Intersubunit Bridges Research Articles

High-resolution yeast actin structures indicate the molecular mechanism of actin filament stiffening by cations

Actin filament assembly and the regulation of its mechanical properties are fundamental processes essential for eukaryotic cell function. Residue E167 in vertebrate actins forms an inter-subunit salt bridge with residue K61 of the adjacent subunit. Saccharomyces cerevisiae actin filaments are more flexible than vertebrate filaments and have an alanine at this position (A167). Substitution of this alanine for a glutamic acid (A167E) confers Saccharomyces cerevisiae actin filaments with salt-dependent stiffness similar to vertebrate actins. We developed an optimized cryogenic electron microscopy workflow refining sample preparation and vitrification to obtain near-atomic resolution structures of wild-type and A167E mutant Saccharomyces cerevisiae actin filaments. The difference between these structures allowed us to pinpoint the potential binding site of a filament-associated cation that controls the stiffness of the filaments in vertebrate and A167E Saccharomyces cerevisiae actins. Through an analysis of previously published high-resolution reconstructions of vertebrate actin filaments, along with a newly determined high-resolution vertebrate actin structure in the absence of potassium, we identified a unique peak near residue 167 consistent with the binding of a magnesium ion. Our findings show how magnesium can contribute to filament stiffening by directly bridging actin subunits and allosterically affecting the orientation of the DNase-I binding loop of actin, which plays a regulatory role in modulating actin filament stiffness and interactions with regulatory proteins.

Absence of calcium-sensing receptor basal activity due to inter-subunit disulfide bridges

G protein-coupled receptors naturally oscillate between inactive and active states, often resulting in receptor constitutive activity with important physiological consequences. Among the class C G protein-coupled receptors that typically sense amino-acids and their derivatives, the calcium sensing receptor (CaSR) tightly controls blood calcium levels. Its constitutive activity has not yet been studied. Here, we demonstrate the importance of the inter-subunit disulfide bridges in maintaining the inactive state of CaSR, resulting in undetectable constitutive activity, unlike the other class C receptors. Deletion of these disulfide bridges results in strong constitutive activity that is abolished by mutations preventing amino acid binding. It shows that this inter-subunit disulfide link is necessary to limit the agonist effect of amino acids on CaSR. Furthermore, human genetic mutations deleting these bridges and associated with hypocalcemia result in elevated CaSR constitutive activity. These results highlight the physiological importance of fine tuning the constitutive activity of G protein-coupled receptors.

Mechanistic insights into the alternative ribosome recycling by HflXr.

During stress conditions such as heat shock and antibiotic exposure, ribosomes stall on messenger RNAs, leading to inhibition of protein synthesis. To remobilize ribosomes, bacteria use rescue factors such as HflXr, a homolog of the conserved housekeeping GTPase HflX that catalyzes the dissociation of translationally inactive ribosomes into individual subunits. Here we use time-resolved cryo-electron microscopy to elucidate the mechanism of ribosome recycling by Listeria monocytogenes HflXr. Within the 70S ribosome, HflXr displaces helix H69 of the 50S subunit and induces long-range movements of the platform domain of the 30S subunit, disrupting inter-subunit bridges B2b, B2c, B4, B7a and B7b. Our findings unveil a unique ribosome recycling strategy by HflXr which is distinct from that mediated by RRF and EF-G. The resemblance between HflXr and housekeeping HflX suggests that the alternative ribosome recycling mechanism reported here is universal in the prokaryotic kingdom.

Evolving precision: rRNA expansion segment 7S modulates translation velocity and accuracy in eukaryal ribosomes.

