Ribosomal Antibiotics: Contemporary Challenges.

Most ribosomal antibiotics obstruct distinct ribosomal functions. In selected cases, in addition to paralyzing vital ribosomal tasks, some ribosomal antibiotics are involved in cellular regulation. Owing to the global rapid increase in the appearance of multi-drug resistance in pathogenic bacterial strains, and to the extremely slow progress in developing new antibiotics worldwide, it seems that, in addition to the traditional attempts at improving current antibiotics and the intensive screening for additional natural compounds, this field should undergo substantial conceptual revision. Here, we highlight several contemporary issues, including challenging the common preference of broad-range antibiotics; the marginal attention to alterations in the microbiome population resulting from antibiotics usage, and the insufficient awareness of ecological and environmental aspects of antibiotics usage. We also highlight recent advances in the identification of species-specific structural motifs that may be exploited for the design and the creation of novel, environmental friendly, degradable, antibiotic types, with a better distinction between pathogens and useful bacterial species in the microbiome. Thus, these studies are leading towards the design of "pathogen-specific antibiotics," in contrast to the current preference of broad range antibiotics, partially because it requires significant efforts in speeding up the discovery of the unique species motifs as well as the clinical pathogen identification.

Structure of trigger factor binding domain in biologically homologous complex with eubacterial ribosome reveals its chaperone action.

Trigger factor (TF), the first chaperone in eubacteria to encounter the emerging nascent chain, binds to the large ribosomal subunit in the vicinity of the protein exit tunnel opening and forms a sheltered folding space. Here, we present the 3.5-A crystal structure of the physiological complex of the large ribosomal subunit from the eubacterium Deinococcus radiodurans with the N-terminal domain of TF (TFa) from the same organism. For anchoring, TFa exploits a small ribosomal surface area in the vicinity of proteins L23 and L29, by using its "signature motif" as well as additional structural elements. The molecular details of TFa interactions reveal that L23 is essential for the association of TF with the ribosome and may serve as a channel of communication with the nascent chain progressing in the tunnel. L29 appears to induce a conformational change in TFa, which results in the exposure of TFa hydrophobic patches to the opening of the ribosomal exit tunnel, thus increasing its affinity for hydrophobic segments of the emerging nascent polypeptide. This observation implies that, in addition to creating a protected folding space for the emerging nascent chain, TF association with the ribosome prevents aggregation by providing a competing hydrophobic environment and may be critical for attaining the functional conformation necessary for chaperone activity.

Inhibition of peptide bond formation by pleuromutilins: the structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin.

Tiamulin, a prominent member of the pleuromutilin class of antibiotics, is a potent inhibitor of protein synthesis in bacteria. Up to now the effect of pleuromutilins on the ribosome has not been determined on a molecular level. The 3.5 A structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin provides for the first time a detailed picture of its interactions with the 23S rRNA, thus explaining the molecular mechanism of the antimicrobial activity of the pleuromutilin class of antibiotics. Our results show that tiamulin is located within the peptidyl transferase center (PTC) of the 50S ribosomal subunit with its tricyclic mutilin core positioned in a tight pocket at the A-tRNA binding site. Also, the extension, which protrudes from its mutilin core, partially overlaps with the P-tRNA binding site. Thereby, tiamulin directly inhibits peptide bond formation. Comparison of the tiamulin binding site with other PTC targeting drugs, like chloramphenicol, clindamycin and streptogramins, may facilitate the design of modified or hybridized drugs that extend the applicability of this class of antibiotics.

Ribosomal crystallography: a flexible nucleotide anchoring tRNA translocation, facilitates peptide-bond formation, chirality discrimination and antibiotics synergism.

The linkage between internal ribosomal symmetry and transfer RNA (tRNA) positioning confirmed positional catalysis of amino-acid polymerization. Peptide bonds are formed concurrently with tRNA-3' end rotatory motion, in conjunction with the overall messenger RNA (mRNA)/tRNA translocation. Accurate substrate alignment, mandatory for the processivity of protein biosynthesis, is governed by remote interactions. Inherent flexibility of a conserved nucleotide, anchoring the rotatory motion, facilitates chirality discrimination and antibiotics synergism. Potential tRNA interactions explain the universality of the tRNA CCA-end and P-site preference of initial tRNA. The interactions of protein L2 tail with the symmetry-related region periphery explain its conservation and its contributions to nascent chain elongation.

Parametric description of cardiac vagal control.

Heart rate variability (HRV) indices that reflect the magnitude of respiratory sinus arrhythmia (RSA) are commonly applied as non-invasive measures of cardiac vagal control. Recently, however, serious doubts have been raised about the accuracy and validity of such assessments. To evaluate these methods, we derived a theoretical model for the dependence of mean heart rate and RSA on gradual vagal blockade by atropine, and compared its predictions to actual experimental results. The experiment involved the injection of nine consecutive intravenous bolus doses of atropine to eight young healthy male subjects. Seven-minute recordings of ECG and respiration were made for each atropine dose. The heart rate (HR) signal was derived from the ECG recording, and mean heart rate and the power of the high frequency peak of HRV (which measures the magnitude of RSA) were computed. The experimental data were fitted to the model's equations, and optimal values were obtained for the model's parameters. A tight agreement is observed between the theoretical fitted curves and the experimental data. The parameters that were computed from fitting the experimental data to the mean heart rate equation display a surprisingly small variance among the different subjects. The parameters that were computed from fitting the experimental data to the RSA equation, and the resulting shape of these fitted curves, explain many of the conflicting results previously published, and provide a new quantitative insight to cardiac vagal activity.

Do the high-frequency indexes of HRV provide a faithful assessment of cardiac vagal tone? A critical theoretical evaluation.

When spectral analysis of the heart rate (HR) signal is performed, it is quite common to attribute the HF indexes of heart rate variability (HRV) to cardiac vagal control. The paradigm underlying this attribution states that changes in cardiac vagal outflow correspond to a proportional change in respiratory sinus arrhythmia (RSA). However, recent studies have demonstrated that variations in these indexes do not necessarily reflect proportional changes in vagal tone. The current study provides a theoretical evaluation of the relationship between mean HR, RSA, and cardiac vagal tone. This evaluation is based on a theoretical model, which quantifies the differential effects of vagal blockade by a competitive muscarinic antagonist on the HF indexes of HRV. The model relies on several assumptions that reflect the basic physiology of the sinoatrial (SA) node, as well as pharmacological relations that describe agonist/antagonist equilibrium at the SA receptors. The mathematical framework of this model is the integral-pulse-frequency-modulation (IPFM) process, and its derivations lead to a specific expressions for the dependence of HF and mean HR on the level of vagal blockade. These expressions provide a new insight into the relationship between mean HR, RSA, and vagal tone, and explain conflicting experimental results previously published.