molecular recognition of aminoglycoside antibiotics by bacterial defence proteins: nmr study of the...

Molecular Recognition ofAminoglycoside Antibioticsby Ribosomal RNA andResistance Enzymes: AnAnalysis of X-Ray CrystalStructures

Quentin VicensEric Westhof

Institut de BiologieMoleculaire et Cellulaire

du CNRS,Modelisation et simulations

des Acides Nucleiques,UPR 9002,

Universite Louis Pasteur,15 rue Rene Descartes,

67084 Strasbourg Cedex,France

Received 18 February 2003;accepted 18 February 2003

Abstract: The potential of RNA molecules to be used as therapeutic targets by small inhibitors isnow well established. In this fascinating wide-open field, aminoglycoside antibiotics constitute themost studied family of RNA binding drugs. Within the last three years, several x-ray crystalstructures were solved for aminoglycosides complexed to one of their main natural targets in thebacterial cell, the decoding aminoacyl–tRNA site (A site). Other crystallographic structures haverevealed the binding modes of aminoglycosides to the three existing types of resistance-associatedenzymes. The present review summarizes the various aspects of the molecular recognition ofaminoglycosides by these natural RNA or protein receptors. The analysis and the comparisons ofthe detailed interactions offer insights that are helpful in designing new generations of antibiotics.© 2003 Wiley Periodicals, Inc. Biopolymers 70: 42–57, 2003

Keywords: aminoglycoside antibiotic; aminoglycoside modifying enzyme; resistance mechanism;ribosomal RNA; x-ray crystal structure

INTRODUCTION

With the emergence of the x-ray structures of largeintricate RNA assemblies1–5 and RNA–protein com-plexes,6–11 RNA was definitely shown to be a prom-ising drug target.12–14 Beside ribozymes, RNA anti-sense, and RNA interference techniques,15–18 RNAtargeting using low-molecular weight compounds rep-resents a young but dynamic field.19,20 Today, only afew systems are used as models for the study of themolecular recognition of drugs by RNA mole-cules,21,22 with the design of rational drugs as the

ultimate goal.23 Particularly, aminoglycoside antibiot-ics form the most studied family of RNA bindingdrugs.24

Aminoglycosides are water-soluble oligosaccha-rides typically functionalized by 3–6 ammoniumgroups.24,25 Most of the aminoglycosides belong toeither the 4,5-2-deoxystreptamine (DOS) or the 4,6-2-DOS sub-class (Figure 1). Ring I is always attachedon position 4 of the common 2-DOS ring (ring II),although it bears some differences in the number andtype of chemical substituents.26 Supplementary ringsare attached at either position 5 or position 6 of

Correspondence to: Eric Westhof; email: [email protected], Vol. 70, 42–57 (2003)© 2003 Wiley Periodicals, Inc.

42

ring II. The common core therefore contains onlyrings I and II, and is referred to as the neaminemoiety.

In the 1960s, aminoglycosides were shown to in-duce miscoding during protein synthesis,27 andtwenty years later, their binding site was found to belocated at the aminoacyl–tRNA decoding site (A site)on the 16S ribosomal RNA.28 Later, kinetic studiesindicated that aminoglycosides induce and stabilize a

conformation of the A site, which is similar to the oneprovoked by a correct interaction between cognatetRNA and mRNA.29,30 The recent crystal structures(at resolutions between 3.00 and 3.80 A) of the 30Sribosomal particle free or bound to various antibioticmolecules (including the aminoglycoside paromomy-cin), and various tRNA and mRNA analogues, haveoffered the ultimate illustration of this phenome-non.8,31–33

In the 1990s, biochemical results revealed that theA site forms a recurrent motif that can be extractedfrom its natural ribosomal environment while stillmaintaining its ability to interact specifically withaminoglycosides.34–36 This remarkable findingopened the door to several biochemical37,38 and struc-tural39 studies that initiated the characterization at amolecular level of the binding of these molecules toRNA hairpins containing the A site, before crystalstructures became available.