Ribosome-enhancedtranslational miscoding of the genetic code causes protein dysfunction and loss of cellular fitness. During evolution, open reading frame length increased, necessitating mechanisms for enhanced translation fidelity. Indeed, eukaryal ribosomes are more accurate than bacterial counterparts, despite their virtually identical, conserved active centers. During the evolution of eukaryotic organisms ribosome expansions at the rRNA and protein level occurred, which potentially increases the options for translation regulation and cotranslational events. Here we tested the hypothesis that ribosomal RNA expansions can modulate the core function of the ribosome, faithful protein synthesis. We demonstrate that a short expansion segment present in all eukaryotes' small subunit, ES7S, is crucial for accurate protein synthesis as its presence adjusts codon-specific velocities and guarantees high levels of cognate tRNA selection. Deletion of ES7S in yeast enhances mistranslation and causes protein destabilization and aggregation, dramatically reducing cellular fitness. Removal of ES7S did not alter ribosome architecture but altered the structural dynamics of inter-subunit bridges thus affecting A-tRNA selection. Exchanging the yeast ES7S sequence with the human ES7S increases accuracy whereas shortening causes the opposite effect. Our study demonstrates that ES7S provided eukaryal ribosomes with higher accuracy without perturbing the structurally conserved decoding center.

Cryo-EM captures a unique conformational rearrangement in 23S rRNA helices of the Mycobacterium 50S subunit

Structural investigations of the ribosomes isolated from pathogenic and non-pathogenic Mycobacterium species have identified several mycobacteria-specific structural features of ribosomal RNA and proteins. Here, we report structural evidence of a hitherto unknown conformational switch of mycobacterium 23S rRNA helices (H54a and H67-H71). Cryo-electron microscopy (cryo-EM) structures (~3–4 Å) of the M. smegmatis (Msm) log-phase 50S ribosomal subunit revealed conformational variability in H67-H71 region of the 23S rRNA, and manifested that, while H68 possesses the usual stretched conformation in one class of the maps, another one exhibits a bulge-out, fused density of H68-H69 at the inter-subunit surface, indicating an intrinsic dynamics of these rRNA helices. Remarkably, altered conformation of H68 forming a more prominent bulge-out structure at the inter-subunit surface of the 50S subunit due to the conformational rearrangements of 23S rRNA H67-H71 region was clearly visualized in a 3 Å cryo-EM map of the 50S subunit obtained from the stationary phase ribosome dataset. The Msm50S subunit having such bulge-out conformation at the intersubunit surface would be incompatible for associating with the 30S subunit due to its inability to form major inter-subunit bridges. Evidently, availability of active 70S ribosome pool can be modulated by stabilizing either one of the H68 conformation.

Antibiotic thermorubin tethers ribosomal subunits and impedes A-site interactions to perturb protein synthesis in bacteria

Thermorubin (THB) is a long-known broad-spectrum ribosome-targeting antibiotic, but the molecular mechanism of its action was unclear. Here, our precise fast-kinetics assays in a reconstituted Escherichia coli translation system and 1.96 Å resolution cryo-EM structure of THB-bound 70S ribosome with mRNA and initiator tRNA, independently suggest that THB binding at the intersubunit bridge B2a near decoding center of the ribosome interferes with the binding of A-site substrates aminoacyl-tRNAs and class-I release factors, thereby inhibiting elongation and termination steps of bacterial translation. Furthermore, THB acts as an anti-dissociation agent that tethers the ribosomal subunits and blocks ribosome recycling, subsequently reducing the pool of active ribosomes. Our results show that THB does not inhibit translation initiation as proposed earlier and provide a complete mechanism of how THB perturbs bacterial protein synthesis. This in-depth characterization will hopefully spur efforts toward the design of THB analogs with improved solubility and effectivity against multidrug-resistant bacteria.

Insights into the molecular mechanismof translation inhibition by the ribosome-targeting antibiotic thermorubin.

