Louis, MO), and 2 mM MgCl₂ using the hanging-drop technique.After incubation of 1 week at 22°C, small, needle-shapedcrystals that were approximately 0.25 mm long appeared underone condition, consisting of 40% (vol/vol) polyethylene glycol600, 0.1 M sodium acetate (pH 4.5), and 0.2 M magnesium chloride.Additive screens I to III (Hampton Research) were used to enhancecrystal quality. Upon the inclusion of one of the additives,3% (wt/vol) D-(+)-sucrose, the size and shape of the crystalswere considerably improved. The resultant crystals were eggshaped and measured approximately 0.40 mm in length and 0.25mm at the widest part in the middle. These crystals belongedto space group P4₂2₁2, with unit cell dimensions of a = b =80.1 Å and c = 110 Å, and contained one APH(3')-IIIamolecule in each asymmetric unit. Resolution data (2.4 Å)were collected from a single crystal under cryogenic conditionsat beamline X8C of the National Synchrotron Light Source, equippedwith an ADSC Quantum charge-coupled-device detector ( ${lambda}$ = 1.072Å). Intensities were integrated and scaled using the HKLprogram suite (44), giving statistics summarized in Table 1.

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TABLE 1. Diffraction data and refinement statistics

Structure determination and refinement. The structure of APH(3')-IIIa with AMPPNP and butirosin A wassolved by molecular replacement using AMoRe (42), as implementedin the CCP4 suite of programs (11). The structure of APH(3')-IIIa-ADP-kanamycin(18), excluding the ligands and solvent molecules, was usedas the search model. After several cycles of refinement usingthe CNS software (8), AMPPNP, Mg²⁺, and butirosin A moleculeswere placed in the active site based on difference electrondensity maps (2F_o – F_c and F_o – F_c). Ideal stereochemistryapplied to butirosin A during subsequent refinement was basedon that of neomycin B and values from the energy-minimized conformationobtained from the molecular mechanics program MM2 (1, 10), implementedin Chem3D (CambridgeSoft). Examination of initial electron densitymaps showed that the aminoglycoside-binding loop (residues 150to 165) and the hinge region (residues 101 to 106) requiredremodeling. Based on difference electron density maps as wellas simulated annealing omit maps, these sections were rebuiltusing the program O (25). Successive cycles of refinement werealternated with manual intervention, and the addition of solventmolecules was continued until no significant improvement inthe model statistics was observed. Final refinement statisticsare given in Table 1.

Modeling of APH(3')-IIIa ternary complex with amikacin. The initial conformation of amikacin was taken from the crystalstructure of the antibiotic bound to an oligonucleotide containingthe bacterial ribosome A site (Protein Data Bank ID: 2G5Q) (28).Amikacin was then modeled into the aminoglycoside-binding pocketof APH(3')-IIIa, according to the butirosin A ternary complexreported here, by superposing the 2-deoxystreptamine and theprime rings of the aminoglycosides. AMPPNP and the magnesiumions from the butirosin A ternary complex were included in theamikacin model during the simulation.

Molecular dynamics simulations were performed with the GROMACS(version 3.3.1) suite of programs (5, 32), using the GROMACS87 force field. The coordinates of AMPPNP and amikacin weresubmitted to the Dundee PRODRG2 server (52) to generate GROMACStopology files and to add polar hydrogens. The topology fileof amikacin was adjusted in order to institute restraints onthe conformation of the ring structures of the aminoglycoside.A truncated octahedron periodic water box was made and was solvatedusing the simple point charge water model. Twenty-one sodiumcounterions were added to neutralize the overall charge. Afterenergy minimization using the steepest descent method, the systemwas subjected to a 20-ps position-restrained dynamics simulation.The protein atom positions were restrained, as prescribed bythe linear constraint algorithm (22), while the solvent andligands were allowed to relax about the protein. Particle-meshEwald electrostatics (15, 17), in combination with the temperatureand pressure coupling methods of Berendsen et al. (4), wereused. Next, a 100-ps molecular dynamics simulation was carriedout under the same conditions, except the restraints on theatomic positions were removed. An average structure was calculatedand then further refined using a steepest descent and conjugategradient energy minimization. The resultant coordinates canbe found in the supplemental material.

