Letizia Brandi

Mapping the fMet-tRNAfMet binding site of initiation factor IF2

The interaction between fMet‐tRNAfMet and Bacillus stearothermophilus translation initiation factor IF2 has been characterized. We demonstrate that essentially all thermodynamic determinants governing the stability and the specificity of this interaction are localized within the acceptor hexanucleotide fMet‐3′ACCAAC of the initiator tRNA and a fairly small area at the surface of the β‐barrel structure of the 90‐amino acid C‐terminal domain of IF2 (IF2 C‐2). A weak but specific interaction between IF2 C‐2 and formyl‐methionyl was also demonstrated. The surface of IF2 C‐2 interacting with fMet‐tRNAfMet has been mapped using two independent approaches, site‐ directed mutagenesis and NMR spectroscopy, which yielded consistent results. The binding site comprises C668 and G715 located in a groove accommodating the methionyl side‐chain, R700, in the vicinity of the formyl group, Y701 and K702 close to the acyl bond between fMet and tRNAfMet, and the surface lined with residues K702‐S660, along which the acceptor arm of the initiator tRNA spans in the direction 3′ to 5′.

How to cope with the quest for new antibiotics.

Since their introduction in therapy, antibiotics have played an essential role in human society, saving millions of lives, allowing safe surgery, organ transplants, cancer therapy. Antibiotics have also helped to elucidate several biological mechanisms and boosted the birth and growth of pharmaceutical companies, generating profits and royalties. The golden era of antibiotics and the scientific and economical drive of big pharma towards these molecules is long gone, but the need for effective antibiotics is increased as their pipelines dwindle and multi‐resistant pathogenic strains spread. Here we outline some strategies that could help meet this emergency and list promising new targets.

Targets and assays for discovering novel antibacterial agents

The increasing frequency of nosocomial infections due to multi-resistant pathogens exerts a significant toll and calls for novel and better antibiotics. Different approaches can be used in the search for novel antibiotics acting on drug-resistant bacterial pathogens. We present some considerations on valid bacterial targets to be used for searching new antibiotics, and how the information from bacterial genome sequences can assist in choosing the appropriate targets. Other factors to be considered in target selection are the chemical diversity available for screening and its uniqueness. We will conclude discussing our strategy for searching novel antibacterials. This is based on a large collection of microbial extracts as a source of chemical diversity and on the use of specific targets essential for the viability of bacterial pathogens. Two assay strategies have been implemented: a pathway-based assay, where a series of essential bacterial targets is screened in a single assay; and a binding assay, where many targets can be screened individually in the same format.

The antibiotics dityromycin and GE82832 bind protein S12 and block EF-G-catalyzed translocation.

The translocation of mRNA and tRNA through the ribosome is catalyzed by elongation factor G (EF-G), a universally conserved guanosine triphosphate hydrolase (GTPase). The mechanism by which the closely related decapeptide antibiotics dityromycin and GE82832 inhibit EF-G-catalyzed translocation is elucidated in this study. Using crystallographic and biochemical experiments, we demonstrate that these antibiotics bind to ribosomal protein S12 in solution alone as well as within the small ribosomal subunit, inducing long-range effects on the ribosomal head. The crystal structure of the antibiotic in complex with the 70S ribosome reveals that the binding involves conserved amino acid residues of S12 whose mutations result in in vitro and in vivo antibiotic resistance and loss of antibiotic binding. The data also suggest that GE82832/dityromycin inhibits EF-G-catalyzed translocation by disrupting a critical contact between EF-G and S12 that is required to stabilize the posttranslocational conformation of EF-G, thereby preventing the ribosome-EF-G complex from entering a conformation productive for translocation.

Structural and functional characterization of the bacterial translocation inhibitor GE82832

The structure of GE82832, a translocation inhibitor produced by a soil microorganism, is shown to be highly related to that of dityromycin, a bicyclodecadepsipeptide antibiotic discovered long ago whose characterization had never been pursued beyond its structural elucidation. GE82832 and dityromycin were shown to interfere with both aminoacyl‐tRNA and mRNA movement and with the Pi release occurring after ribosome‐ and EF‐G‐dependent GTP hydrolysis. These findings and the unusual ribosomal localization of GE82832/dityromycin near protein S13 suggest that the mechanism of inhibition entails an interference with the rotation of the 30S subunit “head” which accompanies the ribosome‐unlocking step of translocation.