Theoretical work showed that the driving force ofthe positively charged aminoglycosides to the nega-tively charged RNA molecules is dominated by elec-trostatic forces and the displacement of cations fromthe RNA.40,41 Crystal structures of 4,5-2- and 4,6-2-DOS aminoglycosides bound to RNA oligonucleo-tides containing two A sites (solved at 2.40–2.54 Aresolution)42–44 showed precisely that the molecularrecognition of aminoglycosides by the A site reliesnot only on several direct but also on water-mediatedhydrogen bonds. The comparative analysis of variousaminoglycosides bound to the same site is essential todecipher the contribution of each functional group tothe RNA–aminoglycoside interactions. In addition,these structures enable us to rationalize various bio-chemical and microbiological data as well as someresistance and toxicity mechanisms at the molecularlevel.

The present review is organized in the followingway. (a) The molecular mechanisms of the amino-glycoside antibiotic recognition by the ribosomalRNA as understood from the available crystal struc-tures of the various aminoglycoside–A site com-plexes will be summarized. (b) Examples of resis-tance mechanisms that can now be rationalized at amolecular level on the basis of these structures willbe presented. (c) The roles of water molecules andthe specificity of particular interactions will be em-phasized through comparisons of crystal structuresof aminoglycosides bound to resistance enzymesand more generally to those of oligosaccharidesbound to proteins. This broad overview enables amore general understanding of the recognition ofsmall molecules by RNA that will assist the designof new antibiotics.

FIGURE 1 Chemical structures of (a) 4,5-2-DOS and (b)4,6-2-DOS aminoglycosides. The 2-DOS ring is shown inblue.

Recognition of Aminoglycoside Antibiotics 43

MOLECULAR MECHANISMS FOR A-SITE RECOGNITION

Complexes with the 30S Particle:Insights into the Interference with theDecoding Process

At the end of the year 2000, Venki Ramakrishnan andco-workers published the crystal structures of the 30Sribosomal subunit from Thermus thermophilus bothfree8 and in complex with paromomycin.31 Severalstructures were obtained afterwards for the 30S par-ticle bound to paromomycin and to both cognate32

and near-cognate33 tRNA–mRNA complexes (TableI). Inside the 30S particle, paromomycin has only onespecific binding site, the A site. This site is located atthe foot of helix 44, the longest of the 30S particle, onthe face that contacts the 50S particle (Figure 2a).Around 73% of the 16S rRNA nucleotides are con-served between T. thermophilus and Escherichiacoli.45 In particular, the A-site sequence is conserved,except for the 1410–1490 base pair (AOU in E. coliand GAC in T. thermophilus), a difference thatshould not interfere with the tertiary structure of the Asite.45 In the absence of any aminoglycoside ligand,the A site is formed by three Watson–Crick GACpairs, one U � U pair, and an internal loop made ofthree adenines (1408, 1492, and 1493 in E. coli) inwhich adenines 1492 and 1493 fold back within thehelix. Paromomycin binds to the deep groove of the Asite by inserting ring I inside the helix: this puckeredring stacks against G1491 and forms two direct hy-drogen bonds to A1408. The main effect of this in-sertion is to force adenines 1492 and 1493 to fullybulge out from the helix. Alignment within the bound

and free structures of the sugar–phosphate backboneatoms of the nucleotides forming the site [root meansquare (RMS) deviation � 0.6 A] results in a RMSdifference of 6.4 A for adenines 1492 and 1493 (Fig-ure 2b). The resulting extrahelical conformation isstabilized by direct hydrogen bonds from the phos-phate oxygen atoms of the bulged adenines to func-tional groups on rings I and II.

During translation, the ultimate step of aminoacyl–tRNA selection is based on the formation of a mini-helix between the codon of the mRNA and the anti-codon of the cognate aminoacyl–tRNA.46,47 This de-coding mechanism is performed at the A site byadenines 1492 and 149329,35,48–50: when the cognatetRNA–mRNA complex is formed, the two adeninesflip out from the A-site helix and form type I–type IIinteractions51,52 with the first two base pairs of theminihelix.32 This conformational change constitutesthe molecular switch that irreversibly decides on thecontinuation of translation.30,53,54 Aminoglycosideslike paromomycin stabilize a conformation of the Asite that normally occurs only when a cognate tRNA–mRNA complex is bound. As a consequence, thestabilities of near-cognate aminoacyl–tRNA bindingto this site are increased and the kinetics controllingthe translation fidelity are disturbed in such a way thatthe ribosome is not able to discriminate between cog-nate and near- or noncognate tRNA–mRNA com-plexes any more.29,30 Based on the crystal structuresof the various 30S particle complexes, it has beenproposed that the binding of paromomycin to the Asite compensates for the energetic cost associated withthe conformational change of adenines 1492 and1493.32,55 The recent structures of the 30S particle

Table I Statistics of the Thermus thermophilus 30S Particle Structures

Ligand

Number ofNonhydrogen

AtomsaResolutionLimit (A)

R/Rfree

(%)PDB Entry

Code Ref.

