Discovery of natural-product-derived sequanamycins as potent oral anti-tuberculosis agents.

The emergence of drug-resistant tuberculosis has created an urgent need for new anti-tubercular agents. Here, we report the discovery of a series of macrolides called sequanamycins with outstanding in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb). Sequanamycins are bacterial ribosome inhibitors that interact with the ribosome in a similar manner to classic macrolides like erythromycin and clarithromycin, but with binding characteristics that allow them to overcome the inherent macrolide resistance of Mtb. Structures of the ribosome with bound inhibitors were used to optimize sequanamycin to produce the advanced lead compound SEQ-9. SEQ-9 was efficacious in mouse models of acute and chronic TB as a single agent, and it demonstrated bactericidal activity in a murine TB infection model in combination with other TB drugs. These results support further investigation of this series as TB clinical candidates, with the potential for use in new regimens against drug-susceptible and drug-resistant TB.

mRNA reading frame maintenance during eukaryotic ribosome translocation.

One of the most critical steps of protein synthesis is coupled translocation of messenger RNA (mRNA) and transfer RNAs (tRNAs) required to advance the mRNA reading frame by one codon. In eukaryotes, translocation is accelerated and its fidelity is maintained by elongation factor 2 (eEF2)1,2. At present, only a few snapshots of eukaryotic ribosome translocation have been reported3-5. Here we report ten high-resolution cryogenic-electron microscopy (cryo-EM) structures of the elongating eukaryotic ribosome bound to the full translocation module consisting of mRNA, peptidyl-tRNA and deacylated tRNA, seven of which also contained ribosome-bound, naturally modified eEF2. This study recapitulates mRNA-tRNA2-growing peptide module progression through the ribosome, from the earliest states of eEF2 translocase accommodation until the very late stages of the process, and shows an intricate network of interactions preventing the slippage of the translational reading frame. We demonstrate how the accuracy of eukaryotic translocation relies on eukaryote-specific elements of the 80S ribosome, eEF2 and tRNAs. Our findings shed light on the mechanism of translation arrest by the anti-fungal eEF2-binding inhibitor, sordarin. We also propose that the sterically constrained environment imposed by diphthamide, a conserved eukaryotic posttranslational modification in eEF2, not only stabilizes correct Watson-Crick codon-anticodon interactions but may also uncover erroneous peptidyl-tRNA, and therefore contribute to higher accuracy of protein synthesis in eukaryotes.

Accuracy mechanism of eukaryotic ribosome translocation.

Translation of the genetic code into proteins is realized through repetitions of synchronous translocation of messenger RNA (mRNA) and transfer RNAs (tRNA) through the ribosome. In eukaryotes translocation is ensured by elongation factor 2 (eEF2), which catalyses the process and actively contributes to its accuracy1. Although numerous studies point to critical roles for both the conserved eukaryotic posttranslational modification diphthamide in eEF2 and tRNA modifications in supporting the accuracy of translocation, detailed molecular mechanisms describing their specific functions are poorly understood. Here we report a high-resolution X-ray structure of the eukaryotic 80S ribosome in a translocation-intermediate state containing mRNA, naturally modified eEF2 and tRNAs. The crystal structure reveals a network of stabilization of codon-anticodon interactions involving diphthamide1 and the hypermodified nucleoside wybutosine at position 37 of phenylalanine tRNA, which is also known to enhance translation accuracy2. The model demonstrates how the decoding centre releases a codon-anticodon duplex, allowing its movement on the ribosome, and emphasizes the function of eEF2 as a 'pawl' defining the directionality of translocation3. This model suggests how eukaryote-specific elements of the 80S ribosome, eEF2 and tRNAs undergo large-scale molecular reorganizations to ensure maintenance of the mRNA reading frame during the complex process of translocation.

Structure of and influence of a tick complement inhibitor on human complement component 5.