Thermorubin (THR) is an aromatic anthracenopyranone antibiotic active against both Gram-positive and Gram-negative bacteria. It is known to bind to the 70S ribosome at the intersubunit bridge B2a and was thought to inhibit factor-dependent initiation of translation and obstruct the accommodation of tRNAs into the A site. Here, we show that thermorubin causes ribosomes to stall in vivo and in vitro at internal and termination codons, thereby allowing the ribosome to initiate protein synthesis and translate at least a few codons before stalling. Our biochemical data show that THR affects multiple steps of translation elongation with a significant impact on the binding stability of the tRNA in the A site, explaining premature cessation of translation. Our high-resolution crystal and cryo-EM structures of the 70S-THR complex show that THR can co-exist with P- and A-site tRNAs, explaining how ribosomes can elongate in the presence of the drug. Remarkable is the ability of THR to arrest ribosomes at the stop codons. Our data suggest that by causing structural re-arrangements in the decoding center, THR interferes with the accommodation of tRNAs or release factors into the ribosomal A site.

Modeling of Electron Hole Transport within a Small Ribosomal Subunit

—Synchronized operation of various parts of the ribosome during protein synthesis implies the presence of a coordinating pathway, however, this is still unknown. We have recently suggested that such a pathway can be based on charge transport along the transfer and ribosomal RNA molecules and localization of the charges in functionally important areas of the ribosome. In the current study, using density functional theory calculations, we show that charge carriers (electron holes) can efficiently migrate within the central element of the small ribosomal subunit—the h44 helix. Monte-Carlo modeling revealed that electron holes tend to localize in the functionally important areas of the h44 helix, near the decoding center and intersubunit bridges. On the basis of the results obtained, we suggest that charge transport and localization within the h44 helix could coordinate intersubunit ratcheting with other processes occurring during protein synthesis.

Cryo-EM structure of the ancient eukaryotic ribosome from the human parasite Giardia lamblia.

Giardiasis is a disease caused by the protist Giardia lamblia. As no human vaccines have been approved so far against it, and resistance to current drugs is spreading, new strategies for combating giardiasis need to be developed. The G. lamblia ribosome may provide a promising therapeutic target due to its distinct sequence differences from ribosomes of most eukaryotes and prokaryotes. Here, we report the cryo-electron microscopy structure of the G. lamblia (WB strain) ribosome determined at 2.75 Å resolution. The ribosomal RNA is the shortest known among eukaryotes, and lacks nearly all the eukaryote-specific ribosomal RNA expansion segments. In contrast, the ribosomal proteins are typically eukaryotic with some species-specific insertions/extensions. Most typical inter-subunit bridges are maintained except for one missing contact site. Unique structural features are located mainly at the ribosome’s periphery. These may be exploited as target sites for the design of new compounds that inhibit selectively the parasite’s ribosomal activity.

Interplay between an ATP-binding cassette F protein and the ribosome from Mycobacterium tuberculosis

EttA, energy-dependent translational throttle A, is a ribosomal factor that gates ribosome entry into the translation elongation cycle. A detailed understanding of its mechanism of action is limited due to the lack of high-resolution structures along its ATPase cycle. Here we present the cryo-electron microscopy (cryo-EM) structures of EttA from Mycobacterium tuberculosis (Mtb), referred to as MtbEttA, in complex with the Mtb 70S ribosome initiation complex (70SIC) at the pre-hydrolysis (ADPNP) and transition (ADP-VO4) states, and the crystal structure of MtbEttA alone in the post-hydrolysis (ADP) state. We observe that MtbEttA binds the E-site of the Mtb 70SIC, remodeling the P-site tRNA and the ribosomal intersubunit bridge B7a during the ribosomal ratcheting. In return, the rotation of the 30S causes conformational changes in MtbEttA, forcing the two nucleotide-binding sites (NBSs) to alternate to engage each ADPNP in the pre-hydrolysis states, followed by complete engagements of both ADP-VO4 molecules in the ATP-hydrolysis transition states. In the post-hydrolysis state, the conserved ATP-hydrolysis motifs of MtbEttA dissociate from both ADP molecules, leaving two nucleotide-binding domains (NBDs) in an open conformation. These structures reveal a dynamic interplay between MtbEttA and the Mtb ribosome, providing insights into the mechanism of translational regulation by EttA-like proteins.

Walking around Ribosomal Small Subunit: A Possible "Tourist Map" for Electron Holes.