None 51,848 3.05 20.8/25.2 1FJF 8Paromomycin, stretptomycin, spectinomycin 51,735 3.00 22.5/25.8 1FJG 31Paromomycin 51,880 3.31 22.9/28.2 1IBK 32mRNA, cognate tRNAPhe analogue 52,109 3.31 23.1/28.6 1IBMmRNA, cognate tRNAPhe analogue and

paromomycin 52,250 3.11 23.2/27.5 1IBLmRNA, near–cognate tRNALeu2 analogue 51,757 3.80 24.2/31.2 1N34 33mRNA, near-cognate tRNASer analogue 51,680 3.65 26.0/32.4 1N36mRNA, near-cognate tRNALeu2 analogue

and paromomycin 52,275 3.00 22.7/27.0 1N32mRNA, near-cognate tRNASer analogue and

paromomycin 52,140 3.35 22.5/28.4 1N33

a Number of protein � RNA � metal atoms.

44 Vicens and Westhof

complexes remarkably show that the overall structuralchange, normally observed for the ribosome only afterthe binding of the cognate tRNA–mRNA complex,occurs likewise after the binding of a near-cognatetRNA–mRNA complex when paromomycin ispresent.33 These structures have revealed the mecha-nism of action of aminoglycosides during proteinsynthesis at a molecular level.

Complexes with OligonucleotidesContaining the A Site Reveal theSpecificity of the Interactions and theRoles of Solvent Molecules

Crystal structures were solved in our group at 2.40–2.54 A resolution for paromomycin,43 and for two4,6-2-DOS derivatives, tobramycin44 and geneticin,42

bound to chemically synthesized RNA fragments con-

taining two A sites (Table II). In these three struc-tures, the oligoribonucleotides assemble into a doublehelix containing two A-site pockets, each one bindingone molecule of aminoglycoside. Due to a particularpacking environment that mimicks tertiary interac-tions naturally occurring in the ribosome, the confor-mations of the A site and the aminoglycosides areboth remarkably similar to those observed for thesame site bound to paromomycin in the crystal struc-ture of the 30S particle (Figure 3a).31,42,43 Therefore,in each structure, the common neamine part binds insuch a way that adenines 1492 and 1493 are extrudedfrom the helix and bulge out to the extent alreadyobserved in the 30S particle structure bound to paro-momycin (RMS deviation � 2.0 A) (Figure 3a,b).31

The particular conformation of the aminoglyco-side–A-site complex is stabilized by a precise set ofspecific interactions (Figure 3c). The puckered ring I

FIGURE 2 Crystal structure of the 30S particle. (a) View of the 50S-facing side, with the 16SrRNA and the proteins shown in grey and red, respectively.8 The A site (green box) is located at thefoot of helix 44 (bold). The conserved A-site sequence and its corresponding numbering in E. coliare specified. (b) Superimposition (based on the sugar–phosphate backbone atoms) of the A site free(grey)8 and bound to paromomycin (gold)31 inside the 30S particle.


(a) stacks over and forms definite C—H–� interac-tions to G1491, and (b) forms a pseudo-base pair withtwo direct H-bonds to the Watson–Crick sites ofA1408 (Figure 3d). The neamine core makes six di-rect or water-bridged H-bonds to phosphate oxygenatoms of adenines 1492 and 1493. Additionally, ap-proximately a dozen of direct or water-bridged H-bonds are formed to the three GAC pairs(1405A1496; 1407A1494; 1409A1491) and to theU1406 � U1495 pair. Consequently, water moleculesplay an important role in modulating the structuralproperties of the site and of the aminoglycoside, sothat each type of aminoglycoside makes an equivalentnumber of H-bonds to the RNA in order to bind witha similar affinity,56 regardless of some variability inchemical structure. For example, a direct H-bond ob-served from the additional ring III to G1491 in theparomomycin complex is replaced by water-bridgedH-bonds from the neamine moiety in the tobramycincomplex (Figure 4a). Vice versa, the direct H-bondsobserved from the additional ring III to G1405 in thetobramycin complex are replaced by water-bridgedH-bonds from ring II in the paromomycin complex(Figure 4b). It is interesting to notice that in the latterexample, the water molecules at the interface betweenthe RNA and the antibiotic are located at positionssimilar to those normally occupied around GAC pairsin naked RNA (see Refs. 57 and 58 for comparisons).

