30S subunit recognition and G1405 modification by the aminoglycoside-resistance 16S ribosomal RNA methyltransferase RmtC.

Acquired ribosomal RNA (rRNA) methylation has emerged as a significant mechanism of aminoglycoside resistance in pathogenic bacterial infections. Modification of a single nucleotide in the ribosome decoding center by the aminoglycoside-resistance 16S rRNA (m7G1405) methyltransferases effectively blocks the action of all 4,6-deoxystreptamine ring-containing aminoglycosides, including the latest generation of drugs. To define the molecular basis of 30S subunit recognition and G1405 modification by these enzymes, we used a S-adenosyl-L-methionine analog to trap the complex in a postcatalytic state to enable determination of a global 3.0 Å cryo-electron microscopy structure of the m7G1405 methyltransferase RmtC bound to the mature Escherichia coli 30S ribosomal subunit. This structure, together with functional analyses of RmtC variants, identifies the RmtC N-terminal domain as critical for recognition and docking of the enzyme on a conserved 16S rRNA tertiary surface adjacent to G1405 in 16S rRNA helix 44 (h44). To access the G1405 N7 position for modification, a collection of residues across one surface of RmtC, including a loop that undergoes a disorder-to order transition upon 30S subunit binding, induces significant distortion of h44. This distortion flips G1405 into the enzyme active site where it is positioned for modification by two almost universally conserved RmtC residues. These studies expand our understanding of ribosome recognition by rRNA modification enzymes and present a more complete structural basis for future development of strategies to inhibit m7G1405 modification to resensitize bacterial pathogens to aminoglycosides.

Structural analysis of noncanonical translation initiation complexes.

Translation initiation is a highly regulated, multi-step process that is critical for efficient and accurate protein synthesis. In bacteria, initiation begins when mRNA, initiation factors, and a dedicated initiator fMet-tRNAfMet bind the small (30S) ribosomal subunit. Specific binding of fMet-tRNAfMet in the peptidyl (P) site is mediated by the inspection of the fMet moiety by initiation factor IF2 and of three conserved G-C base pairs in the tRNA anticodon stem by the 30S head domain. Tandem A-minor interactions form between 16S ribosomal RNA nucleotides A1339 and G1338 and tRNA base pairs G30-C40 and G29-C41, respectively. Swapping the G30-C40 pair of tRNAfMet with C-G (called tRNAfMet M1) reduces discrimination against the noncanonical start codon CUG in vitro, suggesting crosstalk between the gripping of the anticodon stem and recognition of the start codon. Here, we solved electron cryomicroscopy structures of Escherichia coli 70S initiation complexes containing the fMet-tRNAfMet M1 variant paired to the noncanonical CUG start codon, in the presence or absence of IF2 and the non-hydrolyzable GTP analog GDPCP, alongside structures of 70S initiation complexes containing this tRNAfMet variant paired to the canonical bacterial start codons AUG, GUG, and UUG. We find that the M1 mutation weakens A-minor interactions between tRNAfMet and 16S nucleotides A1339 and G1338, with IF2 strengthening the interaction of G1338 with the tRNA minor groove. These structures suggest how even slight changes to the recognition of the fMet-tRNAfMet anticodon stem by the ribosome can impact the start codon selection.

Ratchet, swivel, tilt and roll: a complete description of subunit rotation in the ribosome.

Protein synthesis by the ribosome requires large-scale rearrangements of the 'small' subunit (SSU; ∼1 MDa), including inter- and intra-subunit rotational motions. However, with nearly 2000 structures of ribosomes and ribosomal subunits now publicly available, it is exceedingly difficult to design experiments based on analysis of all known rotation states. To overcome this, we developed an approach where the orientation of each SSU head and body is described in terms of three angular coordinates (rotation, tilt and tilt direction) and a single translation. By considering the entire RCSB PDB database, we describe 1208 fully-assembled ribosome complexes and 334 isolated small subunits, which span >50 species. This reveals aspects of subunit rearrangements that are universal, and others that are organism/domain-specific. For example, we show that tilt-like rearrangements of the SSU body (i.e. 'rolling') are pervasive in both prokaryotic and eukaryotic (cytosolic and mitochondrial) ribosomes. As another example, domain orientations associated with frameshifting in bacteria are similar to those found in eukaryotic ribosomes. Together, this study establishes a common foundation with which structural, simulation, single-molecule and biochemical efforts can more precisely interrogate the dynamics of this prototypical molecular machine.

