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Crystal Structure of the Homo sapiens Cytoplasmic Ribosomal Decoding Site Complexed with Apramycin.

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Zuschriften
Molecular Recognition
DOI: 10.1002/ange.200600354
Crystal Structure of the Homo sapiens
Cytoplasmic Ribosomal Decoding Site
Complexed with Apramycin**
Although the molecular mechanisms of aminoglycosideinduced miscoding in bacterial cells are now well-investigated, the origins of aminoglycoside toxicity in eukaryotic
cells are still debated.[14] Indeed, these compounds are all
toxic to mammals,[1, 15, 16] and the effects in humans are
attributed to the binding of aminoglycosides to the Homo
sapiens cytoplasmic or mitochondrial A sites[17?20] owing to
sequence similarities with those of bacteria (Figure 1 a).
Jiro Kondo, Boris Franois,
Alexandre Urzhumtsev, and
Eric Westhof*
Aminoglycosides are positively charged
oligosaccharides with several ammonium
and hydroxy groups[1] that bind specifically
to bacterial 16S aminoacyl?tRNA (t = transfer) decoding sites (A sites), thereby disturbing the fidelity of the tRNA selection step
during protein synthesis.[2, 3] Various complexes can be formed between 4,5- and 4,6disubstituted aminoglycosides and the 30S
ribosomal particles[4?7] or oligonucleotides
containing the bacterial A-site fragment;[8?12]
3D structures of these complexes were solved
by X-ray crystallographic analysis. Comparative structural analysis showed that a) the
Figure 1. a) Secondary structures of the H. sapiens 18S and the bacterial 16S ribosomal
puckered sugar ring I inserts into the A-site
decoding A sites. The nucleotides of H. sapiens that are different from those in the
helix, stacks onto the G1491 residue, and
bacterial A site are in red. b) Secondary structure of the RNA duplex found in the crystal.
forms two hydrogen bonds with the univerThe unobserved 5?-overhanging uracil residues are in light gray. Geometric nomenclature
sally conserved A1408 residue; b) the central
and symbols for nucleic acid base pairs are according to those in reference [41].
ring II (2-deoxystreptamine; 2-DOS) forms
c) Stereoview of the RNA duplex in complex with apramycin. A1491, A1492, and A1493
are in green, blue, and red, respectively.
four conserved hydrogen bonds, N1 HиииO4(U1495), N3 HиииN7(G1494), N3 HиииO2P(G1494), and N3 HиииO1P(A1493). These
six hydrogen bonds help to maintain the A site in a state in
Apramycin, formerly called nebramycin factor 2,[21] is
which the residues A1492 and A1493 are fully bulged out,
produced by Streptomyces tenebrarius.[22] This antibiotic,
thus allowing them to pack their sugar edges into the shallow/
officially listed as an aminocyclitol because its unusual
minor groove of the first two Watson?Crick base pairs of the
structure contains a bicyclic moiety (Figure 2),[23] is also
codon?anticodon minihelix bound to the 30S ribosome (the
classified as an aminoglycoside owing to its physicochemical
A-minor motif).[13] This conformational state induces misproperties.[24, 25] Apramycin acts against Gram-negative bac[5]
reading of the codon.
teria such as Escherichia coli and Gram-positive bacteria such
as Staphylococcus aureus[24, 25] by inhibiting protein synthesis
after binding to the bacterial A site.[26] This site-specific
[*] Dr. J. Kondo, Dr. B. Fran7ois, Prof. E. Westhof
binding of apramycin was recently revealed by X-ray
Institut de Biologie Mol<culaire et Cellulaire
crystallographic analysis.[27] Apramycin has good potential
Universit< Louis Pasteur
15 rue Ren< Descartes, 67084 Strasbourg (France)
Fax: (+ 33) 3-8860-2218
E-mail: e.westhof@ibmc.u-strasbg.fr
Prof. A. Urzhumtsev
Facult< des Sciences
Universit< Henri Poincar<
Vandoeuvre-lEs-Nancy, 54506 Nancy (France)
[**] J.K. is supported by the Japan Society for the Promotion of Science.
We are grateful to Dr. B. Masquida, Dr. A. C. Vaiana, Dr. P. Auffinger,
and Prof. P. S. Ho for helpful discussions and suggestions. We
thank the European Synchrotron Radiation Facility for the use of
their facilities and acknowledge the staff of beamline ID14-4.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Figure 2. Chemical structure of apramycin.