The antibiotic Furvina® targets the P-site of 30S ribosomal subunits and inhibits translation initiation displaying start codon bias

Furvina®, also denominated G1 (MW 297), is a synthetic nitrovinylfuran [2-bromo-5-(2-bromo-2-nitrovinyl)-furan] antibiotic with a broad antimicrobial spectrum. An ointment (Dermofural®) containing G1 as the only active principle is currently marketed in Cuba and successfully used to treat dermatological infections. Here we describe the molecular target and mechanism of action of G1 in bacteria and demonstrate that in vivo G1 preferentially inhibits protein synthesis over RNA, DNA and cell wall synthesis. Furthermore, we demonstrate that G1 targets the small ribosomal subunit, binds at or near the P-decoding site and inhibits its function interfering with the ribosomal binding of fMet-tRNA during 30S initiation complex (IC) formation ultimately inhibiting translation. Notably, this G1 inhibition displays a bias for the nature (purine vs. pyrimidine) of the 3′-base of the codon, occurring efficiently only when the mRNA directing 30S IC formation and translation contains the canonical AUG initiation triplet or the rarely found AUA triplet, but hardly occurs when the mRNA start codon is either one of the non-canonical triplets AUU or AUC. This codon discrimination by G1 is reminiscent, though of opposite type of that displayed by IF3 in its fidelity function, and remarkably does not occur in the absence of this factor.

Initiation of protein synthesis: a target for antimicrobials

Background: Translation initiation is a basic and universal biological process that employs significantly different components and displays substantially different mechanisms in bacterial, archaeal and eukaryotic cells. A large amount of detailed mechanistic and structural information on the bacterial translation initiation apparatus has been uncovered in recent years. Objective: to understand which translation initiation steps could represent a novel or underexploited target for the discovery of new and specific antibacterial drugs. Methods: Brief descriptions of the properties and mechanism of action of the major antibiotics that have a documented direct inhibitory effect on bacterial translation initiation are presented. Results/conclusions: Considerations and predictions concerning a future scenario for research and identification of bacterial translation initiation inhibitors are presented.

Inhibition of translation initiation complex formation by GE81112 unravels a 16S rRNA structural switch involved in P-site decoding.

Significance Eubacterial protein synthesis entails formation of an unlocked preinitiation complex consisting of the 30S ribosomal subunit, initiation factors, mRNA, and initiator tRNA. A conformational change in the subunit accompanies mRNA–tRNA codon–anticodon base-pairing generating a locked 30S complex. If correctly formed, this complex associates with the 50S ribosomal subunit forming a 70S complex, and the initiation factors are ejected. We show that the translational inhibitor GE81112 targets this essential step, hampering formation of a canonical codon–anticodon interaction and stalling the 30S in an unlocked state. Moreover, in the presence of GE81112 three rRNA helices, h44/h45/h24a, are stabilized in a disengaged conformation, suggesting that their conformation is associated with tRNA/mRNA decoding and transition of the 30S from unlocked to locked state. In prokaryotic systems, the initiation phase of protein synthesis is governed by the presence of initiation factors that guide the transition of the small ribosomal subunit (30S) from an unlocked preinitiation complex (30S preIC) to a locked initiation complex (30SIC) upon the formation of a correct codon–anticodon interaction in the peptidyl (P) site. Biochemical and structural characterization of GE81112, a translational inhibitor specific for the initiation phase, indicates that the main mechanism of action of this antibiotic is to prevent P-site decoding by stabilizing the anticodon stem loop of the initiator tRNA in a distorted conformation. This distortion stalls initiation in the unlocked 30S preIC state characterized by tighter IF3 binding and a reduced association rate for the 50S subunit. At the structural level we observe that in the presence of GE81112 the h44/h45/h24a interface, which is part of the IF3 binding site and forms ribosomal intersubunit bridges, preferentially adopts a disengaged conformation. Accordingly, the findings reveal that the dynamic equilibrium between the disengaged and engaged conformations of the h44/h45/h24a interface regulates the progression of protein synthesis, acting as a molecular switch that senses and couples the 30S P-site decoding step of translation initiation to the transition from an unlocked preIC to a locked 30SIC state.

The fMet-tRNA binding domain of translational initiation factor IF2: role and environment of its two Cys residues.

Mutations of the cysteines (positions 668 and 714) were generated in the IF2 C domain of Bacillus stearothermophilus translation initiation factor IF2. The corresponding proteins were characterized functionally and structurally. Most (yet not all) amino acid replacements at both positions resulted in severe reduction of the fMet‐tRNA binding activity of IF2 C without grossly altering its structure. Our work demonstrates that: (a) both Cys residues are buried within an hydrophobic core and not accessible to protonation or chemical substitution, (b) neither Cys is functionally essential and (c) both Cys residues are located near the active site, probably without participating directly in fMet‐tRNA binding.

Novel assays and novel strains – promising routes to new antibiotics?

There is a need to develop novel antibiotics for treating infections caused by multiresistant pathogens. Notwithstanding a plethora of novel targets and intensive high-throughput screening, conventional chemistry has yet to deliver these badly needed new drugs. Microorganisms have provided many of the existing antibiotics, but there is a general feeling that the large majority of compounds have already been discovered. Novel assays, used to screen common microbes, can provide novel structural scaffolds for antibiotic discovery. However, the highest impact may come from unexplored microbial sources. Fortunately, there is plenty of previously undescribed, antibiotic-producing bacteria in the environment.