David Bulkley

Revisiting the structures of several antibiotics bound to the bacterial ribosome.

The increasing prevalence of antibiotic-resistant pathogens reinforces the need for structures of antibiotic-ribosome complexes that are accurate enough to enable the rational design of novel ribosome-targeting therapeutics. Structures of many antibiotics in complex with both archaeal and eubacterial ribosomes have been determined, yet discrepancies between several of these models have raised the question of whether these differences arise from species-specific variations or from experimental problems. Our structure of chloramphenicol in complex with the 70S ribosome from Thermus thermophilus suggests a model for chloramphenicol bound to the large subunit of the bacterial ribosome that is radically different from the prevailing model. Further, our structures of the macrolide antibiotics erythromycin and azithromycin in complex with a bacterial ribosome are indistinguishable from those determined of complexes with the 50S subunit of Haloarcula marismortui, but differ significantly from the models that have been published for 50S subunit complexes of the eubacterium Deinococcus radiodurans. Our structure of the antibiotic telithromycin bound to the T. thermophilus ribosome reveals a lactone ring with a conformation similar to that observed in the H. marismortui and D. radiodurans complexes. However, the alkyl-aryl moiety is oriented differently in all three organisms, and the contacts observed with the T. thermophilus ribosome are consistent with biochemical studies performed on the Escherichia coli ribosome. Thus, our results support a mode of macrolide binding that is largely conserved across species, suggesting that the quality and interpretation of electron density, rather than species specificity, may be responsible for many of the discrepancies between the models.

Structural Basis for the Rescue of Stalled Ribosomes: Structure of YaeJ Bound to the Ribosome

Ribosome Rescue Ribosomes stall when they reach the end of defective messenger RNAs (mRNAs). In bacteria, the most-studied ribosomal rescue pathway involves a ribonucleoprotein complex comprising tmRNA (which acts as both transfer RNA and mRNA) and the protein SmpB. In an alternative pathway, some Gram-negative bacteria contain proteins that achieve tmRNA-independent rescue. Now, Neubauer et al. (p. 1366) present the structure of the Thermus thermophilus ribosome bound to a fragment of tmRNA, SmpB, and elongation factor Tu, and Gagnon et al. (p. 1370) report the structure of the T. thermophilus ribosome in complex with an initiator tRNA, a short mRNA fragment, and the rescue factor YaeJ. Though the two rescue systems are very different, both involve a protein tail that binds in the mRNA channel. This orients the rescue apparatus to facilitate switching translation to a different message in the tmRNA system or hydrolysis of peptidyl tRNA by YaeJ. Two crystal structures show the molecular bases for two pathways that rescue ribosomes that have stalled on defective messenger RNAs. In bacteria, the hybrid transfer-messenger RNA (tmRNA) rescues ribosomes stalled on defective messenger RNAs (mRNAs). However, certain gram-negative bacteria have evolved proteins that are capable of rescuing stalled ribosomes in a tmRNA-independent manner. Here, we report a 3.2 angstrom–resolution crystal structure of the rescue factor YaeJ bound to the Thermus thermophilus 70S ribosome in complex with the initiator tRNAifMet and a short mRNA. The structure reveals that the C-terminal tail of YaeJ functions as a sensor to discriminate between stalled and actively translating ribosomes by binding in the mRNA entry channel downstream of the A site between the head and shoulder of the 30S subunit. This allows the N-terminal globular domain to sample different conformations, so that its conserved GGQ motif is optimally positioned to catalyze the hydrolysis of peptidyl-tRNA. This structure gives insights into the mechanism of YaeJ function and provides a basis for understanding how it rescues stalled ribosomes.

Crystal structure of elongation factor 4 bound to a clockwise ratcheted ribosome

Better blood thinner, without bleeding Blood thinners prevent heart attacks and strokes by making it harder for blood to clot, but these drugs can put patients at risk of dangerous bleeding. Now Moeckle et al. describe an enzyme that can prevent clots without this perilous side effect. They engineered the enzyme apyrase to remove the pro-clotting molecule ADP from the blood quickly. In dogs and mice with heart attacks, apyrase stopped blood cells from aggregating, the first step in forming a clot. At the highest dose, the animals suffered less heart damage and did not bleed excessively. In comparison, clopidogrel, a blood thinner used currently in patients, protected the heart less well and did cause excessive bleeding. Science, this issue p. 684 In an EF4-ribosome complex, the ribosome is in a state that remodels the decoding center. Elongation factor 4 (EF4/LepA) is a highly conserved guanosine triphosphatase translation factor. It was shown to promote back-translocation of tRNAs on posttranslocational ribosome complexes and to compete with elongation factor G for interaction with pretranslocational ribosomes, inhibiting the elongation phase of protein synthesis. Here, we report a crystal structure of EF4–guanosine diphosphate bound to the Thermus thermophilus ribosome with a P-site tRNA at 2.9 angstroms resolution. The C-terminal domain of EF4 reaches into the peptidyl transferase center and interacts with the acceptor stem of the peptidyl-tRNA in the P site. The ribosome is in an unusual state of ratcheting with the 30S subunit rotated clockwise relative to the 50S subunit, resulting in a remodeled decoding center. The structure is consistent with EF4 functioning either as a back-translocase or a ribosome sequester.