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. 2006, 118, 3388 ?3392
Angewandte
Chemie
as an antibiotic; unfortunately, its use in mammals is limited
by the relatively high risk of toxicity.[28?31] In fact, it is only
used in veterinary medicine and never in humans.[31] Massspectrometric studies with RNA model fragments showed
that apramycin binds to the eukaryotic 18S ribosomal A site
with a higher affinity (Kd = 0.5 mm) than to the bacterial 16S
site (Kd = 2 0.20 mm).[32] This result strongly suggests that the
binding of apramycin to the eukaryotic cytoplasmic A site
may be the cause of decreasing translation fidelity and
inhibition of translocation of the eukaryotic ribosome. We
determined the 3D structure of the H. sapiens 18S cytoplasmic A site complexed with apramycin at 2.8-@ resolution by
X-ray crystallography.
The asymmetrical loop of the H. sapiens cytoplasmic
A site was inserted between Watson?Crick pairs in RNA
fragments designed to fold as a duplex with two A sites
(Figure 1 b). Such RNA fragments were previously used as
successful models for crystallographic studies to determine
the specific interactions between bacterial A sites and aminoglycosides.[8?12] It was shown that A sites inserted into RNA
fragments present the same footprint as that of the full 16S
rRNA (r = ribosomal).[33?36]
The secondary and overall crystal structure of the RNA
fragment in complex with apramycin is shown in Figure 1 b
and c. Two noncrystallographic-symmetry-related RNA
strands form a pseudosymmetrical duplex. The root-meansquare deviation (RMSD) between two A sites is 0.006 @.
The apramycin molecules bind specifically to the deep/major
groove of both A sites (Figures 1 c and 3). A hexahydrated
magnesium ion is held between the two phosphate backbones
(Figure 3) and is in contact with the atoms of both apramycin
and RNA (Figure 4 a). At the center of the duplex, four
contiguous Watson?Crick G=C base pairs are formed, and
Figure 3. Stereoview of the H. sapiens cytoplasmic A site in complex
with apramycin from two different angles. A1491 (green) and A1493
(red) are protruding, whereas A1492 (blue) is inside the A-site helix.
Angew. Chem. 2006, 118, 3388 ?3392
each G/G stack interacts with a hexammine cobalt cation
(Figure 1 c). Three Watson?Crick G=C base pairs close the
stem at both ends. Two of the four overhanging U residues are
involved in crystal-packing interactions, with the other two
being disordered in the solvent channel. Hereafter, we focus
our description on one A site using the numbering of
H. sapiens cytoplasmic 18S rRNA.
At both ends of the internal loop of the cytoplasmic
A site, four canonical Watson?Crick pairs, C1404=G1497,
G1405=C1496, C1407=G1494, and U1410 A1490, are formed
(Figure 4 b, c, e, h). The U1406 residue forms a symmetric base
pair with U1495 involving the Watson?Crick edges (Figure 4 d). The G1408 residue, which is universally conserved in
all eukaryotic cytoplasmic A sites, does not form a base pair,
but makes two contacts with the phosphate anionic oxygen
atom of A1493, that is, N1 HиииO2P(A1493) (3.3 @) and N2
HиииO2P(A1493) (2.9 @) (Figure 4 f). Consequently, the
A1493 residue protrudes into the solvent region but is not
involved in any crystal-packing interaction (Figures 3 and 4 f).
The C1409 residue forms the Watson?Crick/sugar-edge base
pair with A1492 with three hydrogen bonds, O2иииH O2?(A1492) (3.2 @), N3иииH O2?(A1492) (3.1 @), and N4
HиииN3(A1492) (3.3 @) (Figure 4 g). The A1492 residue also
interacts with the phosphate backbone of A1491 by using its
Hoogsteen edge through N6 HиииO1P(A1491) (3.5 @) (Figure 4 g). The A1491 residue protrudes and does not contact a
symmetrical molecule (Figures 3 and 4 g).
Apramycin binds to the deep/major groove of the A site
and makes 14 direct and seven water-mediated contacts to the
base and phosphate oxygen atoms of the RNA (Figure 4 a).