50S subunit recognition and modification by the Mycobacterium tuberculosis ribosomal RNA methyltransferase TlyA.

Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. Loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2'-O-methylations within two structurally distinct contexts. Here, we report the structure of a mycobacterial 50S subunit-TlyA complex trapped in a postcatalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. Together with complementary functional analyses, this structure reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144, which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. Base flipping may thus be a common strategy among rRNA methyltransferase enzymes, even in cases where the target site is accessible without such structural reorganization. Finally, functional studies with 30S subunit suggest that the same TlyA interaction surface is employed to recognize this second substrate, but with distinct dependencies on essential conserved residues.

Structural basis for reduced ribosomal A-site fidelity in response to P-site codon-anticodon mismatches.

Rapid and accurate translation is essential in all organisms to produce properly folded and functional proteins. mRNA codons that define the protein-coding sequences are decoded by tRNAs on the ribosome in the aminoacyl (A) binding site. The mRNA codon and the tRNA anticodon interaction is extensively monitored by the ribosome to ensure accuracy in tRNA selection. While other polymerases that synthesize DNA and RNA can correct for misincorporations, the ribosome is unable to correct mistakes. Instead, when a misincorporation occurs, the mismatched tRNA-mRNA pair moves to the peptidyl (P) site and, from this location, causes a reduction in the fidelity at the A site, triggering post-peptidyl transfer quality control. This reduced fidelity allows for additional incorrect tRNAs to be accepted and for release factor 2 (RF2) to recognize sense codons, leading to hydrolysis of the aberrant peptide. Here, we present crystal structures of the ribosome containing a tRNALys in the P site with a U•U mismatch with the mRNA codon. We find that when the mismatch occurs in the second position of the P-site codon-anticodon interaction, the first nucleotide of the A-site codon flips from the mRNA path to engage highly conserved 16S rRNA nucleotide A1493 in the decoding center. We propose that this mRNA nucleotide mispositioning leads to reduced fidelity at the A site. Further, this state may provide an opportunity for RF2 to initiate premature termination before erroneous nascent chains disrupt the cellular proteome.

The structural basis for mRNA recognition and cleavage by the ribosome-dependent endonuclease RelE.

Translational control is widely used to adjust gene expression levels. During the stringent response in bacteria, mRNA is degraded on the ribosome by the ribosome-dependent endonuclease, RelE. The molecular basis for recognition of the ribosome and mRNA by RelE and the mechanism of cleavage are unknown. Here, we present crystal structures of E. coli RelE in isolation (2.5 A) and bound to programmed Thermus thermophilus 70S ribosomes before (3.3 A) and after (3.6 A) cleavage. RelE occupies the A site and causes cleavage of mRNA after the second nucleotide of the codon by reorienting and activating the mRNA for 2'-OH-induced hydrolysis. Stacking of A site codon bases with conserved residues in RelE and 16S rRNA explains the requirement for the ribosome in catalysis and the subtle sequence specificity of the reaction. These structures provide detailed insight into the translational regulation on the bacterial ribosome by mRNA cleavage.

Druggable differences: Targeting mechanistic differences between trans-translation and translation for selective antibiotic action.

Bacteria use trans-translation to rescue stalled ribosomes and target incomplete proteins for proteolysis. Despite similarities between tRNAs and transfer-messenger RNA (tmRNA), the key molecule for trans-translation, new structural and biochemical data show important differences between translation and trans-translation at most steps of the pathways. tmRNA and its binding partner, SmpB, bind in the A site of the ribosome but do not trigger the same movements of nucleotides in the rRNA that are required for codon recognition by tRNA. tmRNA-SmpB moves from the A site to the P site of the ribosome without subunit rotation to generate hybrid states, and moves from the P site to a site outside the ribosome instead of to the E site. During catalysis, transpeptidation to tmRNA appears to require the ribosomal protein bL27, which is dispensable for translation, suggesting that this protein may be conserved in bacteria due to trans-translation. These differences provide insights into the fundamental nature of trans-translation, and provide targets for new antibiotics that may have decrease cross-reactivity with eukaryotic ribosomes.

Oxidation alters the architecture of the phenylalanyl-tRNA synthetase editing domain to confer hyperaccuracy.