Water molecules also help modulate the interac-tions involving ring II and the U1406 � U1495 pair.Whereas one water molecule is present in each groovein the paromomycin complex (the one in the deepgroove bridging an H-bond from O6 of ring II to O4of U1406), only the one in the shallow groove ispresent in the tobramycin complex, and none is ob-served in the geneticin complex (Figure 5). Ring III isattached on position 6 of ring II in tobramycin andgeneticin, thereby occupying the position of the watermolecule in the deep groove of the paromomycincomplex. Furthermore, ring III of geneticin containsthe hydroxyl group O4� in such a position that it formsa hydrogen bond to O2P of U1406, thereby decreas-ing the C1�–C1� distance between the two uridinesand preventing the binding of a water molecule in theshallow groove (Figure 5c).42 To summarize, the

characteristic adaptability of the A site to accommo-date different aminoglycosides arises from (a) thelarger than 98% conservation of the nucleotides thatinteract with the neamine moiety, (b) the flexibility ofthe adenine bulge and of the U1406 � U1495 pair, and(c) the presence of several water molecules at theaminoglycoside–RNA interface.

MOLECULAR BASIS FOR THREERESISTANCE MECHANISMS

Resistance to aminoglycosides arises in most of thebacterial cells from mainly three mechanisms: (a)chemical modification of aminoglycosides by inacti-vating enzymes, (b) chemical modification, and (c)point mutations of A-site nucleotides.59,60 The chem-ical inactivation of aminoglycosides constitutes themost prevalent resistance mechanism.25 About fiftyinactivating enzymes have been identified and classi-fied into three distinct families, according to thechemical reaction performed.59,61 The ANT, AAC,and APH catalyze respectively the transfer of a nucle-otidyl, an acetyl, or a phosphoryl group on a func-tional group of the aminoglycoside (Figure 6a). Thesestructural modifications reduce the antibiotic activi-ty62,63 by decreasing the binding affinity of the ami-noglycoside for the A site.59,64,65 Interestingly, theaminoglycoside functional groups targeted by the in-activating enzymes are located essentially on theneamine part, at the positions that are common to thevarious aminoglycosides and that form the direct H-bonds to adenine 1408 and to the phosphate oxygenatoms of the bulged adenines 1492 and 1493 (Figures3c and 6a). The addition of a chemical group on sucha position prevents the insertion of ring I into thegroove, a state that controls and stabilizes the bulgingout conformation of adenines 1492 and 1493. Accord-ingly, modified aminoglycosides lose their specificantibiotic properties.

Chemical alterations of precise A-site nucleotideshave also been discovered to provoke resistance.25

Remarkably, enzymatic methylations of either the N1atom of adenine 1408 or the N7 atom of guanine 1405are sufficient to confer high level resistance to ami-

Table II Statistics of the Synthetic A-Site Oligonucleotide Structures

LigandNumber of NonHydrogen


R/Rfree

(%)PDB Entry

Code Ref.

Paromomycin 1038 2.50 20.6/24.7 1J7T 43Tobramycin 1040 2.54 21.4/26.4 1LC4 44Geneticin 1073 2.40 22.4/25.0 1MWL 42

a Number of RNA � ligand � water oxygen atoms.


FIGURE 3 Crystal structures of three aminoglycosides bound to the A site. (a) Superimposition(based on the neamine moiety) of the A site bound to paromomycin in the A site oligonucleotide (gold)43

and in the 30S particle (green).31 The three adenines 1408, 1492, and 1493 are shown in bold. (b)Superimposition (based on the neamine moiety) of the A site bound to paromomycin (gold),43 totobramycin (light blue),44 and to geneticin (magenta).42 Solvent molecules are omitted for clarity. (c)Close up showing the conserved hydrogen bonds involving the neamine moiety in each complex. (d)Detailed view of the pseudo-base pair (in bold) formed between ring I and adenine 1408 in each complex.