To provide insight into the structural and functional properties of human complement component 5 (C5), we determined its crystal structure at a resolution of 3.1 A. The core of C5 adopted a structure resembling that of C3, with the domain arrangement at the position corresponding to the C3 thioester being very well conserved. However, in contrast to C3, the convertase cleavage site in C5 was ordered and the C345C domain flexibly attached to the core of C5. Binding of the tick C5 inhibitor OmCI to C5 resulted in stabilization of the global conformation of C5 but did not block the convertase cleavage site. The structure of C5 may render possible a structure-based approach for the design of new selective complement inhibitors.

Purification and characterization of native human elongation factor 2.

Human elongation factor 2 is the translocase that is responsible for the movement of tRNA from the A- to P- and P- to E-site on the ribosome during the elongation phase of translation. Being a vital factor of protein biosynthesis, its function is highly controlled and regulated. It has been implicated in numerous diseases and pathologies, and as such it is important to have a source for isolated pure and active protein for biomedical and biochemical studies. Here we report development of a purification protocol for native human elongation factor 2 from HEK-293S cells. The resulting protein is active, pure, has an intact diphtamide and is obtainable in yields suitable for functional and structural studies.

A new understanding of the decoding principle on the ribosome.

During protein synthesis, the ribosome accurately selects transfer RNAs (tRNAs) in accordance with the messenger RNA (mRNA) triplet in the decoding centre. tRNA selection is initiated by elongation factor Tu, which delivers tRNA to the aminoacyl tRNA-binding site (A site) and hydrolyses GTP upon establishing codon-anticodon interactions in the decoding centre. At the following proofreading step the ribosome re-examines the tRNA and rejects it if it does not match the A codon. It was suggested that universally conserved G530, A1492 and A1493 of 16S ribosomal RNA, critical for tRNA binding in the A site, actively monitor cognate tRNA, and that recognition of the correct codon-anticodon duplex induces an overall ribosome conformational change (domain closure). Here we propose an integrated mechanism for decoding based on six X-ray structures of the 70S ribosome determined at 3.1-3.4 Å resolution, modelling cognate or near-cognate states of the decoding centre at the proofreading step. We show that the 30S subunit undergoes an identical domain closure upon binding of either cognate or near-cognate tRNA. This conformational change of the 30S subunit forms a decoding centre that constrains the mRNA in such a way that the first two nucleotides of the A codon are limited to form Watson-Crick base pairs. When U·G and G·U mismatches, generally considered to form wobble base pairs, are at the first or second codon-anticodon position, the decoding centre forces this pair to adopt the geometry close to that of a canonical C·G pair. This by itself, or with distortions in the codon-anticodon mini-helix and the anticodon loop, causes the near-cognate tRNA to dissociate from the ribosome.

Structural basis for potent inhibitory activity of the antibiotic tigecycline during protein synthesis.

Here we present an X-ray crystallography structure of the clinically relevant tigecycline antibiotic bound to the 70S ribosome. Our structural and biochemical analysis indicate that the enhanced potency of tigecycline results from a stacking interaction with nucleobase C1054 within the decoding site of the ribosome. Single-molecule fluorescence resonance energy transfer studies reveal that, during decoding, tigecycline inhibits the initial codon recognition step of tRNA accommodation and prevents rescue by the tetracycline-resistance protein TetM.

Cryo-EM structure of the inactive ribosome complex accumulated in chick embryo cells in cold-stress conditions.

Here, we present the high-resolution structure of the Gallus gallus 80S ribosome obtained from cold-treated chicken embryos. The translationally inactive ribosome complex contains elongation factor eEF2 with GDP, SERPINE1 mRNA binding protein 1 (SERBP1) and deacylated tRNA in the P/E position, showing common features with complexes already described in mammals. Modeling of most expansion segments of G. gallus 28S ribosomal RNA allowed us to identify specific features in their structural organization and to describe areas where a marked difference between mammalian and avian ribosomes could shed light on the evolution of the expansion segments. This study provides the first structure of an avian ribosome, establishing a model for future structural and functional studies on the translational machinery in Aves.

Structural aspects of RimP binding on small ribosomal subunit from Staphylococcus aureus.

