Tatsuya Maehigashi

Doc toxin is a kinase that inactivates elongation factor Tu.

Background: Doc toxin, of the phd-doc toxin-antitoxin system, belongs to the Fic protein family found in all domains of life. Results: Doc inactivates elongation factor Tu by phosphorylation of a single amino acid. Conclusion: This phosphorylation event inhibits protein synthesis and thereby arrests cell growth. Significance: The phosphorylation activity of Doc toxin represents a new catalytic activity for members of the Fic protein family. The Doc toxin from bacteriophage P1 (of the phd-doc toxin-antitoxin system) has served as a model for the family of Doc toxins, many of which are harbored in the genomes of pathogens. We have shown previously that the mode of action of this toxin is distinct from the majority derived from toxin-antitoxin systems: it does not cleave RNA; in fact P1 Doc expression leads to mRNA stabilization. However, the molecular triggers that lead to translation arrest are not understood. The presence of a Fic domain, albeit slightly altered in length and at the catalytic site, provided a clue to the mechanism of P1 Doc action, as most proteins with this conserved domain inactivate GTPases through addition of an adenylyl group (also referred to as AMPylation). We demonstrated that P1 Doc added a single phosphate group to the essential translation elongation factor and GTPase, elongation factor (EF)-Tu. The phosphorylation site was at a highly conserved threonine, Thr-382, which was blocked when EF-Tu was treated with the antibiotic kirromycin. Therefore, we have established that Fic domain proteins can function as kinases. This distinct enzymatic activity exhibited by P1 Doc also solves the mystery of the degenerate Fic motif unique to the Doc family of toxins. Moreover, we have established that all characterized Fic domain proteins, even those that phosphorylate, target pivotal GTPases for inactivation through a post-translational modification at a single functionally critical acceptor site.

Growth-regulating Mycobacterium tuberculosis VapC-mt4 toxin is an isoacceptor-specific tRNase

Toxin-antitoxin (TA) systems are implicated in the downregulation of bacterial cell growth associated with stress survival and latent tuberculosis infection, yet the activities and intracellular targets of these TA toxins are largely uncharacterized. Here, we use a specialized RNA-seq approach to identify targets of a Mycobacterium tuberculosis VapC TA toxin, VapC-mt4 (also known as VapC4), which have eluded detection using conventional approaches. Distinct from the one other characterized VapC toxin in M. tuberculosis that cuts 23S rRNA at the sarcin-ricin loop, VapC-mt4 selectively targets three of the 45 M. tuberculosis tRNAs (tRNA(Ala2), tRNA(Ser26) and tRNA(Ser24)) for cleavage at, or adjacent to, their anticodons, resulting in the generation of tRNA halves. While tRNA cleavage is sometimes enlisted as a bacterial host defense mechanism, VapC-mt4 instead alters specific tRNAs to inhibit translation and modulate growth. This stress-linked activity of VapC-mt4 mirrors basic features of eukaryotic tRNases that also generate tRNA halves and inhibit translation in response to stress.

Structure of the basal components of a bacterial transporter

Proteins SpoIIQ and SpoIIIAH interact through two membranes to connect the forespore and the mother cell during endospore development in the bacterium Bacillus subtilis. SpoIIIAH consists of a transmembrane segment and an extracellular domain with similarity to YscJ proteins. YscJ proteins form large multimeric rings that are the structural scaffolds for the assembly of type III secretion systems in Gram-negative bacteria. The predicted ring-forming motif of SpoIIIAH and other evidence led to the model that SpoIIQ and SpoIIIAH form the core components of a channel or transporter through which the mother cell nurtures forespore development. Therefore, to understand the roles of SpoIIIAH and SpoIIQ in channel formation, it is critical to determine whether SpoIIIAH adopts a ring-forming structural motif, and whether interaction of SpoIIIAH with SpoIIQ would preclude ring formation. We report a 2.8-Å resolution structure of a complex of SpoIIQ and SpoIIIAH. SpoIIIAH folds into the ring-building structural motif, and modeling shows that the structure of the SpoIIQ–SpoIIIAH complex is compatible with forming a symmetrical oligomer that is similar to those in type III systems. The inner diameters of the two most likely ring models are large enough to accommodate several copies of other integral membrane proteins. SpoIIQ contains a LytM domain, which is found in metalloendopeptidases, but lacks residues important for metalloprotease activity. Other LytM domains appear to be involved in protein–protein interactions. We found that the LytM domain of SpoIIQ contains an accessory region that interacts with SpoIIIAH.