High fidelity during protein synthesis is accomplished by aminoacyl-tRNA synthetases (aaRSs). These enzymes ligate an amino acid to a cognate tRNA and have proofreading and editing capabilities that ensure high fidelity. Phenylalanyl-tRNA synthetase (PheRS) preferentially ligates a phenylalanine to a tRNAPhe over the chemically similar tyrosine, which differs from phenylalanine by a single hydroxyl group. In bacteria that undergo exposure to oxidative stress such as Salmonella enterica serovar Typhimurium, tyrosine isomer levels increase due to phenylalanine oxidation. Several residues are oxidized in PheRS and contribute to hyperactive editing, including against mischarged Tyr-tRNAPhe, despite these oxidized residues not being directly implicated in PheRS activity. Here, we solve a 3.6 Å cryo-electron microscopy structure of oxidized S. Typhimurium PheRS. We find that oxidation results in widespread structural rearrangements in the ß-subunit editing domain and enlargement of its editing domain. Oxidization also enlarges the phenylalanyl-adenylate binding pocket but to a lesser extent. Together, these changes likely explain why oxidation leads to hyperaccurate editing and decreased misincorporation of tyrosine. Taken together, these results help increase our understanding of the survival of S. Typhimurium during human infection.

Disruption of evolutionarily correlated tRNA elements impairs accurate decoding.

Bacterial transfer RNAs (tRNAs) contain evolutionarily conserved sequences and modifications that ensure uniform binding to the ribosome and optimal translational accuracy despite differences in their aminoacyl attachments and anticodon nucleotide sequences. In the tRNA anticodon stem-loop, the anticodon sequence is correlated with a base pair in the anticodon loop (nucleotides 32 and 38) to tune the binding of each tRNA to the decoding center in the ribosome. Disruption of this correlation renders the ribosome unable to distinguish correct from incorrect tRNAs. The molecular basis for how these two tRNA features combine to ensure accurate decoding is unclear. Here, we solved structures of the bacterial ribosome containing either wild-type [Formula: see text] or [Formula: see text] containing a reversed 32-38 pair on cognate and near-cognate codons. Structures of wild-type [Formula: see text] bound to the ribosome reveal 23S ribosomal RNA (rRNA) nucleotide A1913 positional changes that are dependent on whether the codon-anticodon interaction is cognate or near cognate. Further, the 32-38 pair is destabilized in the context of a near-cognate codon-anticodon pair. Reversal of the pairing in [Formula: see text] ablates A1913 movement regardless of whether the interaction is cognate or near cognate. These results demonstrate that disrupting 32-38 and anticodon sequences alters interactions with the ribosome that directly contribute to misreading.

Ribosomal ambiguity (ram) mutations promote the open (off) to closed (on) transition and thereby increase miscoding.

Decoding is thought to be governed by a conformational transition in the ribosome-open (off) to closed (on)-that occurs upon codon-anticodon pairing in the A site. Ribosomal ambiguity (ram) mutations increase miscoding and map to disparate regions, consistent with a role for ribosome dynamics in decoding, yet precisely how these mutations act has been unclear. Here, we solved crystal structures of 70S ribosomes harboring 16S ram mutations G299A and G347U in the absence A-site tRNA (A-tRNA) and in the presence of a near-cognate anticodon stem-loop (ASL). In the absence of an A-tRNA, each of the mutant ribosomes exhibits a partially closed (on) state. In the 70S-G347U structure, the 30S shoulder is rotated inward and intersubunit bridge B8 is disrupted. In the 70S-G299A structure, the 30S shoulder is rotated inward and decoding nucleotide G530 flips into the anti conformation. Both of these mutant ribosomes adopt the fully closed (on) conformation in the presence of near-cognate A-tRNA, just as they do with cognate A-tRNA. Thus, these ram mutations act by promoting the open (off) to closed (on) transition, albeit in somewhat distinct ways. This work reveals the functional importance of 30S shoulder rotation for productive aminoacylated-tRNA incorporation.

Monomeric YoeB toxin retains RNase activity but adopts an obligate dimeric form for thermal stability.