noglycosides.66 In the crystal structures of the variousaminoglycoside–A-site complexes, ring I forms apseudo-base pair with A1408. The addition of amethyl group at position N1 of A1408 would preventthe formation of the direct H-bond from the hydroxylor the ammonium at the exocyclic 6� position. Fur-thermore, the methylation adds a positive charge onthe N1 atom that makes a repulsion with a 6�-ammo-nium group on ring I. As observed for resistancemechanisms by chemical inactivation, the aminogly-coside loses its ability to insert ring I within the siteand thereby its antibiotic effectiveness. Interestingly,the methylation at the N7 of G1405 gives resistanceselectively against antibiotics belonging to the 4,6-2-DOS subclass.66 The comparisons of the structures ofthe tobramycin- and paromomycin–A-site complexesdenotes that whereas ring III of tobramycin forms adirect H-bond to N7 of G1405, paromomycin onlyforms water-bridged H-bonds to that base (Figure 4b).Therefore, the addition of a methyl group and a pos-

itive charge at the N7 of G1405 will prevent thebinding of ring III, as discussed for the methylation atN1 of A1408 that prevents the binding of ring I.

A third resistance mechanism by which bacteriadecrease the affinity of aminoglycosides for the ribo-somal RNA is the mutation of a specific A-sitebase.67–71 The crystal structures suggest that eachmutation either changes the geometry of the base pair

FIGURE 5 Geometries of the U1495 � U1406 pair boundto aminoglycosides. Interactions are shown in surface rep-resentation with (a) paromomycin,43 (b) tobramycin,44 and(c) geneticin42 colored as in Figure 3b (ring I is omitted forclarity). Water molecules are shown in green. The C1�–C1�distances are specified.

FIGURE 4 Modulation of the aminoglycoside recogni-tion by water molecules. Detailed interactions to (a) guanine1491 and (b) guanine 1405. The orientation of paromomy-cin (gold)43 and tobramycin (light blue)44 is the same asin Figure 3b. The carbon atoms of the guanines and thesolvent molecules are colored according to the boundaminoglycoside.


so that the aminoglycoside cannot bind or aims atpresenting base atoms in the deep groove which donot favor any interaction with the antibiotic. For ex-ample, the single A1408G mutation is sufficient toconfer selectively a strong resistance to aminoglyco-sides containing a 6�-ammonium group, like tobramy-cin.69,72 A model of the A1408G mutation based onthe crystal structure shows that the mutation of A to Gwould disturb the formation of the pseudo-base pairbetween ring I and residue 1408. The functionalgroups on ring I do not match the Watson–Crick sitesof the guanine to form direct H-bonds, and the N1 andN2 amino groups of the guanine form repulsionagainst the 6�-ammonium group of the aminoglyco-side (Figure 6b). Noteworthy, a 6�-hydroxyl group (asin geneticin and paromomycin) would avoid repulsionwith the amino groups of G1408 and would be able toform H-bonds equally to donor or acceptor sites.

COMPARISONS WITHAMINOGLYCOSIDE- ANDCARBOHYDRATE–PROTEINCOMPLEXES

Crystal Structures of Aminoglycoside–Resistance Enzyme Complexes

In order to improve our understanding of the molec-ular recognition of aminoglycosides, it is worth com-

paring the x-ray structures of aminoglycosides boundto the A site with those of aminoglycosides bound toresistance enzymes. To date, the crystal structures ofthree different resistance enzymes, an ANT(4�/4�), anAAC(2�), and an APH(3�) (that add chemical sub-stituents on positions 4� or 4�, 2� and 3� respectively),have been solved in complex with 4,5- and 4,6-2-DOSaminoglycosides, as well as with the various sub-strates required for the catalysis of the chemical re-actions, such as acetylcoenzyme A and ADP (TableIII).73–75 Whereas APH(3�) forms a monomer con-taining one active site, ANT(4�/4�) and AAC(2�) bothform dimers, therefore possessing two active sites. InANT(4�/4�), the aminoglycoside binding sites are lo-cated deep inside the enzyme, while in the case of theAAC(2�), they are located 40 A apart on the outersurface. Additionally, the structures of the AAC(2�)75

and the ANT(4�/4�)76 have been solved without anyaminoglycoside substrate. The comparison betweenthe free and the bound structures shows that bindingof the aminoglycoside does not provoke any confor-mational change in the protein other than the one thatoccurs locally in the binding site.