Protein structure accession number. Coordinates have been deposited in the Protein Data Bank underaccession number 3H8P.

RESULTS AND DISCUSSION

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RESULTS AND DISCUSSION

Overall structure of butirosin A-bound APH(3')-IIIa. To date, seven crystal structures of APH(3')-IIIa have beendetermined. They include the AMPPNP and butirosin A-bound structurereported here, the apo form (9), the ADP- and AMPPNP-bound structures(9, 24), the ternary complexes with ADP and kanamycin A or neomycinB (18), and the inactive form of the enzyme inhibited by anankyrin repeat protein (27). These structures confirm the overallrigidity of APH(3')-IIIa, as no gross domain movements can bedetected upon the binding of natural ligands in the active site.However, four segments have been consistently observed to displaydiffering conformations in the various APH(3')-IIIa structures.These segments are residues 21 to 26, 100 to 112, 147 to 170,and 226 to 238. A detailed description has been reported previously(9, 18). In brief, segments encompassing residues 100 to 112and 226 to 238 are inherently flexible, whereas the conformationof the other two segments is dependent on the nature of thebound ligand. Residues 21 to 26 are associated with nucleotidebinding and residues 147 to 170 are associated with aminoglycosidebinding.

Nucleotide binding. Unlike the kanamycin A and neomycin B structures, which havea bound nucleotide in the form of ADP, the butirosin A ternarycomplex was crystallized with an ATP analogue, AMPPNP. The AMPPNPmolecule can be placed unambiguously into the electron densityfound in the cleft between the N and C termini of the APH(3')-IIIa-butirosinA complex in a location and orientation identical to those ofthe other nucleotide-bound APH(3')-IIIa complexes (Fig. 1).In addition, residues 21 to 26, which form a lid over the phosphatemoieties of the bound nucleotide, adopt the same general conformationin all the structures in complex with a nucleotide. However,in contrast to the AMPPNP binary structure of APH(3')-IIIa (9),an additional interaction is observed between the main chainamide of Met26 and a ${gamma}$ -phosphate oxygen. This observation isin line with the postulated role of Met26 in stabilizing thetransfer of the phosphoryl group (49).

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FIG. 1. (A) Cartoon representation of the crystal structure of APH(3')-IIIa in complex with AMPPNP and butirosin A. Magnesium ions are shown in black. (B) Simulated annealing F_o – F_c omit map for AMPPNP, Mg ions, and butirosin A, contoured at 2.0 ${sigma}$ .

Aminoglycoside binding. Butirosin A occupies the acidic substrate-binding pocket inthe same manner as neomycin B, utilizing subsites A and C tomake contact with the enzyme, where subsite A holds the core2-deoxystreptamine and the prime rings and subsite C accommodatesthe ribose substituted at position 5 of the central ring (18)(Fig. 1 and 2). In the presence of AMPPNP, a hydrogen bond isobserved between a ${gamma}$ -phosphate oxygen and the 4' OH of butirosinA. This interaction plausibly contributes to the proper alignmentof the 3' OH for catalysis. Phosphoryl transfer can occur inan entirely associative or dissociative manner. The associativemechanism is distinguished by the small reaction coordinatedistance and with evidence of a trigonal bipyramidal intermediate,corroborating with a nucleophilic attack that precedes leavinggroup bond breakage. At the other extreme, phosphoryl transfercan take place via a dissociative mechanism, where the nucleophilicattack follows leaving group bond breakage. This is characterizedby the large reaction coordinate distance, generating a fullmetaphosphate structure (7, 35, 36). The structure reveals thatthe 3' OH of the aminoglycoside is located 3.8 Å fromthe ${gamma}$ -phosphorus atom. Although evidence so far supports a dissociativephosphoryl transfer mechanism for APH(3')-IIIa (7), the distanceof 3.8 Å observed here appears to be more consistent witha transition state of greater associative properties (46). Nonetheless,it has been noted that most enzymes are unlikely to adopt afully associative or dissociative mechanism. The actual mechanismmay lie anywhere between the two extremes (39). The crystalstructure reported here could be a representation of the groundstate of the enzyme, with both ligands bound, prior to any bondbreakage. The distances and positions of the ligands may undergoadjustment as the reaction proceeds.

