Abstract OR-4: New Antibiotic Binding Site on the 30S Ribosomal Subunit

Background: Antibiotic resistance becomes one of the main problems of modern medicine; therefore, the development of new antibacterial compounds is absolutely necessary. The ribosome is the target for a lot of different antibiotics; there are several main binding sites on the ribosome – decoding center, peptidyl-transferase center, and ribosome exit tunnel. Modification or mutation of nucleotides in these sites could make cells resistant to structurally different antibiotics. Methods: pDualrep2 reporter system was used for detection of the protein synthesis inhibitors in cultural broths of new soil bacteria. By means of a cell-free translation system, the inhibitory activity and mechanism of action of Auraplanin were estimated. CryoEM data collection was performed on a Titan Krios operated at 300 kV, equipped with a Falcon II direct electron detector. Results: In this work, we have found a new inhibitor of protein synthesis, which binds in a completely new binding site. This compound is produced by Actinoplanes sp. VKM Ac-2862 and by Cryo-EM study of its complex with E.coli ribosome, it was shown, that it binds close to 560 loop of 30S ribosomal subunit. The new compound is a derivative of tetramic acid and we called it Auraplanin, because of bright orange color of the producer strain. Structural data are in good agreement with genetic results – resistant mutations were located close determined binding site. Substitutions C564G, G558U, and G566A significantly increase minimal inhibitory concentration, all these mutations were not detected previously. We also observed resistant mutation in ribosomal protein S4, this mutation was previously identified as error-prone. Interestingly, ribosomal ambiguity mutations, G299A and G347U, also increased resistance to Auraplanin. Conclusion: On the basis of the genetic, structural and biochemical studies we hypothesized that Auraplanin acts prevent the transfer from an open to a closed conformation of 30S subunit, in contrast to streptomycin, which promotes the formation of a closed state.

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Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit

The Journal of Cell Biology ◽

10.1083/jcb.201001124 ◽

2010 ◽

Vol 189 (7) ◽

pp. 1079-1086 ◽

Cited By ~ 49

Author(s):

Jayati Sengupta ◽

Cyril Bussiere ◽

Jesper Pallesen ◽

Matthew West ◽

Arlen W. Johnson ◽

...

Keyword(s):

Binding Site ◽

Nuclear Export ◽

Large Subunit ◽

Ribosomal Subunit ◽

Adaptor Protein ◽

Structural Data ◽

Release Mechanism ◽

Nuclear Pore ◽

Subunit Joining

The nucleocytoplasmic shuttling protein Nmd3 is an adaptor for export of the 60S ribosomal subunit from the nucleus. Nmd3 binds to nascent 60S subunits in the nucleus and recruits the export receptor Crm1 to facilitate passage through the nuclear pore complex. In this study, we present a cryoelectron microscopy (cryo-EM) reconstruction of the 60S subunit in complex with Nmd3 from Saccharomyces cerevisiae. The density corresponding to Nmd3 is directly visible in the cryo-EM map and is attached to the regions around helices 38, 69, and 95 of the 25S ribosomal RNA (rRNA), the helix 95 region being adjacent to the protein Rpl10. We identify the intersubunit side of the large subunit as the binding site for Nmd3. rRNA protection experiments corroborate the structural data. Furthermore, Nmd3 binding to 60S subunits is blocked in 80S ribosomes, which is consistent with the assigned binding site on the subunit joining face. This cryo-EM map is a first step toward a molecular understanding of the functional role and release mechanism of Nmd3.

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Negamycin Binds to the Wall of the Nascent Chain Exit Tunnel of the 50S Ribosomal Subunit

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00455-07 ◽

2007 ◽

Vol 51 (12) ◽

pp. 4462-4465 ◽

Cited By ~ 27

Author(s):

Susan J. Schroeder ◽

Gregor Blaha ◽

Peter B. Moore

Keyword(s):

Protein Synthesis ◽

Small Molecule ◽

Ribosomal Subunit ◽

Small Molecule Inhibitor ◽

Single Site ◽

Haloarcula Marismortui ◽

Molecule Inhibitor ◽

Nascent Chain ◽

Exit Tunnel ◽

Inhibitor Of Protein Synthesis

ABSTRACT Negamycin, a small-molecule inhibitor of protein synthesis, binds the Haloarcula marismortui 50S ribosomal subunit at a single site formed by highly conserved RNA nucleotides near the cytosolic end of the nascent chain exit tunnel. The mechanism of antibiotic action and the function of this unexplored tunnel region remain intriguingly elusive.