Despite several decades of research, the physics underlying translation—protein synthesis at the ribosome—remains poorly studied. For instance, the mechanism coordinating various events occurring in distant parts of the ribosome is unknown. Very recently, we suggested that this allosteric mechanism could be based on the transport of electric charges (electron holes) along RNA molecules and localization of these charges in the functionally important areas; this assumption was justified using tRNA as an example. In this study, we turn to the ribosome and show computationally that holes can also efficiently migrate within the whole ribosomal small subunit (SSU). The potential sites of charge localization in SSU are revealed, and it is shown that most of them are located in the functionally important areas of the ribosome—intersubunit bridges, Fe4S4 cluster, and the pivot linking the SSU head to its body. As a result, we suppose that hole localization within the SSU can affect intersubunit rotation (ratcheting) and SSU head swiveling, in agreement with the scenario of electronic coordination of ribosome operation. We anticipate that our findings will improve the understanding of the translation process and advance molecular biology and medicine.

Cryo-electron microscopy structure of the 70S ribosome from Enterococcus faecalis

Enterococcus faecalis is a gram-positive organism responsible for serious infections in humans, but as with many bacterial pathogens, resistance has rendered a number of commonly used antibiotics ineffective. Here, we report the cryo-EM structure of the E. faecalis 70S ribosome to a global resolution of 2.8 Å. Structural differences are clustered in peripheral and solvent exposed regions when compared with Escherichia coli, whereas functional centres, including antibiotic binding sites, are similar to other bacterial ribosomes. Comparison of intersubunit conformations among five classes obtained after three-dimensional classification identifies several rotated states. Large ribosomal subunit protein bL31, which forms intersubunit bridges to the small ribosomal subunit, assumes different conformations in the five classes, revealing how contacts to the small subunit are maintained throughout intersubunit rotation. A tRNA observed in one of the five classes is positioned in a chimeric pe/E position in a rotated ribosomal state. The 70S ribosome structure of E. faecalis now extends our knowledge of bacterial ribosome structures and may serve as a basis for the development of novel antibiotic compounds effective against this pathogen.

Conformational activation of ribosome recycling by intra- and inter-molecular dynamics of RRF

Ribosome recycling is the final step of the cyclic process of translation, where the post-termination complex (PoTC) is disassembled by the concerted action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in the sub-second time range. Since, however, both the RRF and PoTC display highly dynamic action during this process, it is difficult to assess the molecular details of the interactions between the factors and the ribosome that are essential for rapid subunit separation. Here we characterized the molecular dynamics of RRF and PoTC by combined use of molecular dynamics simulations, single molecule fluorescence detection and single-particle cryo-EM analysis, with time resolutions in the sub-millisecond to minute range. We found that RRF displays two-layer dynamics: intra- and inter-molecular dynamics during ribosome splitting. The intra-molecular dynamics exhibits two different configurations of RRF: ‘bent’ and ‘extended’. A single-site mutant of RRF increases its propensity to the ‘extended’ conformation and leads to a higher binding affinity of RRF to the PoTC. The inter-molecular dynamics between RRF and EF-G in the PoTC reveals that the domain IV of EF-G pushes against the domain II of RRF, triggering the disruption of the major inter-subunit bridge B2a, and catalyzes the splitting.

Structural basis of the dominant inheritance of hypermethioninemia associated with the Arg264His mutation in the MAT1A gene.