Interestingly, the analysis of the structures of theaminoglycoside–enzyme complexes reveals severalcommon features in the molecular recognition mech-anisms of aminoglycosides by proteins and by theA-site RNA. Regardless of the type of the bindingenzyme, the conformations of the aminoglycoside

FIGURE 6 Mechanisms of resistance to aminoglycosides. (a) Principal sites of chemical inacti-vation of 4,6-2-DOS aminoglycosides (i.e., kanamycin B).59 The enzymes are indicated by theirabbreviation followed by the number of the modified position. (b) Interactions involving ring I oftobramycin and adenine 1408 (top).44 Model of the A1408G mutation resulting from a superim-position of guanine coordinates on A1408 (bottom). Hydrogen atoms are displayed for a betterunderstanding.


rings are similar to those observed in solution77,78 orfor aminoglycosides bound to the A site.31,43 Remark-ably, the neamine part keeps an orientation similar in4,5- and 4,6-2-DOS aminoglycosides, except for thebinding to ANT(4�/4�) (Figure 7). However, whereasrings II and III of the 4,6-2-DOS derivatives keepsimilar relative orientations due to the presence of twoconserved intramolecular hydrogen bonds (Table IV),rings II and III of the 4,5-2-DOS compounds do not.For example, ring III of ribostamycin bound toAAC(2�) is turned over compared to ring III of paro-

momycin bound to the A site (Figure 7a). Regardlessof the aminoglycoside subclass, the relative geometryof the neamine moiety constitutes the only essentialpart for the binding to both resistance enzymes andthe ribosomal A site. However, this result is notunexpected, since (a) the neamine part corresponds tothe conserved common core, and (b) the enzymebinding sites are able to recognize various aminogly-coside substrates.

Most of the amino acid residues forming the bind-ing pocket are acidic (Table IV): four glutamic acids

Table III Statistics of Resistance Enzyme Structures

Enzyme LigandNumber of Nonhydrogen


R/Rfree

(%)PDB Entry

Code Ref.

ANT (4�/4�) None 506 3.00 18.9/— 1KAN (76)Kanamycin A 4231 2.50 16.8/— 1KNY (74)

APH(3�) Kanamycin A 2297 2.40 23.4/29.1 1L8T (73)Neomycin 2285 2.70 23.0/30.8 1L8U

AAC(2�) None 3213 1.60 17.4/20.1 1M44 (75)Kanamycin A 3366 1.80 17.9/20.2 1M4ITobramycin 3210 1.50 17.4/21.1 1M4DRibostamycin 3314 1.80 16.5/20.2 1M4G

a Number of protein � ligand � water oxygen � metal atoms.

FIGURE 7 Conformations of aminoglycosides bound to the A site and to resistance enzymes. (a)Superimposition of paromomycin bound to the A site (gold),43 ribostamycin bound to AAC(2�)(magenta)75 and neomycin bound to APH(3�) (grey).73 (b) Superimposition of tobramycin bound tothe A site (light blue)44 and kanamycin A bound to ANT(4�/4�) (pink).74 The superimpositions arebased on the 2-DOS ring atoms. Water molecules directly bound to the antibiotics are coloredaccording to the aminoglycoside and those located at similar positions are indicated by arrows.


for the ANT(4�/4�); four aspartic acids, three glutamicacids, and the carboxy-terminal group for theAPH(3�); five aspartic acids, one glutamic acid, andthe carboxy-terminal group for the AAC(2�) (Figure8). In addition, the binding site of APH(3�) contains abasic amino acid that interacts with hydroxyl groups

of ring I. As a result, each hydroxyl and ammoniumgroup of the aminoglycoside interacts with similarcarbonyl and amine functional groups in the A siteand in resistance enzymes (Table IV). Most of thesecontacts involve functional groups of rings I and II.Particularly, the atoms N1 and N3 on the 2-DOS ring

Table IV H-Bonds Involving the Neamine Moiety in Either the A-Site or the Enzyme Binding Sitea

a The groups positively or negatively charged at the pH of the experiment are indicated in blue and red, respectively. The polar groups areshown in green. “W” stands for the oxygen atom of a water molecule.


have similar environments. In each RNA or proteincomplex, N3 is the only completely dehydrated am-monium group, whereas N1 is less dehydrated andmakes a conserved intramolecular hydrogen bond to

ring III in the 4,6-2-DOS subgroup. N3 forms threedirect H-bonds to the acceptor groups N7(G1491),O1P(A1493), and O2P(G1494) in the crystal struc-tures of the A-site complexes solved at pH 6.4, and

FIGURE 8 Detailed views of the aminoglycoside binding sites in resistance enzymes. (a)Kanamycin A (pink) and an ATP analogue bound to ANT(4�/4�).74 (b) Kanamycin A (pink) and anADP molecule bound to APH(3�).73 (c) Tobramycin (light blue) and a coenzyme A molecule boundto AAC(2�).75 Antibiotics are oriented as in Figure 7. Water molecules and magnesium ions areshown as red and green spheres, respectively.


three direct H-bonds to carbonyl groups in theAPH(3�) complexes crystallized at pH � 9.0 (TableIV; Figures 3c and 8b).

