Investigation of radezolid interaction with non-canonical chloramphenicol binding site by molecular dynamics simulations.

Radezolid is a promising antibiotic of oxazolidinone family, which is able to overcome effect of some linezolid resistance mechanisms of bacterial ribosomes. The structure of the radezolid complex with ribosomes was never published but, by analogy with linezolid, it is considered to prevent the binding of aminoacyl-tRNA to the A-site of the ribosome large subunit. However, as with linezolid, it can be assumed that radezolid binds to the alternative binding site existing in the A,A/P,P-ribosome. In the present article we have investigated this issue by molecular dynamics simulations and proposed the structure of the radezolid complex with a E. coli ribosome, which is consistent with available data of biochemical investigations of radezolid action.

Investigation of Allosteric Effect of 2,8-Dimethylation of A2503 in E. coli 23S rRNA by Molecular-Dynamics Simulations.

Ribosome is a molecular machine that synthesizes all cellular proteins. It also is a target of about half of the clinically used antibiotics. Adaptive chemical modification of ribosomal RNAs residues is one of the ways to provide resistance to certain antibiotics. A curious example of such modification is 2,8-dimethylation of A2503 in 23S rRNA, which induces resistance to phenols, linkosamides, oxazolidinones, pleuromutilins, and certain macrolides. In this article the effect of 2,8-dimethylation of A2503 on conformation and mobility of RNA residues of the 70S E. coli ribosome was investigated employing molecular dynamics simulations method. Significant alterations were detected both in the immediate environment of the 2503 23S rRNA residue and in the nucleotides located deeper in the nascent peptide exit tunnel (NPET), which are known to be involved in signal transmission from the antibiotics bound in the NPET to the peptidyl transferase center. These alterations shift the ribosome towards the A/A, P/P-state from the conformationally different state - P/P, E/E one in our case. The obtained results allow us to conclude that the effect of m2m8A2503 modification involves additional stabilization of the A/A, P/P-state favoring the peptidyl transferase reaction (PTR) contrary to antibiotics that inhibit PTR.

A noncanonical binding site of linezolid revealed via molecular dynamics simulations.

Linezolid, an antibiotic of oxazolidinone family, is a translation inhibitor. The mechanism of its action that consists in preventing the binding of aminoacyl-tRNA to the A-site of the large subunit of a ribosome was embraced on the basis of the X-ray structural analysis of the linezolid complexes with vacant bacterial ribosomes. However, the known structures of the linezolid complexes with bacterial ribosomes poorly explain the linezolid selectivity in suppression of protein biosynthesis, depending on the amino acid sequence of the nascent peptide. In the present study the most probable structure of the linezolid complex with a E. coli ribosome in the A,A/P,P-state that is in line with the results of biochemical studies of linezolid action has been obtained by molecular dynamics simulation methods.

A noncanonical binding site of chloramphenicol revealed via molecular dynamics simulations.

Chloramphenicol, an antibiotic belonging to the family of amphenicols, is an inhibitor of translation. On the basis of X-ray structural analysis of the binding of chloramphenicol to free bacterial ribosomes, the chloramphenicol action mechanism that consists in preventing the binding of aminoacyl-tRNA to the A-site of the large subunit of the ribosome was adopted. However, the known structures of chloramphenicol complexes with bacterial ribosomes poorly explain the results of the experiments on the chemical modification of 23S rRNA, the resistance to chloramphenicol caused by mutations in 23S rRNA and, which is particularly important, the selectivity of chloramphenicol in suppression of translation, depending on the amino acid sequence of the nascent peptide. In the present study the putative structure of the chloramphenicol complex with a bacterial ribosome in the A,A/P,P-state has been obtained by molecular dynamics simulations methods. The proposed structure of the complex allows us to explain the results of biochemical studies of the interaction of chloramphenicol with the bacterial ribosome.

Structural Insight into Interaction between C20 Phenylalanyl Derivative of Tylosin and Ribosomal Tunnel.