Chromosomally-encoded toxin-antitoxin complexes are ubiquitous in bacteria and regulate growth through the release of the toxin component typically in a stress-dependent manner. Type II ribosome-dependent toxins adopt a RelE-family RNase fold and inhibit translation by degrading mRNAs while bound to the ribosome. Here, we present biochemical and structural studies of the Escherichia coli YoeB toxin interacting with both a UAA stop and an AAU sense codon in pre- and post-mRNA cleavage states to provide insights into possible mRNA substrate selection. Both mRNAs undergo minimal changes during the cleavage event in contrast to type II ribosome-dependent RelE toxin. Further, the 16S rRNA decoding site nucleotides that monitor the mRNA in the aminoacyl(A) site adopt different orientations depending upon which toxin is present. Although YoeB is a RelE family member, it is the sole ribosome-dependent toxin that is dimeric. We show that engineered monomeric YoeB is active against mRNAs bound to both the small and large subunit. However, the stability of monomeric YoeB is reduced ∼20°C, consistent with potential YoeB activation during heat shock in E. coli as previously demonstrated. These data provide a molecular basis for the ability of YoeB to function in response to thermal stress.

Mechanism of tRNA-mediated +1 ribosomal frameshifting.

Accurate translation of the genetic code is critical to ensure expression of proteins with correct amino acid sequences. Certain tRNAs can cause a shift out of frame (i.e., frameshifting) due to imbalances in tRNA concentrations, lack of tRNA modifications or insertions or deletions in tRNAs (called frameshift suppressors). Here, we determined the structural basis for how frameshift-suppressor tRNASufA6 (a derivative of tRNAPro) reprograms the mRNA frame to translate a 4-nt codon when bound to the bacterial ribosome. After decoding at the aminoacyl (A) site, the crystal structure of the anticodon stem-loop of tRNASufA6 bound in the peptidyl (P) site reveals ASL conformational changes that allow for recoding into the +1 mRNA frame. Furthermore, a crystal structure of full-length tRNASufA6 programmed in the P site shows extensive conformational rearrangements of the 30S head and body domains similar to what is observed in a translocation intermediate state containing elongation factor G (EF-G). The 30S movement positions tRNASufA6 toward the 30S exit (E) site disrupting key 16S rRNA-mRNA interactions that typically define the mRNA frame. In summary, this tRNA-induced 30S domain change in the absence of EF-G causes the ribosome to lose its grip on the mRNA and uncouples the canonical forward movement of the tRNAs during elongation.

Importance of a tRNA anticodon loop modification and a conserved, noncanonical anticodon stem pairing in tRNACGGProfor decoding

Modification of anticodon nucleotides allows tRNAs to decode multiple codons, expanding the genetic code. Additionally, modifications located in the anticodon loop, outside the anticodon itself, stabilize tRNA­codon interactions, increasing decoding fidelity. Anticodon loop nucleotide 37 is 3' to the anticodon and, in tRNACGGPro, is methylated at the N1 position in its nucleobase (m1G37). The m1G37 modification in tRNACGGPro stabilizes its interaction with the codon and maintains the mRNA frame. However, it is unclear how m1G37 affects binding at the decoding center to both cognate and +1 slippery codons. Here, we show that the tRNACGGProm1G37 modification is important for the association step during binding to a cognate CCG codon. In contrast, m1G37 prevented association with a slippery CCC-U or +1 codon. Similar analyses of frameshift suppressor tRNASufA6, a tRNACGGPro derivative containing an extra nucleotide in its anticodon loop that undergoes +1 frameshifting, reveal that m1G37 destabilizes interactions with both the cognate CCG and slippery codons. One reason for this destabilization is the disruption of a conserved U32·A38 nucleotide pairing in the anticodon stem through insertion of G37.5. Restoring the tRNASufA6 U32·A37.5 pairing results in a high-affinity association on the slippery CCC-U codon. Further, an X-ray crystal structure of the 70S ribosome bound to tRNASufA6 U32·A37.5 at 3.6 Å resolution shows a reordering of the anticodon loop consistent with the findings from the high-affinity measurements. Our results reveal how the tRNA modification at nucleotide 37 stabilizes interactions with the mRNA codon to preserve the mRNA frame.

Functionally critical residues in the aminoglycoside resistance-associated methyltransferase RmtC play distinct roles in 30S substrate recognition.