Ring II (2-DOS) is oriented flat against the base-pair edges in
the deep/major groove of the upper side of the A site
(Figure 3) and interacts with C1404=G1497, G1405=C1496,
and C1407=G1494 through four direct and two watermediated contacts (Figure 4 a?c, e). The N3 atom of ring II
is not involved in any interaction with the A site (Figure 4 a).
The central bicyclic ring I is perpendicular to the central part
of the A site (Figure 3) and makes seven direct and five watermediated contacts with RNA atoms (Figure 4 a). The G1408
residue forms two hydrogen-bond interactions with sugar
ring I through O6(G1408)иииH C3? and O6(G1408)иииH N2?
by using the Watson?Crick edge (Figure 4 f). The N2? atom
also interacts with the phosphate backbone of A1493,
maintaining it in a bulged-out conformation (Figure 4 f). A
hexahydrated magnesium ion is wedged between two phosphate-ribose backbones and bridges ring I (O5? and O6?) to
the phosphate oxygen atoms of the C1404, A1492, and G3
residues (Figure 4 a). Ring III recognizes the Watson?Crick/
sugar-edge C1409 A1492 base pair from the deep/major
groove and forms the O5??иииH N6(A1492) hydrogen bond
with the Watson?Crick edge of A1492 (Figure 4 g). This
interaction forces A1492 inside the RNA duplex.
The binding mode of apramycin to the H. sapiens
cytoplasmic A site is surprisingly different from that observed
in the analogous bacterial complex.[27] The orientation of
apramycin in the bacterial A site is rotated back to front with
respect to that in the H. sapiens cytoplasmic A site (see
Supporting Information). Apramycin penetrates the internal
loop of the bacterial A site from the deep/major to the
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Zuschriften
Figure 4. Description and analysis of the contacts between apramycin and the H. sapiens cytoplasmic A site. a) Structure adopted by the
apramycin molecule inside the H. sapiens cytoplasmic A site. Ring numbers (I?III) and atom names are specified. The H. sapiens cytoplasmic
numbering is used for the RNA atoms; W = water molecule, Mg = magnesium ion. Direct and water-mediated contacts between apramycin and
the minimal A site are shown as red and black dashed lines, respectively. b?h) Atomic details of the contacts involving each base pair of the
minimal A site interacting with apramycin. A1491, A1492, and A1493 are in green, blue, and red, respectively. Hydrogen bonds are represented by
black dashed lines. The C HиииO hydrogen bonds are also indicated.
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2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. 2006, 118, 3388 ?3392
Angewandte
Chemie
shallow/minor groove and forces both A1492 and A1493 to
bulge out. The number of direct contacts between apramycin
and the bacterial A site (nine) is slightly less than that
observed in the H. sapiens cytoplasmic complex (14) (Figure 4 a and Supporting Information). This result agrees with
mass-spectrometric studies, which show that apramycin binds
to the eukaryotic 18S ribosomal A site in the absence of
magnesium ions with a higher affinity (Kd = 0.5 mm) than to
the bacterial site (Kd = 2 0.20 mm).[32] Ring II makes three
direct contacts with U1406 U1495, C1407=G1494, and the
protruding A1493 of the bacterial A site through the N1 and
N3 atoms. The O5 atom, which interacts with G1494 in the
cytoplasmic A site (Figure 4 a, e), is not bound to the bacterial
A site. Ring I stacks onto G1491 of the Watson?Crick C1409=
G1491 base pair and forms a sugar?base-pair interaction with
the universally conserved A1408 of the bacterial A site with
O5?иииH N6(A1408) and O6? HиииN1(A1408). In contrast, in
the H. sapiens cytoplasmic complex, O5? and O6? are bound to
three of the six hydration water molecules of the magnesium
ion. The A1492 and A1493 residues of the bacterial A site are
thus forced to protrude from the RNA helix. The N2? atom,
which binds to G1408 of the cytoplasmic A site (Figure 4 a, f),
is free from any interactions in the bacterial complex. In other
words, ring I forms sugar?base-pair interactions with the
bacterial A1408 and the cytoplasmic G1408 residues through
different edges. The terminal ring III, which forms a sugar?