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Erythromycin- and Chloramphenicol-Induced Ribosomal Assembly Defects Are Secondary Effects of Protein Synthesis Inhibition

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00870-08 ◽

2008 ◽

Vol 53 (2) ◽

pp. 563-571 ◽

Cited By ~ 45

Author(s):

Triinu Siibak ◽

Lauri Peil ◽

Liqun Xiong ◽

Alexander Mankin ◽

Jaanus Remme ◽

...

Keyword(s):

Protein Synthesis ◽

Ribosomal Subunit ◽

Protein Synthesis Inhibition ◽

Large Ribosomal Subunit ◽

Secondary Effects ◽

Protein Synthesis Inhibitors ◽

Erythromycin Resistance ◽

Ribosomal Assembly ◽

Direct Binding ◽

Assembly Defect

ABSTRACT Several protein synthesis inhibitors are known to inhibit ribosome assembly. This may be a consequence of direct binding of the antibiotic to ribosome precursor particles, or it could result indirectly from loss of coordination in the production of ribosomal components due to the inhibition of protein synthesis. Here we demonstrate that erythromycin and chloramphenicol, inhibitors of the large ribosomal subunit, affect the assembly of both the large and small subunits. Expression of a small erythromycin resistance peptide acting in cis on mature ribosomes relieves the erythromycin-mediated assembly defect for both subunits. Erythromycin treatment of bacteria expressing a mixture of erythromycin-sensitive and -resistant ribosomes produced comparable effects on subunit assembly. These results argue in favor of the view that erythromycin and chloramphenicol affect the assembly of the large ribosomal subunit indirectly.

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Activity of RX-04 Pyrrolocytosine Protein Synthesis Inhibitors against Multidrug-Resistant Gram-Negative Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00689-18 ◽

2018 ◽

Vol 62 (8) ◽

Cited By ~ 2

Author(s):

Anna Vickers ◽

Shazad Mushtaq ◽

Neil Woodford ◽

Michel Doumith ◽

David M. Livermore

Keyword(s):

Pseudomonas Aeruginosa ◽

Protein Synthesis ◽

Acinetobacter Baumannii ◽

Ribosomal Subunit ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Protein Synthesis Inhibitors ◽

Gram Negative ◽

Content Type ◽

Active Analog

ABSTRACT Pyrrolocytosines RX-04A to -D are designed to bind to the bacterial 50S ribosomal subunit differently from currently used antibiotics. The four analogs had broad anti-Gram-negative activity: RX-04A—the most active analog—inhibited 94.7% of clinical Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa at 0.5 to 4 μg/ml, with no MICs of >8 μg/ml. MICs for multidrug-resistant (MDR) carbapenemase producers were up to 2-fold higher than those for control strains; values were highest for one Serratia isolate with porin and efflux lesions. mcr-1 did not affect MICs.

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Fragments of the Nonlytic Proline-Rich Antimicrobial Peptide Bac5 KillEscherichia coliCells by Inhibiting Protein Synthesis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00534-18 ◽

2018 ◽

Vol 62 (8) ◽

Cited By ~ 10

Author(s):

Mario Mardirossian ◽

Quentin Barrière ◽

Tatiana Timchenko ◽

Claudia Müller ◽

Sabrina Pacor ◽

...

Keyword(s):

Protein Synthesis ◽

Antimicrobial Peptides ◽

Binding Site ◽

Mode Of Action ◽

Antimicrobial Agents ◽

Bacterial Membrane ◽

Main Mode ◽

Content Type ◽

Inhibitor Of Protein Synthesis

ABSTRACTUnlike most antimicrobial peptides (AMPs), the main mode of action of the subclass of proline-rich antimicrobial peptides (PrAMPs) is not based on disruption of the bacterial membrane. Instead, PrAMPs exploit the inner membrane transporters SbmA and YjiL/MdtM to pass through the bacterial membrane and enter the cytosol of specific Gram-negative bacteria, where they exert an inhibitory effect on protein synthesis. Despite sharing a high proline and arginine content with other characterized PrAMPs, the PrAMP Bac5 has a low sequence identity with them. Here we investigated the mode of action of three N-terminal Bac5 fragments, Bac5(1-15), Bac5(1-25), and Bac5(1-31). We show that Bac5(1-25) and Bac5(1-31) retained excellent antimicrobial activity towardEscherichia coliand low toxicity toward eukaryotic cells, whereas Bac5(1-15) was inactive. Bac5(1-25) and Bac5(1-31) inhibited bacterial protein synthesisin vitroandin vivo. Competition assays suggested that the binding site of Bac5 is within the ribosomal tunnel, where it prevents the transition from the initiation to the elongation phase of translation, as reported for other PrAMPs, such as the bovine PrAMP Bac7. Surprisingly, unlike Bac7, Bac5(1-25) exhibited species-specific inhibition, being an excellent inhibitor of protein synthesis onE. coliribosomes but a poor inhibitor onThermus thermophilusribosomes. This indicates that while Bac5 most likely has an overlapping binding site with Bac7, the mode of interaction is distinct, suggesting that Bac5 fragments may be interesting alternative lead compounds for the development of new antimicrobial agents.