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FIG. 2. (A) Cartoon representation of the aminoglycoside-binding site of the butirosin A ternary complex of APH(3')-IIIa. Residues interacting with butirosin A are drawn as gray sticks, and their hydrogen bond interactions with butirosin A are shown as dashed lines. The color scheme is the same as that shown in Fig. 1. (B) Schematic representation of the hydrogen bonding interactions between butirosin A and APH(3')-IIIa. The distance between the ${gamma}$ -phosphate of the nucleotide and the reactive 3'-hydroxyl of the aminoglycoside substrate is also indicated. The asterisks denote the amino acid residues that underwent a conformation change in the amikacin-bound model of APH(3')-IIIa.

Although the binding properties of butirosin and neomycin arelargely analogous, the enzyme's antibiotic-binding loop adoptsdramatically different conformations. The conformation of thisloop differs, depending on the presence or absence as well asthe structure of the antibiotic substrates. The antibiotic-bindingloop located between ${alpha}$ -helices A and B has been shown to be flexiblein the absence of a bound aminoglycoside substrate (9). However,upon the binding of kanamycin A and neomycin B, the loop undergoesa significant conformation change (18). This segment folds overtoward the antibiotic substrate, and several residues in thissection form interactions with the substrate, thereby completingthe aminoglycoside-binding pocket. However, in the presenceof butirosin A, the loop assumes an open conformation, akinto that observed in the binary enzyme structures, in order tocreate room for the AHB group (Fig. 3B and C). Consequently,the antibiotic-binding pocket cannot be completed in the samemanner, and some stabilizing interactions are forfeited. Theopen loop conformation in the presence of an N1-substitutedaminoglycoside was not anticipated, based on the informationobtained from our previous studies. This structure illustratesthe importance of nonspecific electrostatic and van der Waalsinteractions for aminoglycoside binding to APH(3')-IIIa.

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FIG. 3. (A) Chemical structure of several aminoglycoside antibiotics. (B to E) Superpositions of APH(3')-IIIa, shown in ribbon representation, bound with various ligands, drawn as sticks. The nucleotide, magnesium ions, aminoglycoside, and antibiotic-binding loops are highlighted in color. (B) APH(3')-IIIa bound with butirosin A shown in dark gray/green is superposed on the neomycin B complex colored in light gray/cyan. (C) The butirosin A ternary complex is shown in dark gray/green, and the AMPPNP binary structure is shown in light gray/magenta. (D) The butirosin A-bound structure shown in dark gray/green is overlaid with the amikacin-bound model shown in light gray/yellow. (E) The kanamycin A ternary complex is shown in dark gray/red, and the APH(3')-IIIa model bound with amikacin is shown in light gray/yellow. The RMSD value of the ${alpha}$ -carbon atoms for residues 150 to 165 of the butirosin A ternary complex and the binary structure is 1.63 Å, whereas this value is 4.23 Å between the APH(3')-IIIa-AMPPNP-butirosin A and APH(3')-IIIa-ADP-kanamycin A or -neomycin B structures.

The difference in the conformation of the antibiotic-bindingloop between the butirosin A ternary complex and the kanamycinA as well as the neomycin B ternary complexes is reflected intheir root mean square deviation (RMSD) values in this region(Fig. 3). Moreover, the temperature factors in this region ofthe butirosin A-bound complex are comparable to those in thenucleotide-bound enzyme structures, which are noticeably higherthan those in the kanamycin A and neomycin B ternary structures.The average B factor in the antibiotic-binding loop for theADP-bound and butirosin A-bound enzymes is 1.4 standard deviationsabove the mean, whereas the corresponding values for kanamycinA- and neomycin B-bound complexes are 0.4 and 1.0, respectively.The open loop conformation can be ascribed to the AHB at N1of the central ring, which hinders the loop from approachingand forming as many interactions with the aminoglycoside.