Structural insights into +1 frameshifting promoted by expanded or modification-deficient anticodon stem loops

Significance Biological fitness is dependent on the accurate flow of genetic information from DNA to mRNA to protein. Breakdown in ribosome translational fidelity is detrimental because of its central role in the production of proteins. Altering the 3-base genetic code usually results in the expression of aberrant or nonsense proteins that are degraded. Here, we describe molecular snapshots of the ribosome in the process of decoding a 4-base codon by a frameshift suppressor tRNA that results in a +1-nt shift of the mRNA reading frame. Conformational dynamics of the anticodon stem loop seem to drive remodeling of the tRNA–mRNA interaction to promote the +1 movement, which we predict occurs after accommodation of the tRNA onto the ribosome. Maintenance of the correct reading frame on the ribosome is essential for accurate protein synthesis. Here, we report structures of the 70S ribosome bound to frameshift suppressor tRNASufA6 and N1-methylguanosine at position 37 (m1G37) modification-deficient anticodon stem loopPro, both of which cause the ribosome to decode 4 rather than 3 nucleotides, resulting in a +1 reading frame. Our results reveal that decoding at +1 suppressible codons causes suppressor tRNASufA6 to undergo a rearrangement of its 5′ stem that destabilizes U32, thereby disrupting the conserved U32–A38 base pair. Unexpectedly, the removal of the m1G37 modification of tRNAPro also disrupts U32–A38 pairing in a structurally analogous manner. The lack of U32–A38 pairing provides a structural correlation between the transition from canonical translation and a +1 reading of the mRNA. Our structures clarify the molecular mechanism behind suppressor tRNA-induced +1 frameshifting and advance our understanding of the role played by the ribosome in maintaining the correct translational reading frame.

Structure of the Proteus vulgaris HigB-(HigA)2-HigB toxin-antitoxin complex.

Background: Toxin-antitoxin (TA) systems play a crucial role in bacterial survival during stress. Results: Structures of the P. vulgaris HigBA complex reveal novel structural features such as the HigB and HigA interaction and the solvent accessibility of the HigB active site. Conclusion: Antitoxin HigA interacts with toxin HigB in a novel manner. Significance: Our results emphasize that antitoxins are a structurally diverse class of proteins. Bacterial toxin-antitoxin (TA) systems regulate key cellular processes to promote cell survival during periods of stress. During steady-state cell growth, antitoxins typically interact with their cognate toxins to inhibit activity presumably by preventing substrate recognition. We solved two x-ray crystal structures of the Proteus vulgaris tetrameric HigB-(HigA)2-HigB TA complex and found that, unlike most other TA systems, the antitoxin HigA makes minimal interactions with toxin HigB. HigB adopts a RelE family tertiary fold containing a highly conserved concave surface where we predict its active site is located. HigA does not cover the solvent-exposed HigB active site, suggesting that, in general, toxin inhibition is not solely mediated by active site hindrance by its antitoxin. Each HigA monomer contains a helix-turn-helix motif that binds to its own DNA operator to repress transcription during normal cellular growth. This is distinct from antitoxins belonging to other superfamilies that typically only form DNA-binding motifs upon dimerization. We further show that disruption of the HigB-(HigA)2-HigB tetramer to a HigBA heterodimer ablates operator binding. Taken together, our biochemical and structural studies elucidate the novel molecular details of the HigBA TA system.

Mechanisms of Toxin Inhibition and Transcriptional Repression by Escherichia coli DinJ-YafQ

Background: Toxin-antitoxin complexes autoregulate transcription depending upon growth conditions. Results: DinJ-YafQ structure was determined, and minimal requirements for transcriptional autorepression were identified. Conclusion: The dinJyafQ operon is regulated in a novel manner by either DinJ-YafQ- or LexA-mediated repression. Significance: Our results reveal new mechanistic insights into the action of DinJ-YafQ as a transcriptional repressor. Bacteria encounter environmental stresses that regulate a gene expression program required for adaptation and survival. Here, we report the 1.8-Å crystal structure of the Escherichia coli toxin-antitoxin complex YafQ-(DinJ)2-YafQ, a key component of the stress response. The antitoxin DinJ dimer adopts a ribbon-helix-helix motif required for transcriptional autorepression, and toxin YafQ contains a microbial RNase fold whose proposed active site is concealed by DinJ binding. Contrary to previous reports, our studies indicate that equivalent levels of transcriptional repression occur by direct interaction of either YafQ-(DinJ)2-YafQ or a DinJ dimer at a single inverted repeat of its recognition sequence that overlaps with the −10 promoter region. Surprisingly, multiple YafQ-(DinJ)2-YafQ complexes binding to the operator region do not appear to amplify the extent of repression. Our results suggest an alternative model for transcriptional autorepression that may be novel to DinJ-YafQ.

Reorganization of an intersubunit bridge induced by disparate 16S ribosomal ambiguity mutations mimics an EF-Tu-bound state

After four decades of research aimed at understanding tRNA selection on the ribosome, the mechanism by which ribosomal ambiguity (ram) mutations promote miscoding remains unclear. Here, we present two X-ray crystal structures of the Thermus thermophilus 70S ribosome containing 16S rRNA ram mutations, G347U and G299A. Each of these mutations causes miscoding in vivo and stimulates elongation factor thermo unstable (EF-Tu)-dependent GTP hydrolysis in vitro. Mutation G299A is located near the interface of ribosomal proteins S4 and S5 on the solvent side of the subunit, whereas G347U is located 77 Å distant, at intersubunit bridge B8, close to where EF-Tu engages the ribosome. Despite these disparate locations, both mutations induce almost identical structural rearrangements that disrupt the B8 bridge—namely, the interaction of h8/h14 with L14 and L19. This conformation most closely resembles that seen upon EF-Tu⋅GTP⋅aminoacyl-tRNA binding to the 70S ribosome. These data provide evidence that disruption and/or distortion of B8 is an important aspect of GTPase activation. We propose that, by destabilizing B8, G299A and G347U reduce the energetic cost of attaining the GTPase-activated state and thereby decrease the stringency of decoding. This previously unappreciated role for B8 in controlling the decoding process may hold relevance for many other ribosomal mutations known to influence translational fidelity.