Macrolides are clinically important antibiotics that inhibit protein biosynthesis on ribosomes by binding to ribosomal tunnel. Tylosin belongs to the group of 16-membered macrolides. It is a potent inhibitor of translation whose activity is largely due to reversible covalent binding of its aldehyde group with the base of A2062 in 23S ribosomal RNA. It is known that the conversion of the aldehyde group of tylosin to methyl or carbinol groups dramatically reduces its inhibitory activity. However, earlier we obtained several derivatives of tylosin having comparable activity in spite of the fact that the aldehyde group of tylosin in these compounds was substituted with an amino acid or a peptide residue. Details of the interaction of these compounds with the ribosome that underlies their high inhibitory activity were not known. In the present work, the structure of the complex of tylosin derivative containing in position 20 the residue of ethyl ester of 2-imino(oxy)acetylphenylalanine with the tunnel of the E. coli ribosome was identified by means of molecular dynamics simulations, which could explain high biological activity of this compound.

Investigation of Ribosomes Using Molecular Dynamics Simulation Methods.

The ribosome as a complex molecular machine undergoes significant conformational changes while synthesizing a protein molecule. Molecular dynamics simulations have been used as complementary approaches to X-ray crystallography and cryoelectron microscopy, as well as biochemical methods, to answer many questions that modern structural methods leave unsolved. In this review, we demonstrate that all-atom modeling of ribosome molecular dynamics is particularly useful in describing the process of tRNA translocation, atomic details of behavior of nascent peptides, antibiotics, and other small molecules in the ribosomal tunnel, and the putative mechanism of allosteric signal transmission to functional sites of the ribosome.

Molecular Dynamics Investigation of a Mechanism of Allosteric Signal Transmission in Ribosomes.

The ribosome is a molecular machine that synthesizes all cellular proteins via translation of genetic information encoded in polynucleotide chain of messenger RNA. Transition between different stages of the ribosome working cycle is strictly coordinated by changes in structure and mutual position both of subunits of the ribosome and its ligands. Therein, information regarding structural transformations is transmitted between functional centers of the ribosome through specific signals. Usually, functional centers of ribosomes are located at a distance reaching up to several tens of angstroms, and it is believed that such signals are transduced allosterically. In our study, we attempted to answer the question of how allosteric signal can be transmitted from one of the so-called sensory elements of ribosomal tunnel (RT) to the peptidyl transferase center (PTC). A segment of RT wall from the E. coli ribosome composed of nucleotide residues A2058, A2059, m(2)A2503, G2061, A2062, and C2063 of its 23S rRNA was examined by molecular dynamics simulations. It was found that a potential signal transduction pathway A2058-C2063 acted as a dynamic ensemble of interdependent conformational states, wherein cascade-like changes can occur. It was assumed that structural rearrangement in the A2058-C2063 RT segment results in reversible inactivation of PTC due to a strong stacking contact between functionally important U2585 residue of the PTC and nucleotide residue C2063. A potential role for the observed conformational transition in the A2058-C2063 segment for regulating ribosome activity is discussed.

Modeling Interactions of Erythromycin Derivatives with Ribosomes.

Using a method of static simulation, a series of erythromycin A analogs was designed with aldehyde functions introduced instead of one of the methyl substituents in the 3'-N-position of the antibiotic that was potentially capable of forming a covalent bond with an amino group of one of the nucleotide residues of the 23S rRNA in the ribosomal exit tunnel. Similar interaction is observed for antibiotics of the tylosin series, which bind tightly to the large ribosomal subunit and demonstrate high antibacterial activity. Binding of novel erythromycin derivatives with the bacterial ribosome was investigated with the method of fluorescence polarization. It was found that the erythromycin analog containing a 1-methyl-3-oxopropyl group in the 3'-N-position demonstrates the best binding. Based on the ability to inhibit protein biosynthesis, it is on the same level as erythromycin, and it is significantly better than desmethyl-erythromycin. Molecular dynamic modeling of complexes of the derivatives with ribosomes was conducted to explain the observed effects.