Most of the hydrogen bond interactions made between APH(3')-IIIaand butirosin A, kanamycin A, or neomycin B, notably those bondsmade with the core moiety (the central 2-deoxystreptamine andthe prime rings) of the aminoglycosides, are conserved. However,since the antibiotic-binding loop adopts an open conformationin order to accommodate the AHB, differences in the patternsof hydrogen bond interactions are observed (Fig. 2). For example,the side chain of Glu157 no longer makes contacts with the coremoiety of butirosin A but is now involved in hydrogen bondingof the hydroxyl of the butyryl group. In addition, the higherlevel of flexibility in the aminoglycoside-binding loop, asindicated by elevated temperature factor values, may resultin less-stable interactions with the aminoglycoside substrate.This may account for the lower binding affinity (higher K_m)of butirosin A relative to those of kanamycin A and neomycinB. The APH(3')-IIIa-AMPPNP-butirosin A structure illustratesthe importance of the pliable aminoglycoside-binding loop inthe recognition and binding of structurally diverse aminoglycosidesubstrates.

Model of amikacin binding to APH(3')-IIIa. Amikacin is the first semisynthetic aminoglycoside with an N1-substitutedAHB. It differs from butirosin A in that it is a derivativeof kanamycin A, a 4,6-disubstituted aminoglycoside. In contrastto butirosin A, which lacks good pharmaceutical properties,amikacin has had great success and remains an effective treatmentfor serious infections, especially those caused by pathogensresistant to other aminoglycosides such as kanamycin, tobramycin,and gentamicin (2). Although amikacin can be inactivated byAPH(3')-IIIa in vitro, only a low level of resistance is detectedin vivo. Nevertheless, synergy of amikacin with a cell wall-activeantibiotic is eliminated in S. aureus and enterococcal isolatesexpressing APH(3')-IIIa (55). Attempts were made to crystallizeAPH(3')-IIIa with amikacin without success. In the absence ofa crystal structure, we have modeled amikacin into the antibiotic-bindingpocket of APH(3')-IIIa, based on our knowledge of butirosinA binding, in order to speculate how a 4,6-disubstituted aminoglycosidewith an AHB substitution at N1 is accommodated in the enzyme.

The conformation of amikacin was taken from the crystal structureof an RNA fragment solved with a bound amikacin (28). When theamikacin is overlaid with kanamycin A and butirosin A from theAPH(3')-IIIa ternary structures, it is evident that their conformationsare remarkably similar. This similarity has previously beenillustrated by the comparison of APH(3')-IIIa-bound kanamycinA and neomycin B (18), ribosome-bound paromomycin I, RNA fragment-boundtobramycin (53), and APH(3')-IIa-bound kanamycin A (43). Basedon this knowledge, it can be deduced that the ring structuresof amikacin bound to APH(3')-IIIa should not deviate significantlyfrom those found in the RNA fragment-bound structure. Therefore,the conformation of the ring structures of amikacin was maintainedby imposing restraints on the dihedral angles during the courseof molecular dynamics. The resulting model shows that AMPPNP,magnesium ions, and amikacin remain in their respective bindingsites, adopting positions and orientations similar to theirequivalents found in the other aminoglycoside-bound ternarystructures (Fig. 3D and E). Although the AHB group of amikacinis in a different conformation than that of butirosin A, themodeled aminoglycoside is able to fit in the binding site withoutany significant movement in the antibiotic-binding loop andthe rest of the enzyme compared to the butirosin A-bound structure.The overall and the main chain RMSD between the butirosin-boundAPH(3')-IIIa and the amikacin-bound model of the enzyme are1.2 Å and 0.8 Å, respectively. These values arein the same range as those between the kanamycin A- and neomycinB-bound ternary structures of APH(3')-IIIa (1.2 Å and0.4 Å, respectively). This result is not unexpected, sincesimilar types and magnitudes of movements in the protein wouldbe expected of binding a 4,5-disubstituted and a 4,6-disubstitutedaminoglycoside. However, the side chains of Glu157 and Glu262,which make direct contact with the AHB group of butirosin, adoptdifferent rotamers in order to accommodate the AHB group ofamikacin. Based on this model, it can be inferred that isepamicinwould bind to APH(3')-IIIa in the same manner as amikacin. Sinceisepamicin is substituted at N1 with a 4-amino-2-hydroxypropionylgroup containing one less carbon than the butyryl group in amikacin,isepamicin should be able to be accommodated by the enzyme withoutany difficulty. Although the other semisynthetic N1-substitutedaminoglycosides arbekacin and netilmicin are not substratesof APH(3')-IIIa since they lack the 3'-hydroxyl functional group,they are competitive inhibitors of the enzyme (38). It can alsobe extrapolated that arbekacin and netilmicin, which are substitutedwith an AHB group and an ethyl group at N1, respectively, wouldalso be able to bind to APH(3')-IIIa.

