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Understanding the origins of bacterial resistance to aminoglycosides through molecular dynamics mutational study of the ribosomal A-site.

Romanowska J, McCammon JA, Trylska J - PLoS Comput. Biol. (2011)

Bottom Line: In this study, we performed multiple molecular dynamics simulations of the mutated A-site RNA fragment in explicit solvent to analyze changes in the physicochemical features of the A-site that were introduced by substitutions of specific bases.We found that the specific mutations affect the shape and dynamics of the binding cleft as well as significantly alter its electrostatic properties.The most pronounced changes were observed in the U1406C∶U1495A mutant, where important hydrogen bonds between the RNA and paromomycin were disrupted.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Faculty of Physics, University of Warsaw, Warsaw Poland. jrom@icm.edu.pl

ABSTRACT
Paromomycin is an aminoglycosidic antibiotic that targets the RNA of the bacterial small ribosomal subunit. It binds in the A-site, which is one of the three tRNA binding sites, and affects translational fidelity by stabilizing two adenines (A1492 and A1493) in the flipped-out state. Experiments have shown that various mutations in the A-site result in bacterial resistance to aminoglycosides. In this study, we performed multiple molecular dynamics simulations of the mutated A-site RNA fragment in explicit solvent to analyze changes in the physicochemical features of the A-site that were introduced by substitutions of specific bases. The simulations were conducted for free RNA and in complex with paromomycin. We found that the specific mutations affect the shape and dynamics of the binding cleft as well as significantly alter its electrostatic properties. The most pronounced changes were observed in the U1406C∶U1495A mutant, where important hydrogen bonds between the RNA and paromomycin were disrupted. The present study aims to clarify the underlying physicochemical mechanisms of bacterial resistance to aminoglycosides due to target mutations.

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RMSF [Ångstrom] per residue.PAR denotes paromomycin; (*) the base is cytosine (C) in U1406C/U1495A and U1406C/U1495A_PAR; (**) the base is adenine (A) in G1491A and G1491A_PAR, and uracil (U) in G1491U and G1491U_PAR; (***) this base is adenine (A) in U1406C/U1495A and U1406C/U1495A_PAR, and cytosine (C) in U1495C and U1495C_PAR. The plot also shows the RMSF for the original, prokaryotic A-site structure [33]. Two graphs for each simulation depict RMSF of two symmetric fragments of the structure.
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pcbi-1002099-g002: RMSF [Ångstrom] per residue.PAR denotes paromomycin; (*) the base is cytosine (C) in U1406C/U1495A and U1406C/U1495A_PAR; (**) the base is adenine (A) in G1491A and G1491A_PAR, and uracil (U) in G1491U and G1491U_PAR; (***) this base is adenine (A) in U1406C/U1495A and U1406C/U1495A_PAR, and cytosine (C) in U1495C and U1495C_PAR. The plot also shows the RMSF for the original, prokaryotic A-site structure [33]. Two graphs for each simulation depict RMSF of two symmetric fragments of the structure.

Mentions: We analyzed the flexibility of the entire model A-site by calculating the average root mean square deviation (RMSD) of atomic positions and root mean square fluctuations (RMSF) of each nucleotide as well as paromomycin. The average RMSD from the initial structure, that was calculated for all heavy atoms, did not exceed 2.9 Å in every simulation (Figure S2). Previous studies have shown that paromomycin stabilizes both the wild-type [30], [33] and the A1408G mutated [33] A-site RNA structure (for base numbering see Figure 1a; throughout the paper the E. coli numbering convention of the A-site is used). In this study, we observed a similar stabilizing effect by the presence of paromomycin on the single point mutant structures G1491U and G1491A. A slightly less pronounced effect of paromomycin was also observed in the U1495C simulation. The stabilizing effects are reflected by the RMSF and RMSD values, which are shown in Figure 2 and Figure S2, respectively. For example, in the structure that contains the G1491U mutation without the drug, the bases that were in proximity to the mutated site as well as on the opposite strand of the RNA helix (i.e., A1408 and C1409) showed larger fluctuations, particularly in one section of the RNA fragment. In the presence of the antibiotic, all of the residues became more conformationally restrained.


