Crystal structures of complexes between aminoglycosides and decoding A site oligonucleotides: role of the number of rings and positive charges in the specific binding leading to miscoding

Abstract

The crystal structures of six complexes between aminoglycoside antibiotics (neamine, gentamicin C1A, kanamycin A, ribostamycin, lividomycin A and neomycin B) and oligonucleotides containing the decoding A site of bacterial ribosomes are reported at resolutions between 2.2 and 3.0 A. Although the number of contacts between the RNA and the aminoglycosides varies between 20 and 31, up to eight direct hydrogen bonds between rings I and II of the neamine moiety are conserved in the observed complexes. The puckered sugar ring I is inserted into the A site helix by stacking against G1491 and forms a pseudo base pair with two H-bonds to the Watson-Crick sites of the universally conserved A1408. This central interaction helps to maintain A1492 and A1493 in a bulged-out conformation. All these structures of the minimal A site RNA complexed to various aminoglycosides display crystal packings with intermolecular contacts between the bulging A1492 and A1493 and the shallow/minor groove of Watson-Crick pairs in a neighbouring helix. In one crystal, one empty A site is observed. In two crystals, two aminoglycosides are bound to the same A site with one bound specifically and the other bound in various ways in the deep/major groove at the edge of the A sites.

Figure 1

Chemical structures of the three aminoglycoside subclasses studied in this work. The common 2-deoxystreptamine ring is coloured in blue and the substituted positions are numbered in bold.

Figure 2

Left: secondary structure of the minimal A site. The standard E.coli numbering is used. Right: secondary structure of the crystallized RNA helix. The two A sites are boxed and the 5′-dangling UU, usually not observed in the density maps, are shaded.

Figure 3

Description and analysis of the contacts between gentamicin C1A and the RNA fragment. (A) Structure adopted by the gentamicin C1A molecule inside the A site of the duplex. Ring numbers (I–III) and atom names are specified. The E.coli numbering is used for the RNA atoms, and ‘W’ stands for water molecule. Hydrogen bonds are shown as dashed lines. Conserved contacts between gentamicin C1A and the minimal A site are shown in red. (B–F) Atomic details of the contacts involving each base pair of the minimal A site interacting with gentamicin C1A.

Figure 4

Description and analysis of the contacts between neomycin B and the RNA fragment. (A) Structure adopted by the neomycin B molecule inside the A site of the duplex. Ring numbers (I–IV) and atom names are specified. The E.coli numbering is used for the RNA atoms, and ‘W’ stands for water molecule. Hydrogen bonds are shown as dashed lines. Conserved contacts between neomycin B and the minimal A site are shown in red. (B–F) Atomic details of the contacts involving each base pair of the minimal A site interacting with neomycin B.

Figure 5

Description and analysis of the contacts between lividomycin A and the RNA fragment. (A) Structure adopted by the lividomycin A molecule inside the A site of the duplex. Ring numbers (I–V) and atom names are specified. The E.coli numbering is used for the RNA atoms, and ‘W’ stands for water molecule. Hydrogen bonds are shown as dashed lines. Conserved contacts between lividomycin A and the minimal A site are shown in red. (B–F) Atomic details of the contacts involving each base pair of the minimal A site interacting with lividomycin A.

Figure 6

Specific and ‘non-specific’ binding modes of aminoglycosides. (A) Overall view of crystallized kanamycin A complex. (B) Overall view of crystallized ribostamycin complex. In both structures, the specifically bound antibiotics are coloured in yellow ball and sticks and the ‘non-specifically’ bound antibiotics are coloured in cyan ball and sticks.

Figure 7

Specific binding of kanamycin A. (A) Structure adopted by the specific kanamycin molecule coloured in yellow ball and sticks inside the A site of the duplex. Ring numbers (I–III) and atom names are specified. The E.coli numbering is used for the RNA atoms, and ‘W’ stands for water molecule. Hydrogen bonds are shown in dashed lines. Conserved contacts between specific kanamycin and the minimal A site are shown in red. (B–F) Atomic details of the contacts involving each base pair of the minimal A site interacting with the two kanamycin molecules. (G) Position of the non-specifically bound kanamycin (coloured in cyan ball and sticks) at the entrance of the deep/major groove of the A site duplex. The specific molecule of kanamycin is coloured in yellow lines. (H) Contacts made by the ‘non-specific’ kanamycin, Ring numbers (I–III) and atom names are specified. The E.coli numbering is used for the RNA atoms, and ‘W’ stands for water molecule. Hydrogen bonds are shown as dashed lines.

Figure 8

Specific and ‘non-specific’ binding of ribostamycin. (A) Structure adopted by the specific ribostamycin molecule coloured in yellow ball and sticks inside the A site of the duplex. Ring numbers (I–III) and atom names are specified. The E.coli numbering is used for the RNA atoms, and ‘W’ stands for water molecule. Hydrogen bonds are shown as dashed lines. (B–C) Conserved contacts between specific kanamycin and the first two base pairs of the minimal A site are shown in red. (D–F) Atomic details of the contacts involving the base pairs of the minimal A site interacting with the two ribostamycin molecules. (G) Position of ribostamycin non-specifically bound (coloured in cyan ball and sticks) at the entrance of the deep/major groove of the duplex. The specifically bound molecule of ribostamycin is coloured in yellow lines. (H) Contacts made by the ‘non-specific’ ribostamycin, Ring numbers (I–III) and atom names are specified. The E.coli numbering is used for the RNA atoms, and ‘W’ stands for water molecule. Hydrogen bonds are shown as dashed lines.

Figure 9

Description and analysis of the contacts between neamine and the RNA fragment. (A) Overall view of the crystallized complex. Site 1 contains one neamine in yellow. In both sites, A1408 are coloured in dark blue, A1492 in green, A1493 in cyan, U1406 in orange (which makes intermolecular contacts with C1405-G1491) and U1495 in magenta [which makes contacts with A1492 of site 2 of a symmetric molecule, see (D)]. (B) Ring numbers (I and II) and atom names are specified. The E.coli numbering is used for the RNA atoms, and ‘W’ stands for water molecule. Hydrogen bonds are shown as dashed lines. Conserved contacts between neamine and the minimal A Site are shown in red. (C–F) Atomic details of the contacts involving each base pair interacting with neamine in site 1.

Figure 10

Description of the empty A site of the crystallized neamine complex. (A) Overall view of site 2. A1408 is coloured in dark blue, A1492 in green, A1493 in cyan, U1406 in orange and U1495 in magenta. (B and C) Atomic details of the base pair interactions into the empty minimal A site. Hydrogen bonds are shown as dashed lines.

Figure 11

Three-dimensional structure of the packing interactions observed in the neomycin B crystal at the central Watson–Crick domain of the duplex. This view shows a central duplex interacting with two symmetry-related duplexes via the bulged adenines. In both A sites, A1492 are coloured in green, A1493 in cyan and red spheres indicate water molecules. Close-up of the interactions between each adenine and its counterpart G = C pair (on the right magenta in site 1, top of central drawing and, on the left orange in site 2, bottom of the central drawing). The hydrogen bonds are represented by black dashed lines.