Implications for future semisynthetic N1-substituted aminoglycosides. The clinical success of amikacin, isepamicin, and arbekacinhave validated the efficacy of aminoglycosides derivated atN1 of the 2-deoxystreptamine ring. Nonetheless, incidents ofresistance to these semisynthetic N1-substituted aminoglycosidesare slowly rising, including those to arbekacin, the most recentaddition to the aminoglycoside antibiotic arsenal (3). Previously,the crystal structures of various aminoglycoside-bound A siteand kanamycin A- or neomycin B-bound ternary complexes of APH(3')-IIIa(18) have contributed to the design of several promising paromomycinderivatives (19-21). The additional information provided bythe butirosin A crystal structure and the amikacin-bound modelof APH(3')-IIIa reported here could be implemented to furtherimprove the prospect of new aminoglycoside derivatives. Ouranalysis suggests that even though enzyme-mediated resistanceto the existing N1-substituted aminoglycosides has developed,further derivation of the AHB tail may still be a viable optionfor generating antibiotics with dramatically reduced susceptibilityto resistance mechanisms (Fig. 4). In both the butirosin-boundstructure and amikacin-bound model of APH(3')-IIIa, the carbonylpoints toward the center of the enzyme. In contrast, the samegroup in the amikacin bound to an RNA fragment (28) points toan open end of the groove and is not involved in any interactionwith the A site. Therefore, it can be deduced that a bulky groupsubstituting the oxygen at this position of the AHB tail wouldgenerate an aminoglycoside capable of evading resistance mediatedby APH(3')-IIIa, while retaining its ability to bind to theA site and precipitate bacterial cell death.

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FIG. 4. Molecular surfaces of the ribosomal A site (A) (28) and APH(3')-IIIa around the amikacin-binding site (B). Amikacin is shown in yellow sticks, and the green sphere represents the bulky group that could be added to replace the carbonyl of the AHB tail. An appropriate substitution at this location should not interfere with the binding of the aminoglycoside derivative to the A site. However, such an addition would not be accommodated by the APH(3')-IIIa resistance enzyme.

Concluding remarks. The significance of the aminoglycoside-binding loop in acceptingand positioning structurally diverse aminoglycosides in thebinding pocket of APH(3')-IIIa is emphasized by the structureof the butirosin ternary complex. While the presence of AHBat N1 of aminoglycosides results in a reduction in binding affinitytoward many resistance factors, butirosin, amikacin, and isepamicinare unable to evade the recognition and inactivation by APH(3')-IIIa.The structure of the butirosin A ternary complex illustratesthat the substrate-binding pocket of APH(3')-IIIa is highlymalleable, due to the flexible substrate-binding loop. The loopaccommodates butirosin A by receding from the core of the aminoglycosideto make space for the AHB group at the central ring. Modelingof the amikacin-bound enzyme suggests that this conformationaladaptation is the basis for conferring resistance to other clinicallyrelevant N1-substituted aminoglycosides. Thus, these studiesprovide structural insights into APH(3')-IIIa-mediated resistanceto AHB-containing aminoglycoside antibiotics. These insightscan be exploited for the development of aminoglycoside derivativeswith reduced sensitivity to antibiotic resistance.

ACKNOWLEDGMENTS

We thank past and present members of the Berghuis lab for theirassistance and suggestions.

This work was supported by grant FRN13107 (A.M.B.) from theCanadian Institutes of Health Research. A.M.B. holds a CanadaResearch Chair in Structural Biology.

FOOTNOTES

* Corresponding author. Mailing address: Room 466, Bellini Pavilion, 3649 Promenade Sir William Osler, Montreal, Quebec H3G 0B1, Canada. Phone: (514) 398-8795. Fax: (514) 398-2983. E-mail: albert.berghuis{at}mcgill.ca

Published ahead of print on 11 May 2009.

Supplemental material for this article may be found at http://aac.asm.org/.

REFERENCES

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