The binding of aminoglycosides to resistance en-zymes provokes the departure of several water mole-cules (e.g. six to eight for the AAC(2�).75 About tenwater molecules remain located around the aminogly-cosides bound to APH(3�) and AAC(2�).73,75 Theymediate hydrogen bonds from the antibiotic to theamino acid functional groups, thereby increasing theability of the binding site to accommodate structurallydifferent aminoglycosides. Eight direct and four wa-ter-bridged H-bonds are formed by the neamine moi-ety in A-site as well as in APH(3�) complexes (TableIV). Five direct and six water-bridged H-bonds areobserved in AAC(2�) complexes (Table IV). Twowater molecules are even found to occupy the samepositions in the A-site and AAC(2�) complexes (Fig-ure 7a). The modulation of the structural properties ofthe binding sites is therefore an important parameterfor the molecular recognition by both the A-site and

the enzyme to be taken into account in drug designstrategies.

Crystal Structures ofCarbohydrate–Protein Complexes

Since aminoglycosides are oligosaccharides contain-ing amino-sugar rings, it is interesting to comparetheir binding modes to the resistance enzymes and tothe A-site nucleotides with those of carbohydrate-binding proteins. Carbohydrates have similar chemi-cal scaffolds, but are functionally distinct.79,80 It isnoteworthy that the specific interactions observed forthe aminoglycoside complexes are reminiscent ofthose detected earlier in several structures of oligo-saccharide–protein complexes. The rings constitutingthe oligosaccharides are usually in chair conforma-tions to orient the functional groups on equatorialpositions.81 They also interact either partially or to-tally with the protein, through hydrogen bonds andvan der Waals interactions, in binding pockets mainly

FIGURE 9 Binding modes of carbohydrates to proteins. (a) Stacking of saccharides (black)against aromatic residues in a maltodextrine binding protein.84 (b) Superimposition of L-arabinose(green)86 and D-galactose (pink)87 bound to a carrier protein. The carbon atoms of the amino acidsand the solvent molecules are colored according to the bound saccharide.


constituted by charged and polar amino acids (for afew examples, see Refs. 82 and 83). The stacking ofone or several of the puckered sugar rings constitutingthe oligosaccharide against the aromatic side chains oftyrosine, tryptophan, and phenylalanine residues isfrequently observed (i.e., see Ref. 84), especially inlectins (Figure 9a).81 These interactions are remark-ably similar to those observed for ring I of the ami-noglycosides and the guanine 1491 in the A site(Figure 3d).

Water molecules also help to optimize the numberof potential H-bonds made from the oligosaccharideto the protein.85,86 As observed for the A site and theresistance enzymes complexed to aminoglycosides,water molecules modulate the structural properties ofthe binding sites of carbohydrate-binding proteins, sothat various substrates are accommodated in the samesite. For example, crystal structures were solved at1.7–1.8 A resolution for L-arabinose87 and D-galac-tose88 in complex with a carrier located in theperiplasm of Gram-negative bacteria. The structure ofthe binding site is similar for both substrates, exceptfor the hydroxyl group O5� inserted inside the sugarring. In the L-arabinose complex, O5� forms a hydro-gen bond to the main-chain NH group of Met108 thatis mediated by two water molecules. In the D-galac-tose complex, this interaction is replaced by a hydro-gen bond that involves the exocyclic hydroxyl groupO6� and only one water molecule (Figure 9b).88 Asobserved for aminoglycosides bound to the A site(Figure 4), this strategy allows the protein to binddifferent substrates with the same affinity: both L-arabinose and D-galactose bind to the carrier proteinwith a 10�7M dissociation constant.89

FUTURE PROSPECTS

Towards the Design of New Antibioticsthat Bind to the A Site

The design of new antibacterial agents that bind to theA site is a tough challenge. Active antibiotics have tobe able to (a) be taken up into bacterial cells, (b)interfere selectively with the protein synthesis in bac-