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Interaction between membrane functions and protein synthesis in reticulocytes. An elongation-stage inhibitor of protein synthesis extracted from the reticulocyte membrane

Biochemical Journal ◽

10.1042/bj1800379 ◽

1979 ◽

Vol 180 (2) ◽

pp. 379-387 ◽

Cited By ~ 4

Author(s):

D H Wreschner ◽

M Herzberg

Keyword(s):

Protein Synthesis ◽

Cell Membrane ◽

High Speed ◽

Ribosomal Subunit ◽

Initiation Factor ◽

Membrane Component ◽

Initiation Complex ◽

Initiation Factor 2 ◽

Reticulocyte Lysate ◽

Inhibitor Of Protein Synthesis

A component of the reticulocyte cell membrane was found to inhibit protein synthesis severely in a reticulocyte lysate system. An investigation into the mode of action of the membrane inhibitor revealed the following facts. (1) The binding of the tertiary initiation complex (methionyl-tRNAfMet-Initiation Factor 2-GTP) to the 40S ribosomal subunit was unaffected by the membrane inhibitor. (2) The membrane component did not interfere with the binding of the 40S initiation complex to the AUG initiation codon and subsequent attachment of the 60S ribosomal subunit. (3) Elongation of the peptide chain, as assayed by peptidyl-puromycin formation, was markedly affected by the membrane inhibitor. Surprisingly, the membrane component caused a considerable increase in peptidyl-puromycin formation. (4) Reticulocyte ribosomes that had been reisolated by high-speed centrifugation, after preincubation with the membrane component, were found to be highly defective when assayed in a cell-free protein-synthesizing system. These results indicated that an extract of the reticulocyte cell membrane inhibited protein synthesis by interacting with the ribosome and thus interfered with the correct functions of the elongation stage of protein synthesis. The implications of this conclusion are discussed in the light of data showing that a highly purified preparation of the membrane inhibitor also displayed an endonucleolytic activity highly specific for 28S RNA.

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Expression of Translation Initiation Factors eIF-4E and eIF-2α and a Potential Physiologic Role of Continuous Protein Synthesis in Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0037-1612917 ◽

2001 ◽

Vol 85 (01) ◽

pp. 142-151 ◽

Cited By ~ 34

Author(s):

Anping Han ◽

Linrong Lu ◽

German Pihan ◽

Bruce Woda ◽

Jane-Jane Chen ◽

...

Keyword(s):

Protein Synthesis ◽

Translation Initiation ◽

Ribosomal Subunit ◽

Human Platelets ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Protein Synthesis Inhibitors ◽

Initiation Factors ◽

Translation Initiation Factors ◽

Rate Limiting

SummaryIt is generally believed that platelets do not have a functionally significant protein synthetic machinery. However, our analysis demonstrated that normal bone marrow megakaryocytes express high levels of translation initiation factors eIF-4E and eIF-2α and the expression of these protein synthesis initiation factors is continued in platelets (as determined by immunohistochemistry and Western blot analysis). Both eIF-4E and eIF-2α are key regulators of protein synthesis. The eIF-4E is a rate-limiting part of a multisubunit complex, eIF-4F, that binds to the 5’ cap structure present in virtually all eukaryotic mRNAs, and carries out transfer of mRNAs to ribosomes for translation. Translation initiation factor eIF-2α is also a rate-limiting protein which associates with two other proteins to form an eIF-2 initiation factor complex responsible for the transfer of initiator methionyl-tRNA to the 40S ribosomal subunit. We confirm that expression of eIF-4E and eIF-2α is biologically relevant in that platelets continue protein synthesis, albeit at a 16 times lower rate than WBC (as determined by 35S-labeled amino acid incorporation, SDS-PAGE and scintillation counting). Finally, we determined that protein synthesis inhibitors (puromycin and emetine) attenuate the platelet aggregation response to a combination of ADP and epinephrine, but potentiate the response to collagen. Our data are consistent with the existence of different signal transducing pathways mediating the response to ADP/epinephrine and collagen. We suggest that the ADP/epinephrine response is positively affected by continuously synthesized proteins, while the response to collagen is modulated by continuously produced inhibitory proteins. Taken together, our results suggest that continuous protein synthesis is important for platelet function and its role in platelet physiology and pathophysiology deserves further study.

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