base interaction with the C1409*A1492 base pair of the
cytoplasmic A site (Figure 4 g), recognizes the C1409=G1491
Watson?Crick base pair of the bacterial A site in the shallow/
minor groove through O5??иииH N2(G1491) and O2?иииH
O2?(G1491) sugar?base interactions. This comparative structural analysis shows that apramycin specifically recognizes
both the bacterial and the H. sapiens cytoplasmic A sites, but
uses different interaction modes that involve two changes in
the sequence: a) A1408 (bacterial) and G1408 (cytoplasmic)
for the interactions between ring I and residue 1408; b) the
C1409=G1491 (bacterial) and C1409*A1492 (cytoplasmic)
pairs for the interactions with ring III. Because of the
important contribution of electrostatics to the binding
strength of aminoglycosides to RNA,[37, 38] it is interesting to
compare the contacts made by the positively charged
ammonium groups in the two complexes. In both cases,
three ammonium groups have no contact with the RNA; of
those, N8? (ring I) and N4?? (ring III) are common. While N3
(ring II) has no contact in the H. sapiens cytoplasmic complex
and N2? (ring I) forms several hydrogen bonds, the opposite is
observed in the bacterial complex. The N1 atom (ring II)
forms either a water-mediated or a direct hydrogen bond with
the RNA, albeit not to the same atom. Although the initial
concentrations of magnesium ions in the crystallization drops
are not much different (1.25 mm in the H. sapiens cytoplasmic
and 1.00 mm in the bacterial complex), one magnesium ion is
clearly trapped only in the H. sapiens cytoplasmic complex.
Recently, we reported the crystal structure of the H. sapiens cytoplasmic A site without any bound aminoglycosides.[39]
In that structure, two conformational states, the ?on? (A1492/
A1493 bulging out and A1491 tucked in) and ?off? (A1491/
A1393 bulging out and A1492 tucked in), were observed. The
conformation of the cytoplasmic A site in the present RNA/
Angew. Chem. 2006, 118, 3388 ?3392
apramycin complex is almost the same as the ?off? state of the
free structure (RMSD = 1.0 @). Therefore, it can be concluded that apramycin stabilizes the ?off? state of the
H. sapiens cytoplasmic A site.
In the absence of an eukaryotic cytoplasmic ribosome,
superimposition of the H. sapiens cytoplasmic A site in
complex with apramycin on the crystal structure of the 30S
ribosome in complex with mRNA (m = messenger), tRNA,
and paromomycin[6] yields some insight into the molecular
mechanisms of apramycin toxicity in mammals. The A1493
residue, which monitors the first Watson?Crick base pair of
the codon?anticodon helix, protrudes toward the mRNA/
tRNA complex (Figure 5). However, A1493 is too far from
Figure 5. Stereoview of the superimposition of the H. sapiens cytoplasmic A site complexed with apramycin (PDB ID: 2G5K) on the 30S
ribosomal decoding site (PDB ID: 1IBL).[6] A1491, A1492, and A1493
are colored as in the other figures. Ribosomal protein S12, tRNA, and
mRNA are in green, cyan, and orange, respectively.
the codon?anticodon base pairs to form an A-minor motif.
Since A1492 is inside the A-site helix in the apramycin
complex, it cannot recognize the second Watson?Crick base
pair. Furthermore, the bulged-out A1491 residue is in close
contact with ribosomal protein S12, which is known as a
control element for translocation of the mRNA/tRNA
complex (Figure 5).[40] In the bacterial A site, ring III of
apramycin is in close contact with S12 and may play the same
role as A1491 in the complex of the cytoplasmic A site.[27]
Therefore, in 40S cytoplasmic ribosomes, A1491 may disturb
the local conformation of a cytoplasmic ribosomal protein,
thereby inhibiting translocation of the eukaryotic ribosome.
In a previous paper, we showed how a modification of the
aminoglycoside paromomycin led to a conformational change
and a better binding mode of the drug to the same
conformation of the A site.[11] The present results show how
the same drug binds to different conformations of the A site
following a change of two nucleotides in the sequence. The
observed surprising adaptability of both the binding mode of
the drug (aminoglycosides) and the conformation of the
target (A site) constitutes a challenge for drug design and the
understanding of toxicity.
Received: January 26, 2006
Published online: April 5, 2006
.
Keywords: aminoglycosides и antibiotics и decoding site и
molecular recognition и RNA structures
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.de
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