Understanding the origins of bacterial resistance to aminoglycosides through molecular dynamics mutational study of the ribosomal A-site.

Romanowska J, McCammon JA, Trylska J - PLoS Comput. Biol. (2011)

RMSF [Ångstrom] per residue.PAR denotes paromomycin; (*) the base is cytosine (C) in U1406C/U1495A and U1406C/U1495A_PAR; (**) the base is adenine (A) in G1491A and G1491A_PAR, and uracil (U) in G1491U and G1491U_PAR; (***) this base is adenine (A) in U1406C/U1495A and U1406C/U1495A_PAR, and cytosine (C) in U1495C and U1495C_PAR. The plot also shows the RMSF for the original, prokaryotic A-site structure [33]. Two graphs for each simulation depict RMSF of two symmetric fragments of the structure.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140962&req=5

pcbi-1002099-g002: RMSF [Ångstrom] per residue.PAR denotes paromomycin; (*) the base is cytosine (C) in U1406C/U1495A and U1406C/U1495A_PAR; (**) the base is adenine (A) in G1491A and G1491A_PAR, and uracil (U) in G1491U and G1491U_PAR; (***) this base is adenine (A) in U1406C/U1495A and U1406C/U1495A_PAR, and cytosine (C) in U1495C and U1495C_PAR. The plot also shows the RMSF for the original, prokaryotic A-site structure [33]. Two graphs for each simulation depict RMSF of two symmetric fragments of the structure.
Mentions: We analyzed the flexibility of the entire model A-site by calculating the average root mean square deviation (RMSD) of atomic positions and root mean square fluctuations (RMSF) of each nucleotide as well as paromomycin. The average RMSD from the initial structure, that was calculated for all heavy atoms, did not exceed 2.9 Å in every simulation (Figure S2). Previous studies have shown that paromomycin stabilizes both the wild-type [30], [33] and the A1408G mutated [33] A-site RNA structure (for base numbering see Figure 1a; throughout the paper the E. coli numbering convention of the A-site is used). In this study, we observed a similar stabilizing effect by the presence of paromomycin on the single point mutant structures G1491U and G1491A. A slightly less pronounced effect of paromomycin was also observed in the U1495C simulation. The stabilizing effects are reflected by the RMSF and RMSD values, which are shown in Figure 2 and Figure S2, respectively. For example, in the structure that contains the G1491U mutation without the drug, the bases that were in proximity to the mutated site as well as on the opposite strand of the RNA helix (i.e., A1408 and C1409) showed larger fluctuations, particularly in one section of the RNA fragment. In the presence of the antibiotic, all of the residues became more conformationally restrained.

Bottom Line: In this study, we performed multiple molecular dynamics simulations of the mutated A-site RNA fragment in explicit solvent to analyze changes in the physicochemical features of the A-site that were introduced by substitutions of specific bases.We found that the specific mutations affect the shape and dynamics of the binding cleft as well as significantly alter its electrostatic properties.The most pronounced changes were observed in the U1406C∶U1495A mutant, where important hydrogen bonds between the RNA and paromomycin were disrupted.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Faculty of Physics, University of Warsaw, Warsaw Poland. jrom@icm.edu.pl

ABSTRACT
Paromomycin is an aminoglycosidic antibiotic that targets the RNA of the bacterial small ribosomal subunit. It binds in the A-site, which is one of the three tRNA binding sites, and affects translational fidelity by stabilizing two adenines (A1492 and A1493) in the flipped-out state. Experiments have shown that various mutations in the A-site result in bacterial resistance to aminoglycosides. In this study, we performed multiple molecular dynamics simulations of the mutated A-site RNA fragment in explicit solvent to analyze changes in the physicochemical features of the A-site that were introduced by substitutions of specific bases. The simulations were conducted for free RNA and in complex with paromomycin. We found that the specific mutations affect the shape and dynamics of the binding cleft as well as significantly alter its electrostatic properties. The most pronounced changes were observed in the U1406C∶U1495A mutant, where important hydrogen bonds between the RNA and paromomycin were disrupted. The present study aims to clarify the underlying physicochemical mechanisms of bacterial resistance to aminoglycosides due to target mutations.

Show MeSH
Related in: MedlinePlus