Methylation of the small ribosome subunit rRNA in the ribosomal decoding center results in exceptionally high-level aminoglycoside resistance in bacteria. Enzymes that methylate 16S rRNA on N7 of nucleotide G1405 (m7G1405) have been identified in both aminoglycoside-producing and clinically drug-resistant pathogenic bacteria. Using a fluorescence polarization 30S-binding assay and a new crystal structure of the methyltransferase RmtC at 3.14 Å resolution, here we report a structure-guided functional study of 30S substrate recognition by the aminoglycoside resistance-associated 16S rRNA (m7G1405) methyltransferases. We found that the binding site for these enzymes in the 30S subunit directly overlaps with that of a second family of aminoglycoside resistance-associated 16S rRNA (m1A1408) methyltransferases, suggesting that both groups of enzymes may exploit the same conserved rRNA tertiary surface for docking to the 30S. Within RmtC, we defined an N-terminal domain surface, comprising basic residues from both the N1 and N2 subdomains, that directly contributes to 30S-binding affinity. In contrast, additional residues lining a contiguous adjacent surface on the C-terminal domain were critical for 16S rRNA modification but did not directly contribute to the binding affinity. The results from our experiments define the critical features of m7G1405 methyltransferase-substrate recognition and distinguish at least two distinct, functionally critical contributions of the tested enzyme residues: 30S-binding affinity and stabilizing a binding-induced 16S rRNA conformation necessary for G1405 modification. Our study sets the scene for future high-resolution structural studies of the 30S-methyltransferase complex and for potential exploitation of unique aspects of substrate recognition in future therapeutic strategies.

Structural basis of transcriptional regulation by the HigA antitoxin.

Bacterial toxin-antitoxin systems are important factors implicated in growth inhibition and plasmid maintenance. Type II toxin-antitoxin pairs are regulated at the transcriptional level by the antitoxin itself. Here, we examined how the HigA antitoxin regulates the expression of the Proteus vulgaris higBA toxin-antitoxin operon from the Rts1 plasmid. The HigBA complex adopts a unique architecture suggesting differences in its regulation as compared to classical type II toxin-antitoxin systems. We find that the C-terminus of the HigA antitoxin is required for dimerization and transcriptional repression. Further, the HigA structure reveals that the C terminus is ordered and does not transition between disorder-to-order states upon toxin binding. HigA residue Arg40 recognizes a TpG dinucleotide in higO2, an evolutionary conserved mode of recognition among prokaryotic and eukaryotic transcription factors. Comparison of the HigBA and HigA-higO2 structures reveals the distance between helix-turn-helix motifs of each HigA monomer increases by ~4 Å in order to bind to higO2. Consistent with these data, HigBA binding to each operator is twofold less tight than HigA alone. Together, these data show the HigB toxin does not act as a co-repressor suggesting potential novel regulation in this toxin-antitoxin system.

The structure and function of Mycobacterium tuberculosis MazF-mt6 toxin provide insights into conserved features of MazF endonucleases.

Toxin-antitoxin systems are ubiquitous in prokaryotic and archaeal genomes and regulate growth in response to stress. Escherichia coli contains at least 36 putative toxin-antitoxin gene pairs, and some pathogens such as Mycobacterium tuberculosis have over 90 toxin-antitoxin operons. E. coli MazF cleaves free mRNA after encountering stress, and nine M. tuberculosis MazF family members cleave mRNA, tRNA, or rRNA. Moreover, M. tuberculosis MazF-mt6 cleaves 23S rRNA Helix 70 to inhibit protein synthesis. The overall tertiary folds of these MazFs are predicted to be similar, and therefore, it is unclear how they recognize structurally distinct RNAs. Here we report the 2.7-Å X-ray crystal structure of MazF-mt6. MazF-mt6 adopts a PemK-like fold but lacks an elongated ß1-ß2 linker, a region that typically acts as a gate to direct RNA or antitoxin binding. In the absence of an elongated ß1-ß2 linker, MazF-mt6 is unable to transition between open and closed states, suggesting that the regulation of RNA or antitoxin selection may be distinct from other canonical MazFs. Additionally, a shortened ß1-ß2 linker allows for the formation of a deep, solvent-accessible, active-site pocket, which may allow recognition of specific, structured RNAs like Helix 70. Structure-based mutagenesis and bacterial growth assays demonstrate that MazF-mt6 residues Asp-10, Arg-13, and Thr-36 are critical for RNase activity and likely catalyze the proton-relay mechanism for RNA cleavage. These results provide further critical insights into how MazF secondary structural elements adapt to recognize diverse RNA substrates.

Importance of the E. coli DinJ antitoxin carboxy terminus for toxin suppression and regulated proteolysis.