Ribosome biogenesis is an energy-intense multistep process where even minimal defects can cause severe phenotypes up to cell death. Ribosome assembly is facilitated by biogenesis factors such as ribosome assembly factors. These proteins facilitate the interaction of ribosomal proteins with rRNA and correct rRNA folding. One of these maturation factors is RimP which is required for efficient 16S rRNA processing and 30S ribosomal subunit assembly. Here, we describe the binding mode of Staphylococcus aureus RimP to the small ribosomal subunit and present a 4.2 Å resolution cryo-EM reconstruction of the 30S-RimP complex. Together with the solution structure of RimP solved by NMR spectroscopy and RimP-uS12 complex analysis by EPR, DEER, and SAXS approaches, we show the specificity of RimP binding to the 30S subunit from S. aureus. We believe the results presented in this work will contribute to the understanding of the RimP role in the ribosome assembly mechanism.

Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms.

Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2-GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue.

Structural basis for messenger RNA movement on the ribosome.

Translation initiation is a major determinant of the overall expression level of a gene. The translation of functionally active protein requires the messenger RNA to be positioned on the ribosome such that the start/initiation codon will be read first and in the correct frame. Little is known about the molecular basis for the interaction of mRNA with the ribosome at different states of translation. Recent crystal structures of the ribosomal subunits, the empty 70S ribosome and the 70S ribosome containing functional ligands have provided information about the general organization of the ribosome and its functional centres. Here we compare the X-ray structures of eight ribosome complexes modelling the translation initiation, post-initiation and elongation states. In the initiation and post-initiation complexes, the presence of the Shine-Dalgarno (SD) duplex causes strong anchoring of the 5'-end of mRNA onto the platform of the 30S subunit, with numerous interactions between mRNA and the ribosome. Conversely, the 5' end of the 'elongator' mRNA lacking SD interactions is flexible, suggesting a different exit path for mRNA during elongation. After the initiation of translation, but while an SD interaction is still present, mRNA moves in the 3'-->5' direction with simultaneous clockwise rotation and lengthening of the SD duplex, bringing it into contact with ribosomal protein S2.

E-site drug specificity of the human pathogen Candida albicans ribosome.

Candida albicans is a widespread commensal fungus with substantial pathogenic potential and steadily increasing resistance to current antifungal drugs. It is known to be resistant to cycloheximide (CHX) that binds to the E-transfer RNA binding site of the ribosome. Because of lack of structural information, it is neither possible to understand the nature of the resistance nor to develop novel inhibitors. To overcome this issue, we determined the structure of the vacant C. albicans 80S ribosome at 2.3 angstroms and its complexes with bound inhibitors at resolutions better than 2.9 angstroms using cryo-electron microscopy. Our structures reveal how a change in a conserved amino acid in ribosomal protein eL42 explains CHX resistance in C. albicans and forms a basis for further antifungal drug development.

Cryo-EM structure of the ribosome functional complex of the human pathogen Staphylococcus aureus at 3.2 Å resolution.

Staphylococcus aureus is a bacterial pathogen and one of the leading causes of healthcare-acquired infections in the world. The growing antibiotic resistance of S. aureus obliges us to search for new drugs and treatments. As the majority of antibiotics target the ribosome, knowledge of its detailed structure is crucial for drug development. Here, we report the cryo-EM reconstruction at 3.2 Å resolution of the S. aureus ribosome with P-site tRNA, messenger RNA, and 10 RNA modification sites previously not assigned or visualized. The resulting model is the most precise and complete high-resolution structure to date of the S. aureus 70S ribosome with functional ligands.

Structure and Function of the Bacterial Protein Toxin Phenomycin.