Defining the mRNA recognition signature of a bacterial toxin protein

Significance Bacteria have a tremendous capacity to rapidly adapt their gene expression profiles and metabolic rates through global regulatory responses. Toxin–antitoxin complexes regulate their own expression under exponential growth but inhibit energy-demanding processes like protein synthesis during stress. A majority of toxins display exquisite endonucleolytic specificity for mRNAs but only in the context of the ribosome. The molecular basis for this selectivity is unclear given their simple microbial RNase architecture. Here, we demonstrate the mechanistic determinants for host inhibition of growth B (HigB) toxin selection of mRNA substrates. Moreover, we propose that ribosome-dependent toxins recognize their mRNA substrates primarily through identification of the third nucleotide of the codon, contrary to how tRNAs and other translation factors also recognize the A site. Bacteria contain multiple type II toxins that selectively degrade mRNAs bound to the ribosome to regulate translation and growth and facilitate survival during the stringent response. Ribosome-dependent toxins recognize a variety of three-nucleotide codons within the aminoacyl (A) site, but how these endonucleases achieve substrate specificity remains poorly understood. Here, we identify the critical features for how the host inhibition of growth B (HigB) toxin recognizes each of the three A-site nucleotides for cleavage. X-ray crystal structures of HigB bound to two different codons on the ribosome illustrate how HigB uses a microbial RNase-like nucleotide recognition loop to recognize either cytosine or adenosine at the second A-site position. Strikingly, a single HigB residue and 16S rRNA residue C1054 form an adenosine-specific pocket at the third A-site nucleotide, in contrast to how tRNAs decode mRNA. Our results demonstrate that the most important determinant for mRNA cleavage by ribosome-dependent toxins is interaction with the third A-site nucleotide.

Molecular basis of ribosome recognition and mRNA hydrolysis by the E. coli YafQ toxin

Bacterial type II toxin-antitoxin modules are protein–protein complexes whose functions are finely tuned by rapidly changing environmental conditions. E. coli toxin YafQ is suppressed under steady state growth conditions by virtue of its interaction with its cognate antitoxin, DinJ. During stress, DinJ is proteolytically degraded and free YafQ halts translation by degrading ribosome-bound mRNA to slow growth until the stress has passed. Although structures of the ribosome with toxins RelE and YoeB have been solved, it is unclear what residues among ribosome-dependent toxins are essential for mediating both recognition of the ribosome and the mRNA substrate given their low sequence identities. Here we show that YafQ coordinates binding to the 70S ribosome via three surface-exposed patches of basic residues that we propose directly interact with 16S rRNA. We demonstrate that YafQ residues H50, H63, D67 and H87 participate in acid-base catalysis during mRNA hydrolysis and further show that H50 and H63 functionally complement as general bases to initiate the phosphodiester cleavage reaction. Moreover YafQ residue F91 likely plays an important role in mRNA positioning. In summary, our findings demonstrate the plasticity of ribosome-dependent toxin active site residues and further our understanding of which toxin residues are important for function.

Macrolide-Peptide Conjugates as Probes of the Path of Travel of the Nascent Peptides through the Ribosome.

Despite decades of research on the bacterial ribosome, the ribosomal exit tunnel is still poorly understood. Although it has been suggested that the exit tunnel is simply a convenient route of egress for the nascent chain, specific protein sequences serve to slow the rate of translation, suggesting some degree of interaction between the nascent peptide chain and the exit tunnel. To understand how the ribosome interacts with nascent peptide sequences, we synthesized and characterized a novel class of probe molecules. These peptide–macrolide (or “peptolide”) conjugates were designed to present unique peptide sequences to the exit tunnel. Biochemical and X-ray structural analyses of the interactions between these probes and the ribosome reveal interesting insights about the exit tunnel. Using translation inhibition and RNA structure probing assays, we find the exit tunnel has a relaxed preference for the directionality (N → C or C → N orientation) of the nascent peptides. Moreover, the X-ray crystal structure of one peptolide derived from a positively charged, reverse Nuclear Localization Sequence peptide, bound to the 70S bacterial ribosome, reveals that the macrolide ring of the peptolide binds in the same position as other macrolides. However, the peptide tail folds over the macrolide ring, oriented toward the peptidyl transferase center and interacting in a novel manner with 23S rRNA residue C2442 and His69 of ribosomal protein L4. These data suggest that these peptolides are viable probes for interrogating nascent peptide–exit tunnel interaction.