Methionine adenosyltransferase (MAT) deficiency, characterized by isolated persistent hypermethioninemia (IPH), is caused by mutations in the MAT1A gene encoding MATαl, one of the major hepatic enzymes. Most of the associated hypermethioninemic conditions are inherited as autosomal recessive traits; however, dominant inheritance of hypermethioninemia is caused by an Arg264His (R264H) mutation. This mutation has been confirmed in a screening programme of newborns as the most common mutation in babies with IPH. Arg264 makes an inter-subunit salt bridge located at the dimer interface where the active site assembles. Here, it is demonstrated that the R264H mutation results in greatly reduced MAT activity, while retaining its ability to dimerize, indicating that the lower activity arises from alteration at the active site. The first crystallographic structure of the apo form of the wild-type MATαl enzyme is provided, which shows a tetrameric assembly in which two compact dimers combine to form a catalytic tetramer. In contrast, the crystal structure of the MATαl R264H mutant reveals a weaker dimeric assembly, suggesting that the mutation lowers the affinity for dimer-dimer interaction. The formation of a hetero-oligomer with the regulatory MATβV1 subunit or incubation with a quinolone-based compound (SCR0911) results in the near-full recovery of the enzymatic activity of the pathogenic mutation R264H, opening a clear avenue for a therapeutic solution based on chemical interventions that help to correct the defect of the enzyme in its ability to metabolize methionine.

The structural basis for inhibition of ribosomal translocation by viomycin

Viomycin, an antibiotic that has been used to fight tuberculosis infections, is believed to block the translocation step of protein synthesis by inhibiting ribosomal subunit dissociation and trapping the ribosome in an intermediate state of intersubunit rotation. The mechanism by which viomycin stabilizes this state remains unexplained. To address this, we have determined cryo-EM and X-ray crystal structures of Escherichia coli 70S ribosome complexes trapped in a rotated state by viomycin. The 3.8-Å resolution cryo-EM structure reveals a ribosome trapped in the hybrid state with 8.6° intersubunit rotation and 5.3° rotation of the 30S subunit head domain, bearing a single P/E state transfer RNA (tRNA). We identify five different binding sites for viomycin, four of which have not been previously described. To resolve the details of their binding interactions, we solved the 3.1-Å crystal structure of a viomycin-bound ribosome complex, revealing that all five viomycins bind to ribosomal RNA. One of these (Vio1) corresponds to the single viomycin that was previously identified in a complex with a nonrotated classical-state ribosome. Three of the newly observed binding sites (Vio3, Vio4, and Vio5) are clustered at intersubunit bridges, consistent with the ability of viomycin to inhibit subunit dissociation. We propose that one or more of these same three viomycins induce intersubunit rotation by selectively binding the rotated state of the ribosome at dynamic elements of 16S and 23S rRNA, thus, blocking conformational changes associated with molecular movements that are required for translocation.

Dynamics of uS19 C-Terminal Tail during the Translation Elongation Cycle in Human Ribosomes

Ribosomes undergo multiple conformational transitions during translation elongation. Here, we report the high-resolution cryoelectron microscopy (cryo-EM) structure of the human 80S ribosome in the post-decoding pre-translocation state (classical-PRE) at 3.3-Å resolution along with the rotated (hybrid-PRE) and the post-translocation states (POST). The classical-PRE state ribosome structure reveals a previously unobserved interaction between the C-terminal region of the conserved ribosomal protein uS19 and the A- and P-site tRNAs and the mRNA in the decoding site. In addition to changes in the inter-subunit bridges, analysis of different ribosomal conformations reveals the dynamic nature of this domain and suggests a role in tRNA accommodation and translocation during elongation. Furthermore, we show that disease-associated mutations in uS19 result in increased frameshifting. Together, this structure-function analysis provides mechanistic insights into the role of the uS19 C-terminal tail in the context of mammalian ribosomes.

Structural basis for ribosome recycling by RRF and tRNA.

The bacterial ribosome is recycled into subunits by two conserved proteins, elongation factor G (EF-G) and the ribosome recycling factor (RRF). The molecular basis for ribosome recycling by RRF and EF-G remains unclear. Here, we report the crystal structure of a posttermination Thermus thermophilus 70S ribosome complexed with EF-G, RRF and two transfer RNAs at a resolution of 3.5 Å. The deacylated tRNA in the peptidyl (P) site moves into a previously unsuspected state of binding (peptidyl/recycling, p/R) that is analogous to that seen during initiation. The terminal end of the p/R-tRNA forms nonfavorable contacts with the 50S subunit while RRF wedges next to central inter-subunit bridges, illuminating the active roles of tRNA and RRF in dissociation of ribosomal subunits. The structure uncovers a missing snapshot of tRNA as it transits between the P and exit (E) sites, providing insights into the mechanisms of ribosome recycling and tRNA translocation.