Phenomycin is a bacterial mini-protein of 89 amino acids discovered more than 50 years ago with toxicity in the nanomolar regime toward mammalian cells. The protein inhibits the function of the eukaryotic ribosome in cell-free systems and appears to target translation initiation. Several fundamental questions concerning the cellular activity of phenomycin, however, have remained unanswered. In this paper, we have used morphological profiling to show that direct inhibition of translation underlies the toxicity of phenomycin in cells. We have performed studies of the cellular uptake mechanism of phenomycin, showing that endosomal escape is the toxicity-limiting step, and we have solved a solution phase high-resolution structure of the protein using NMR spectroscopy. Through bioinformatic as well as functional comparisons between phenomycin and two homologs, we have identified a peptide segment, which constitutes one of two loops in the structure that is critical for the toxicity of phenomycin.

Cryo-EM structure of the hibernating Thermus thermophilus 100S ribosome reveals a protein-mediated dimerization mechanism.

In response to cellular stresses bacteria conserve energy by dimerization of ribosomes into inactive hibernating 100S ribosome particles. Ribosome dimerization in Thermus thermophilus is facilitated by hibernation-promoting factor (TtHPF). In this study we demonstrate high sensitivity of Tt100S formation to the levels of TtHPF and show that a 1:1 ratio leads to optimal dimerization. We report structures of the T. thermophilus 100S ribosome determined by cryo-electron microscopy to average resolutions of 4.13 Å and 4.57 Å. In addition, we present a 3.28 Å high-resolution cryo-EM reconstruction of a 70S ribosome from a hibernating ribosome dimer and reveal a role for the linker region connecting the TtHPF N- and C-terminal domains in translation inhibition by preventing Shine-Dalgarno duplex formation. Our work demonstrates that species-specific differences in the dimerization interface govern the overall conformation of the 100S ribosome particle and that for Thermus thermophilus no ribosome-ribosome interactions are involved in the interface.

The structure of bovine complement component 3 reveals the basis for thioester function.

The third component of complement (C3) is a 190 kDa glycoprotein essential for eliciting the complement response. The protein consists of two polypeptide chains (alpha and beta) held together with a single disulfide bridge. The beta-chain is composed of six MG domains, one of which is shared with the alpha-chain. The disulfide bridge connecting the chains is positioned in the shared MG domain. The alpha-chain consists of the anaphylatoxin domain, three MG domains, a CUB domain, an alpha(6)/alpha(6)-barrel domain and the C-terminal C345c domain. An internal thioester in the alpha-chain of C3 (present in C4 but not in C5) is cleaved during complement activation. This mediates covalent attachment of the activated C3b to immune complexes and invading microorganisms, thereby opsonizing the target. We present the structure of bovine C3 determined at 3 Angstroms resolution. The structure shows that the ester is buried deeply between the thioester domain and the properdin binding domain, in agreement with the human structure. This domain interface is broken upon activation, allowing nucleophile access. The structure of bovine C3 clearly demonstrates that the main chain around the thioester undergoes a helical transition upon activation. This rearrangement is proposed to be the basis for the high level of reactivity of the thioester group. A strictly conserved glutamate residue is suggested to function catalytically in thioester proteins. Structure-based design of inhibitors of C3 activation may target a conserved pocket between the alpha-chain and the beta-chain of C3, which appears essential for conformational changes in C3.

Structural analysis of ribosomal RACK1 and its role in translational control.

Receptor for Activated C-Kinase 1 (RACK1) belongs to the WD40 family of proteins, known to act as scaffolding proteins in interaction networks. Accordingly, RACK1 is found to have numerous interacting partners ranging from kinases and signaling proteins to membrane bound receptors and ion channels. Interestingly, RACK1 has also been identified as a ribosomal protein present in all eukaryotic ribosomes. Structures of eukaryotic ribosomes have shown RACK1 to be located at the back of the head of the small ribosomal subunit. This suggests that RACK1 could act as a ribosomal scaffolding protein recruiting regulators of translation to the ribosome, and several studies have in fact found RACK1 to play a role in regulation of translation. To fully understand the role of RACK1 we need to understand whether the many reported interaction partners of RACK1 bind to free or ribosomal RACK1. In this review we provide a structural analysis of ribosome-bound RACK1 to provide a basis for answering this fundamental question. Our analysis shows that RACK1 is tightly bound to the ribosome through highly conserved and specific interactions confirming RACK1 as an integral ribosomal protein. Furthermore, we have analyzed whether reported binding sites for RACK1 interacting partners with a proposed role in translational control are accessible on ribosomal RACK1. Our analysis shows that most of the interaction partners with putative regulatory functions have binding sites that are available on ribosomal RACK1, supporting the role of RACK1 as a ribosomal signaling hub. We also discuss the possible role for RACK1 in recruitment of ribosomes to focal adhesion sites and regulation of local translation during cell spreading and migration.

