Raz Zarivach

Structure of Functionally Activated Small Ribosomal Subunit at 3.3 Å Resolution

The small ribosomal subunit performs the decoding of genetic information during translation. The structure of that from Thermus thermophilus shows that the decoding center, which positions mRNA and three tRNAs, is constructed entirely of RNA. The entrance to the mRNA channel will encircle the message when a latch-like contact closes and contributes to processivity and fidelity. Extended RNA helical elements that run longitudinally through the body transmit structural changes, correlating events at the particles far end with the cycle of mRNA translocation at the decoding region. 96% of the nucleotides were traced and the main fold of all proteins was determined. The latter are either peripheral or appear to serve as linkers. Some may assist the directionality of translocation.

High Resolution Structure of the Large Ribosomal Subunit from a Mesophilic Eubacterium

We describe the high resolution structure of the large ribosomal subunit from Deinococcus radiodurans (D50S), a gram-positive mesophile suitable for binding of antibiotics and functionally relevant ligands. The over-all structure of D50S is similar to that from the archae bacterium Haloarcula marismortui (H50S); however, a detailed comparison revealed significant differences, for example, in the orientation of nucleotides in peptidyl transferase center and in the structures of many ribosomal proteins. Analysis of ribosomal features involved in dynamic aspects of protein biosynthesis that are partially or fully disordered in H50S revealed the conformations of intersubunit bridges in unbound subunits, suggesting how they may change upon subunit association and how movements of the L1-stalk may facilitate the exit of tRNA.

Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria

Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics. Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action. Conversely, drugs are excellent tools for studying the ribosome function. To elucidate the structural basis of ribosome–antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin. We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavity and do not involve any interaction of the drugs with ribosomal proteins. Here we report the details of antibiotic interactions with the components of their binding sites. Our results also show the importance of putative Mg+2 ions for the binding of some drugs. This structural analysis should facilitate rational drug design.

Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3.

The small ribosomal subunit is responsible for the decoding of genetic information and plays a key role in the initiation of protein synthesis. We analyzed by X‐ray crystallography the structures of three different complexes of the small ribosomal subunit of Thermus thermophilus with the A‐site inhibitor tetracycline, the universal initiation inhibitor edeine and the C‐terminal domain of the translation initiation factor IF3. The crystal structure analysis of the complex with tetracycline revealed the functionally important site responsible for the blockage of the A‐site. Five additional tetracycline sites resolve most of the controversial biochemical data on the location of tetracycline. The interaction of edeine with the small subunit indicates its role in inhibiting initiation and shows its involvement with P‐site tRNA. The location of the C‐terminal domain of IF3, at the solvent side of the platform, sheds light on the formation of the initiation complex, and implies that the anti‐association activity of IF3 is due to its influence on the conformational dynamics of the small ribosomal subunit.

Structural basis for the antibiotic activity of ketolides and azalides.

The azalide azithromycin and the ketolide ABT-773, which were derived by chemical modifications of erythromycin, exhibit elevated activity against a number of penicillin- and macrolide-resistant pathogenic bacteria. Analysis of the crystal structures of the large ribosomal subunit from Deinococcus radiodurans complexed with azithromycin or ABT-773 indicates that, despite differences in the number and nature of their contacts with the ribosome, both compounds exert their antimicrobial activity by blocking the protein exit tunnel. In contrast to all macrolides studied so far, two molecules of azithromycin bind simultaneously to the tunnel. The additional molecule also interacts with two proteins, L4 and L22, implicated in macrolide resistance. These studies illuminated and rationalized the enhanced activity of the drugs against specific macrolide-resistant bacteria.

Structural and Functional Discussion of the Tetra-Trico-Peptide Repeat, a Protein Interaction Module

Tetra-trico-peptide repeat (TPR) domains are found in numerous proteins, where they serve as interaction modules and multiprotein complex mediators. TPRs can be found in all kingdoms of life and regulate diverse biological processes, such as organelle targeting and protein import, vesicle fusion, and biomineralization. This review considers the structural features of TPR domains that permit the great ligand-binding diversity of this motif, given that TPR-interacting partners display variations in both sequence and secondary structure. In addition, tools for predicting TPR-interacting partners are discussed, as are the abilities of TPR domains to serve as protein-protein interaction scaffolds in biotechnology and therapeutics.

Structural Insight into the Antibiotic Action of Telithromycin against Resistant Mutants

The crystal structure of the ketolide telithromycin bound to the Deinococcus radiodurans large ribosomal subunit shows that telithromycin blocks the ribosomal exit tunnel and interacts with domains II and V of the 23S RNA. Comparisons to other clinically relevant macrolides provided structural insights into its enhanced activity against macrolide-resistant strains.

Metalloprotease type III effectors that specifically cleave JNK and NF-κB.

Two major arms of the inflammatory response are the NF‐κB and c‐Jun N‐terminal kinase (JNK) pathways. Here, we show that enteropathogenic Escherichia coli (EPEC) employs the type III secretion system to target these two signalling arms by injecting host cells with two effector proteins, NleC and NleD. We provide evidence that NleC and NleD are Zn‐dependent endopeptidases that specifically clip and inactivate RelA (p65) and JNK, respectively, thus blocking NF‐κB and AP‐1 activation. We show that NleC and NleD co‐operate and complement other EPEC effectors in accomplishing maximal inhibition of IL‐8 secretion. This is a remarkable example of a pathogen using multiple effectors to manipulate systematically the host inflammatory response signalling network.

Structural analysis of the essential self-cleaving type III secretion proteins EscU and SpaS.

During infection by Gram-negative pathogenic bacteria, the type III secretion system (T3SS) is assembled to allow for the direct transmission of bacterial virulence effectors into the host cell. The T3SS system is characterized by a series of prominent multi-component rings in the inner and outer bacterial membranes, as well as a translocation pore in the host cell membrane. These are all connected by a series of polymerized tubes that act as the direct conduit for the T3SS proteins to pass through to the host cell. During assembly of the T3SS, as well as the evolutionarily related flagellar apparatus, a post-translational cleavage event within the inner membrane proteins EscU/FlhB is required to promote a secretion-competent state. These proteins have long been proposed to act as a part of a molecular switch, which would regulate the appropriate chronological secretion of the various T3SS apparatus components during assembly and subsequently the transported virulence effectors. Here we show that a surface type II β-turn in the Escherichia coli protein EscU undergoes auto-cleavage by a mechanism involving cyclization of a strictly conserved asparagine residue. Structural and in vivo analysis of point and deletion mutations illustrates the subtle conformational effects of auto-cleavage in modulating the molecular features of a highly conserved surface region of EscU, a potential point of interaction with other T3SS components at the inner membrane. In addition, this work provides new structural insight into the distinct conformational requirements for a large class of self-cleaving reactions involving asparagine cyclization.

Structural analysis of a prototypical ATPase from the type III secretion system.

The type III secretion system (T3SS) ATPase is the conserved and essential inner-membrane component involved in the initial stages of selective secretion of specialized T3SS virulence effector proteins from the bacterial cytoplasm through to the infected host cell, a process crucial to subsequent pathogenicity. Here we present the 1.8-Å-resolution crystal structure of the catalytic domain of the prototypical T3SS ATPase EscN from enteropathogenic Escherichia coli (EPEC). Along with in vitro and in vivo mutational analysis, our data show that the T3SS ATPases share similarity with the F1 ATPases but have important structural and sequence differences that dictate their unique secretory role. We also show that T3SS ATPase activity is dependent on EscN oligomerization and describe the molecular features and possible functional implications of a hexameric ring model.