Toxin-antitoxin genes play important roles in the regulation of bacterial growth during stress. One response to stress is selective proteolysis of antitoxin proteins which releases their cognate toxin partners causing rapid inhibition of growth. The features of toxin-antitoxin complexes that are important to inhibit toxin activity as well as to release the active toxin remain elusive. Furthermore, it is unclear how antitoxins are selected for proteolysis by cellular proteases. Here, we test the minimal structural requirements of the Escherichia coli DinJ antitoxin to suppress its toxin partner, YafQ. We find that DinJ-YafQ complex formation is critically dependent on the last ten C-terminal residues of DinJ. However, deletion of these 10 DinJ residues has little effect on transcriptional autorepression suggesting that the YafQ toxin is not a critical component of the repression complex in contrast to other toxin-antitoxin systems. We further demonstrate that loop 5 preceding these ten C-terminal residues is important for Lon-mediated proteolysis. These results provide important insights into the critical interactions between toxin-antitoxin pairs necessary to inhibit toxin activity and the regulated proteolysis of antitoxins.

mRNA bound to the 30S subunit is a HigB toxin substrate.

Activation of bacterial toxins during stress results in cleavage of mRNAs in the context of the ribosome. These toxins are thought to function as global translational inhibitors yet recent studies suggest each may have distinct mRNA specificities that result in selective translation for bacterial survival. Here we demonstrate that mRNA in the context of a bacterial 30S subunit is sufficient for ribosome-dependent toxin HigB endonucleolytic activity, suggesting that HigB interferes with the initiation step of translation. We determined the X-ray crystal structure of HigB bound to the 30S, revealing that two solvent-exposed clusters of HigB basic residues directly interact with 30S 16S rRNA helices 18, 30, and 31. We further show that these HigB residues are essential for ribosome recognition and function. Comparison with other ribosome-dependent toxins RelE and YoeB reveals that each interacts with similar features of the 30S aminoacyl (A) site yet does so through presentation of diverse structural motifs.

Structural Insights into Oxygen-Dependent Signal Transduction within Globin Coupled Sensors.

In order to respond to external stimuli, bacteria have evolved sensor proteins linking external signals to intracellular outputs that can then regulate downstream pathways and phenotypes. Globin coupled sensor proteins (GCSs) serve to link environmental O2 levels to cellular processes by coupling a heme-containing sensor globin domain to a catalytic output domain. However, the mechanism by which O2 binding activates these proteins is currently unknown. To provide insights into the signaling mechanism, two distinct dimeric complexes of the isolated globin domain of the GCS from Bordetella pertussis ( BpeGlobin) were solved via X-ray crystallography in which differences in ligand-bound states were observed. Both monomers of one dimer contain Fe(II)-O2 states, while the other dimer consists of the Fe(III)-H2O and Fe(II)-O2 states. These data provide the first molecular insights into the heme pocket conformation of the active Fe(II)-O2 form of these enzymes. In addition, heme distortion modes and heme-protein interactions were found to correlate with the ligation state of the globin, suggesting that these conformational changes play a role in O2-dependent signaling. Fourier transform infrared spectroscopy (FTIR) of the full-length GCS from B. pertussis ( BpeGReg) and the closely related GCS from Pectobacterium carotovorum ssp. carotovorum ( PccGCS) confirmed the importance of an ordered water within the heme pocket and two distal residues (Tyr43 and Ser68) as hydrogen-bond donors. Taken together, this work provides mechanistic insights into BpeGReg O2 sensing and the signaling mechanisms of diguanylate cyclase-containing GCS proteins.

Mechanism of endonuclease cleavage by the HigB toxin.

Bacteria encode multiple type II toxin-antitoxin modules that cleave ribosome-bound mRNAs in response to stress. All ribosome-dependent toxin family members structurally characterized to date adopt similar microbial RNase architectures despite possessing low sequence identities. Therefore, determining which residues are catalytically important in this specialized RNase family has been a challenge in the field. Structural studies of RelE and YoeB toxins bound to the ribosome provided significant insights but biochemical experiments with RelE were required to clearly demonstrate which residues are critical for acid-base catalysis of mRNA cleavage. Here, we solved an X-ray crystal structure of the wild-type, ribosome-dependent toxin HigB bound to the ribosome revealing potential catalytic residues proximal to the mRNA substrate. Using cell-based and biochemical assays, we further determined that HigB residues His54, Asp90, Tyr91 and His92 are critical for activity in vivo, while HigB H54A and Y91A variants have the largest effect on mRNA cleavage in vitro Comparison of X-ray crystal structures of two catalytically inactive HigB variants with 70S-HigB bound structures reveal that HigB active site residues undergo conformational rearrangements likely required for recognition of its mRNA substrate. These data support the emerging concept that ribosome-dependent toxins have diverse modes of mRNA recognition.