Structure of TSA2 reveals novel features of the active-site loop of peroxiredoxins.

Saccharomyces cerevisiae TSA2 belongs to the family of typical 2-Cys peroxiredoxins, a ubiquitously expressed family of redox-active enzymes that utilize a conserved peroxidatic cysteine to reduce peroxides. Typical 2-Cys peroxiredoxins have been shown to be involved in protection against oxidative stress and in hydrogen peroxide signalling. Furthermore, several 2-Cys peroxiredoxins, including S. cerevisiae TSA1 and TSA2, are able to switch to chaperone activity upon hyperoxidation of their peroxidatic cysteine. This makes the sensitivity to hyperoxidation of the peroxidatic cysteine a very important determinant for the cellular function of a peroxiredoxin under different cellular conditions. Typical 2-Cys peroxiredoxins exist as dimers, and in the course of the reaction the peroxidatic cysteine forms a disulfide with a resolving cysteine located in the C-terminus of its dimeric partner. This requires a local unfolding of the active site and the C-terminus. The balance between the fully folded and locally unfolded conformations is of key importance for the reactivity and sensitivity to hyperoxidation of the different peroxiredoxins. Here, the structure of a C48S mutant of TSA2 from S. cerevisiae that mimics the reduced state of the peroxidatic cysteine has been determined. The structure reveals a novel conformation for the strictly conserved Pro41, which is likely to affect the delicate balance between the fully folded and locally unfolded conformations of the active site, and therefore the reactivity and the sensitivity to hyperoxidation. Furthermore, the structure also explains the observed difference in the pKa values of the peroxidatic cysteines of S. cerevisiae TSA1 and TSA2 despite their very high sequence identity.

New structural insights into the decoding mechanism: translation infidelity via a G·U pair with Watson-Crick geometry.

Pioneer crystallographic studies of the isolated 30S ribosomal subunit provided the first structural insights into the decoding process. Recently, new crystallographic data on full 70S ribosomes with mRNA and tRNAs have shown that the formation of the tight decoding centre is ensured by conformational rearrangement of the 30S subunit (domain closure), which is identical for cognate or near-cognate tRNA. When a G·U forms at the first or second codon-anticodon positions (near-cognate tRNA), the ribosomal decoding centre forces the adoption of Watson-Crick G·C-like geometry rather than that of the expected Watson-Crick wobble pair. Energy expenditure for rarely occuring tautomeric base required for Watson-Crick G·C-like G·U pair or the repulsion energy due to steric clash within the mismatched base pair could constitute the only cause for efficient rejection of a near-cognate tRNA. Our data suggest that "geometrical mimicry" can explain how wrong aminoacyl-tRNAs with G·U pairs in the codon-anticodon helix forming base pairs with Watson-Crick geometry in the decoding center can be incorporated into the polypeptide chain.

One core, two shells: bacterial and eukaryotic ribosomes.

Ribosomes are universally conserved enzymes that carry out protein biosynthesis. Bacterial and eukaryotic ribosomes, which share an evolutionarily conserved core, are thought to have evolved from a common ancestor by addition of proteins and RNA that bestow different functionalities to ribosomes from different domains of life. Recently, structures of the eukaryotic ribosome, determined by X-ray crystallography, have allowed us to compare these structures to previously determined structures of bacterial ribosomes. Here we describe selected bacteria- or eukaryote-specific structural features of the ribosome and discuss the functional implications of some of them.