Kasugamycin Function in Thermus thermophilus 30S Ribosomal Subunit: Inhibition of Canonical Translation

Edna Kemboi '16 and Jiayu Chen '17


I. Introduction

Kasugamycin (Ksg) is an antibiotic produced by the bacterium Streptomyces kasugaensis. It inhibits proliferation of bacteria by inhibiting initiation of translation by binding to 16s rRNA, mimicking codon nucleotides at the P- site and E sites and blocking initiator t-RNA binding through perturbation of the mRNA-tRNA codon-anticodon interaction during 30S canonical initiation. The structure of Ksg bound to the T. thermophilus 70S ribosome was determined by X-ray crystallography. This tutorial mainly shows the structure of 30S subunit of ribosome of T. thermophilus. E. coli numbering is used throughout the whole tutorial.

II. Structure of Kasugamycin

Ksg is an aminoglycoside antibiotic with the chemical formula C14H28CIN3O10.Click Here for Structure of Ksg It consists of an acetamidiniumcarboxylate group attached to  an inositol ring (ring I) and a hexopyranosyl ring (ring II).

At both binding sites, the hexopyranosyl ring bridges the backbone of 16S rRNAnucleotides. The inositol ring forms hydrogen bonds with oxygen and nitrogen atoms of nucleotides. Furthermore, the acetaminiumcarboxylate group makes a number of contacts that may involve in hydrogen bond network with either nucleotide or amino acid of ribosomal proteins.  Click Here for Schematics of Ksg1 and Ksg2 interactions with the 30S subunit.

III. Ksg Binding Site

The Ksg-binding pocket is composed entirely of 16S rRNA residues line the mRNA channel of the 30S subunit and protect two nucleotides, the universally conserved, A794 and G926 of 16S rRNA. Also, kasugamycin has two binding site: primary and secondary binding sites.

Two binding sites for Ksg are observed, both of which are on the intersubunit side of the 30S subunit. In the primary binding site, Ksg1 sits at the top of helix 44, spanning the region between h24 and h28 of the 16S rRNAand interacts with G926 in h28 through ring I. Ring II rests on the backbone of a single-stranded region (h44-45), connecting h44 and h45, and the tail reaches towards A792 and A794 in h24. The D-inositol ring of Ksg1 forms hydrogen bonds with N1 and N6 of G926 in h28 and O2P of G1504 in the h44-45 linker, and ring II forms a hydrogen bond to O3 of G1505. The kasugamine tail forms hydrogen bonds with N6 of A792 and N1 of A794 in h24 and is within hydrogen-bonding distance of the backbone of A1499 in h44.

In the secondary position, Ksg2 interacts with the base of G693 in h23 by reaching across the mRNA channel (structure does not show) and with the backbone of Gly81-Gly82 in the beta-hairpin extension of the ribosomal protein S7, while the tail of Ksg2 forms hydrogen bonds with the O2 of U788 and O2P of U789 in h24.

IV. mRNA Binding and Inhibition of Translation

Once Ksg binds to ribosome, its binding sites occupy a position overlapping the kink in the mRNA between the P-site and E-site codons.  Specifically, ring I of Ksg1 encroaches on the ribose in the +1 position of the P-site codon, whereas the kasugamine moiety (ring II and tail) reaches toward the phosphateoxygen and ribose in the +2 and +1 positions of the E-site codon, respectively. Ksg2 is positioned so that ring II mimics the base of a nucleotide at second (-2) position of the E-site codon. (The results were based on programming so there was no actual PDB.) Click Here for binding of mRNA

(E-tRNA and Ksg do not show)
Ksg inhibit P-tRNA binding in an indirect way. P-tRNA protects A794 and G926 of rRNA from chemical modification and the kink between the P- and E-site codons of the 16S 3' end mRNA mimic is stabilized by a hydrogen bond to the N1 of G926, suggesting that the protection of this base by P-tRNA indeed results indirectly from the presence of mRNA. Though the position of Ksg1 does not overlap with that of the P-tRNA, it does overlap with both P- and E-codons.  The overlap disrupts the function of mRNA channel and antiSD sequence which stablize binding between tRNA and mRNA, and makes tRNA leave. Ksg2 overlaps E-codon, competes with E-tRNA and therefore reinforces the disrupt effect on binding.

V. Ksg only Inhibit Canonical Translation

Ksg only inhibits the translation initiation of canonical mRNAs but has no effect on that of leaderless mRNA in vivo. This is because the overlap between the binding position of Ksg and canonical mRNAs is quite extensive in the P and E sites, whereas for initiation with leaderless mRNA, which does not have an antiSD sequence, the overlap is reduced to only one nucleotide position. Click Here for comparison

VI. References

Frank Schluenzen, Chie Takemoto, Daniel N Wilson, Tatsuya Kaminishi, Joerg M Harms, Kyoko Hanawa-Suetsugu, Witold Szaflarski, Masahito Kawazoe, Mikako Shirouzo, Knud H Nierhaus, Shigeyuki Yokoyama & Paola Fucini. 2006. The antibiotic kasugamycin mimics mRNA nucleotides to destabilize tRNA binding and inhibit canonical translation initiation. Nature structural & molecular biology Volume 13: 871-878.

Barbara S Schuwirth, J Michael Day, Cathy W Hau, Gary R Janssen, Albert E Dahlberg, Jamie H Doudna Cate, & AntoŽn Vila-Sanjurjo. 2006. Structural analysis of kasugamycin inhibition of translation. Nature structural & molecular biology. Volume 13: 879-886

Daniel N. Wilson. 2009. The A?Z of bacterial translation inhibitors. Critical Reviews in Biochemistry and Molecular Biology, 2009;44(6): 393-433.

Daniel Boehringer, Heather C. O'Farrell, Jason P. Rife and Nenad Ban. 2006. Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis. . J. Biol. Chem. 2012, 287:10453-10459.

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