The antibiotic thermorubin inhibits protein synthesis by binding to inter-subunit bridge B2a of the ribosome

Thermorubin is a small-molecule inhibitor of bacterial protein synthesis, but relatively little is known about the molecular mechanism by which it blocks translation. The structure of the complex between thermorubin and the 70S ribosome from Thermus thermophilus reported here shows that thermorubin interacts with the ribosome in a way that is distinct from any other known class of ribosome inhibitor. Though it is structurally similar to tetracycline, it binds to the ribosome at an entirely different location-the interface between the small and large subunits that is formed by inter-subunit bridge B2a. This region of the ribosome is known to play a role in the initiation of translation, and thus, the binding site we observe is consistent with evidence suggesting that thermorubin inhibits the initiation stage of protein synthesis. The binding of thermorubin induces a rearrangement of two bases on helix 69 of the 23S rRNA, and presumably, this rearrangement blocks the binding of an A-site tRNA, thereby inhibiting peptide bond formation. Due in part to its low solubility in aqueous media, thermorubin has not been used clinically, although it is a potent antibacterial agent with low toxicity (Therapeutic Index>200). The interactions between thermorubin and the ribosome, as well as its adjacency to the observed binding sites of three other antibiotic classes, may enable the design of novel derivatives that share thermorubins mode of action but possess improved pharmacodynamic properties.

The antibiotics dityromycin and GE82832 bind protein S12 and block EF-G-catalyzed translocation.

The translocation of mRNA and tRNA through the ribosome is catalyzed by elongation factor G (EF-G), a universally conserved guanosine triphosphate hydrolase (GTPase). The mechanism by which the closely related decapeptide antibiotics dityromycin and GE82832 inhibit EF-G-catalyzed translocation is elucidated in this study. Using crystallographic and biochemical experiments, we demonstrate that these antibiotics bind to ribosomal protein S12 in solution alone as well as within the small ribosomal subunit, inducing long-range effects on the ribosomal head. The crystal structure of the antibiotic in complex with the 70S ribosome reveals that the binding involves conserved amino acid residues of S12 whose mutations result in in vitro and in vivo antibiotic resistance and loss of antibiotic binding. The data also suggest that GE82832/dityromycin inhibits EF-G-catalyzed translocation by disrupting a critical contact between EF-G and S12 that is required to stabilize the posttranslocational conformation of EF-G, thereby preventing the ribosome-EF-G complex from entering a conformation productive for translocation.

Contribution of Partial Charge Interactions and Base Stacking to the Efficiency of Primer Extension at and beyond Abasic Sites in DNA.

During DNA synthesis, base stacking and Watson-Crick (WC) hydrogen bonding increase the stability of nascent base pairs when they are in a ternary complex. To evaluate the contribution of base stacking to the incorporation efficiency of dNTPs when a DNA polymerase encounters an abasic site, we varied the penultimate base pairs (PBs) adjacent to the abasic site using all 16 possible combinations. We then determined pre-steady-state kinetic parameters with an RB69 DNA polymerase variant and solved nine structures of the corresponding ternary complexes. The efficiency of incorporation for incoming dNTPs opposite an abasic site varied between 2- and 210-fold depending on the identity of the PB. We propose that the A rule can be extended to encompass the fact that DNA polymerase can bypass dA/abasic sites more efficiently than other dN/abasic sites. Crystal structures of the ternary complexes show that the surface of the incoming base was stacked against the PBs interface and that the kinetic parameters for dNMP incorporation were consistent with specific features of base stacking, such as surface area and partial charge-charge interactions between the incoming base and the PB. Without a templating nucleotide residue, an incoming dNTP has no base with which it can hydrogen bond and cannot be desolvated, so that these surrounding water molecules become ordered and remain on the PBs surface in the ternary complex. When these water molecules are on top of a hydrophobic patch on the PB, they destabilize the ternary complex, and the incorporation efficiency of incoming dNTPs is reduced.

Structural Studies of the Functional Complexes of the 50S and 70S Ribosome, a Major Antibiotic Target

Our crystal structure of the Haloarcula marismortui (H.ma.) 50S ribosomal subunit and its complexes with substrates and antibiotics have illuminated the mechanism of peptide bond formation and its inhibition by antibiotics. Our structures of the Thermus thermophilus (T.th.) 70S ribosome complexed with tRNAs, protein factor EF-P or antibiotics have also provided insights into their mechanisms of action. We conclude that the CCA ends of the A- and P-site tRNAs bind to the 70S ribosome as the CCA fragments bind to the 50S subunit; macrolide antibiotics bind to the T.th. 70S ribosome as they bind to the H.ma. 50S subunit; EF-P binds to the 70S ribosome adjacent to and interacting with a P-site tRNA, and cryoEM maps of a 70S ribosome bound to a peptidyl-tRNA containing an arresting sequence shows an extended polypeptide interacting with the tunnel wall.

Structural studies of complexes of the 70S ribosome

During the first decade of our studies of the structural basis of ribosome functions, we concentrated our efforts on the Haloarcula marismortui (H. ma.) 50S ribosomal subunit. This resulted in our obtaining its atomic structure in 2000 from a 2.4 A resolution map (Ban et al., 2000) and the structures of many complexes with substrate analogues (Nissen et al., 2000; Hansen et al., 2002b; Schmeing et al., 2002; Schmeing et al., 2005b) and antibiotics (Hansen et al., 2002a; Hansen et al., 2003; Tu et al., 2005b) in the subsequent five years. More recently our focus has turned to studies of the 70S ribosome and its complexes with either protein factors, tRNAs, antibiotics or a peptidyl-tRNA with a peptide sequence that stalls protein synthesis; these 70S ribosome structures will be the primary results discussed here.