Functional Interactions of Ribosomal Intersubunit Bridges in Saccharomyces cerevisiae.

Ribosomes of Archaea and Eukarya share higher homology with each other than with bacterial ribosomes. For example, there is a set of 35 r-proteins that are specific only for archaeal and eukaryotic ribosomes. Three of these proteins-eL19, eL24, and eL41-participate in interactions between ribosomal subunits. The eukaryote-specific extensions of r-proteins eL19 and eL24 form two intersubunit bridges eB12 and eB13, which are present only in eukaryotic ribosomes. The third r-protein, eL41, forms bridge eB14. Notably, eL41 is found in all eukaryotes but only in some Archaea. It has been shown that bridges eB12 and eB13 are needed for efficient translation, while r-protein eL41 plays a minor role in ribosome function. Here, the functional interactions between intersubunit bridges were studied using budding yeast strains lacking different combinations of the abovementioned bridges/proteins. The growth phenotypes, levels of in vivo translation, ribosome-polysome profiles, and in vitro association of ribosomal subunits were analyzed. The results show a genetic interaction between r-protein eL41 and the eB12 bridge-forming region of eL19, and between r-proteins eL41 and eL24. It was possible to construct viable yeast strains with Archaea-like ribosomes lacking two or three eukaryote-specific bridges. These strains display slow growth and a poor translation phenotype. In addition, bridges eB12 and eB13 appear to cooperate during ribosome subunit association. These results indicate that nonessential structural elements of r-proteins become highly important in the context of disturbed subunit interactions. Therefore, eukaryote-specific bridges may contribute to the evolutionary success of eukaryotic translation machinery.

Structure of Pseudomonas aeruginosa ribosomes from an aminoglycoside-resistant clinical isolate

Resistance to antibiotics has become a major threat to modern medicine. The ribosome plays a fundamental role in cell vitality by the translation of the genetic code into proteins; hence, it is a major target for clinically useful antibiotics. We report here the cryo-electron microscopy structures of the ribosome of a pathogenic aminoglycoside (AG)-resistant Pseudomonas aeruginosa strain, as well as of a nonresistance strain isolated from a cystic fibrosis patient. The structural studies disclosed defective ribosome complex formation due to a conformational change of rRNA helix H69, an essential intersubunit bridge, and a secondary binding site of the AGs. In addition, a stable conformation of nucleotides A1486 and A1487, pointing into helix h44, is created compared to a non-AG-bound ribosome. We suggest that altering the conformations of ribosomal protein uL6 and rRNA helix H69, which interact with initiation-factor IF2, interferes with proper protein synthesis initiation.

Extensive ribosome and RF2 rearrangements during translation termination.

Protein synthesis ends when a ribosome reaches an mRNA stop codon. Release factors (RFs) decode the stop codon, hydrolyze peptidyl-tRNA to release the nascent protein, and then dissociate to allow ribosome recycling. To visualize termination by RF2, we resolved a cryo-EM ensemble of E. coli 70S•RF2 structures at up to 3.3 Å in a single sample. Five structures suggest a highly dynamic termination pathway. Upon peptidyl-tRNA hydrolysis, the CCA end of deacyl-tRNA departs from the peptidyl transferase center. The catalytic GGQ loop of RF2 is rearranged into a long β-hairpin that plugs the peptide tunnel, biasing a nascent protein toward the ribosome exit. Ribosomal intersubunit rotation destabilizes the catalytic RF2 domain on the 50S subunit and disassembles the central intersubunit bridge B2a, resulting in RF2 departure. Our structures visualize how local rearrangements and spontaneous inter-subunit rotation poise the newly-made protein and RF2 to dissociate in preparation for ribosome recycling.