FIGURE 10 Accessible surfaces of A-site RNA and re-sistance enzymes around a 4,6-2-DOS aminoglycoside. To-bramycin (light blue) bound to (a) the A site44 and (b)AAC(2�).75 Kanamycin A (pink) bound to (c) APH(3�)73

and (d) ANT (4�/4�).74 The surfaces were calculated using aspherical probe (1.4 A radius) within a 10 A radius from theaminoglycoside. In each structure the black arrow indicatesring I.


teria by stabilizing a similar A-site conformation, (c)bind selectively to bacterial wild-type and mutatedA-sites, and (d) be poor substrates for resistance en-zymes. To date, given the difficulties associated withde novo design, all the antibiotics designed to targetthe A site are aminoglycoside derivatives.23,90–93

The comparisons of the various crystal structuresof A-site RNA and enzyme complexes reveal that themolecular recognition of aminoglycosides is achievedin a similar way in both systems. (a) The rings haveequivalent conformations and relative orientations(except rings I and II in the ANT(4�/4�) complex andmost of the contacts involve the neamine part. (b) Theadaptability of the binding site to recognize variousaminoglycosides having different 2-DOS substitu-tions comes either from the formation of alternativehydrogen bonds to different nucleotides (in the Asite), or from the flexibility of the side chains liningthe binding pocket (in resistance enzymes). Addition-ally, in one subclass, some water molecules mediatehydrogen bonds to compensate for the direct interac-tions that occur through the additional ring attached ata different position in the other subclass. These sim-ilarities make the task to design new antibiotics evenmore difficult. However, Fong and co-workers haveshown that despite the high correspondence betweenthe binding modes of aminoglycosides to the A siteand to the APH(3�), the faces of the antibiotic thatform van der Waals interactions to the RNA and to theAPH(3�) enzyme are opposite.73 Interestingly, thesame characteristic is observed in the ANT(4�/4�) andthe AAC(2�) complexes (Figure 10). Drug designerscould therefore capitalize on this difference to preventthe binding of new aminoglycoside derivatives toresistance enzymes by obstructing the correspondingface with chemical bulky groups. Ultimately, in orderto decrease the risk that newly developed antibioticsrapidly become substrates of resistance enzymes,molecules without carbohydrate rings could be syn-thetized.75

What Can We Learn from the AvailableStudies on the Choice of RNA Targets?

Aminoglycoside antibiotics target ribosomes in bac-terial cells and interfere with translation by binding tothe aminoacyl–tRNA decoding site (A site) on the16S ribosomal RNA. Kinetic analysis showed that,during decoding, a correct tRNA–mRNA interactioninduces a conformational change of the A site thatpermits the translation.94 Aminoglycosides interferewith the fidelity of this selection step by stabilizing asimilar conformation for incorrect complexes.30 Crys-tallographic structures of the 30S particle with variousligands revealed this process with atomic details: dur-

ing decoding, the A site changes its conformationfrom an “off” conformation (with A1492 and A1493folded in the shallow groove of the A site) to an “on”conformation (with A1492 and A1493 fully bulgingout from the A site).8,32,33 This conformationalchange is necessary to allow A1492 and A1493 tointeract with the minihelix formed by the cognatecodon–anticodon interaction.32 Aminoglycosides lockthe site in the “on” conformation31,43 so that theribosome has lost its ability to discriminate cognate vsnoncognate tRNA–mRNA associations.32,33 By anal-ogy, RNA targets ought to be chosen in regionsswitching between alternative states, with each stateleading to a different biological action. Such a situa-tion was, for example, encountered in the inhibition ofthe yeast tRNAAsp aminoacylation reaction by amino-glycosides: the binding of tobramycin alters the nativeconformation of the tRNA so that binding of thesynthetase becomes less favorable.95

Interestingly, it is now becoming apparent thatseveral important biological mechanisms are similarlyregulated by molecular switches involving distinctconformational states of RNA molecules.96,97 For ex-ample, small effectors promote up- or downregulationof a particular protein expression by binding to the5�-UTR regions of the corresponding mRNA, therebystabilizing a particular “on” or “off” conforma-tion.97,98 Therefore, the understanding and the identi-fication of such control mechanisms should ultimatelyhelp to identify new potential viral or bacterial targetsfor therapeutic intervention.

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