Structural Basis for Interaction of the Ribosome with the Switch Regions of GTP-Bound Elongation Factors

Self Cite

160

141

Ribosome protection proteins (RPPs) confer tetracycline resistance by binding to the ribosome and chasing the drug from its binding site. The current model for the mechanism of action of RPPs proposes that drug release is indirect and achieved via conformational changes within the drug-binding site induced upon binding of the RPP to the ribosome. Here we report a cryo-EM structure of the RPP TetM in complex with the 70S ribosome at 7.2-Å resolution. The structure reveals the contacts of TetM with the ribosome, including interaction between the conserved and functionally critical C-terminal extension of TetM and the decoding center of the small subunit. Moreover, we observe direct interaction between domain IV of TetM and the tetracycline binding site and identify residues critical for conferring tetracycline resistance. A model is presented whereby TetM directly dislodges tetracycline to confer resistance.antibiotic | protein synthesis | RNA | tigecycline | translation

Section: Resultssupporting

confidence: 81%

“…A large additional density within the subunit interface was attributed to TetM (Fig. 1A), as expected from the similarity in location of TetO (14) and EF-G (20)(21)(22) on the ribosome. In the absence of a crystal structure of any RPP, a homology model for TetM was built on the basis of the high sequence similarity between TetM and EF-G (10).…”

Section: Resultssupporting

confidence: 66%

Structural basis for TetM-mediated tetracycline resistance

Dönhöfer

Franckenberg

Wickles

et al. 2012

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160

141

“…1 and Fig. S3), in agreement with conclusions based on the results of numerous other approaches (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37). In some cases, multiconformer analysis may provide information about molecular motions that is not evident from TLS-derived B factor values alone.…”

Section: Resultssupporting

confidence: 88%

Multistart simulated annealing refinement of the crystal structure of the 70S ribosome

Коростелев

Laurberg

Noller

2009

A macromolecular X-ray crystal structure is usually represented as a single static model with a single set of temperature factors representing a simple approximation of motion and disorder of the structure. Multiconformer representations of small proteins have been shown to better describe anisotropic motion and disorder and improve the quality of their electron density maps. Here, we apply multistart simulated annealing crystallographic refinement to a 70S ribosome-RF1 translation termination complex that was recently solved at 3.2 Å resolution. The analysis improves the interpretability of the electron density map of this 2.5-MDa ribonucleoprotein complex and provides insights into its structural dynamics. We also used multistart refinement and conventional Fourier difference maps to address a recent study in which cross-crystal averaging between two crystal forms of the 70S ribosome was used to evaluate reported differences between two ribosome crystal structures solved at 2.8 and 3.7 Å resolution. Our analysis suggests that results obtained from cross-crystal averaging are inherently biased toward the higherresolution dataset.multistart refinement ͉ protein synthesis ͉ ribosome dynamics ͉ rRNA C rystallographic techniques and methods of structure determination have progressed rapidly in recent years, accompanied by an explosion in the amount of structural information about biological molecules and macromolecular assemblies. X-ray crystal structures have traditionally been represented by a single set of atomic coordinates accompanied by a set of temperature factors (B factors) to characterize atomic thermal motion. At most experimentally attainable resolutions, a spherical approximation for thermal motion is used, accounting only for the isotropic component of atomic displacement. It has been shown that the use of a static model with isotropic thermal parameters underestimates the total disorder and conformational variability of macromolecules (1-5). The limitation of the single-model approach is exemplified by several cases where high-resolution data are available. Nearly half the amino acid side chains and even some backbone atoms were found to have alternative conformations in the 0.54 Å structure of crambin and in the 1.0 Å structure of calmodulin (1, 6). To account for anharmonic contributions and conformational variability in X-ray structures, the use of an ensemble of models, as has long been standard practice in structure determination by NMR, has been suggested as a replacement for the traditional single-model approach (7). Multiconformer refinement has been implemented using both real-and reciprocal-space refinement approaches (2,3,(8)(9)(10)(11)(12)(13) and was demonstrated to yield improved agreement with diffraction data in most cases. In the case of molecular replacement phasing, electron density maps are often biased toward the search model; when a structure ensemble is used, however, model bias can be reduced significantly (14).In X-ray structures of the ribosome, the largest asymmetric struct...

“…Besides the high sequence and structure similarities, trGTPases share the way of association with the large ribosomal subunit, close to the A-site. Biochemical and structural studies identified the sarcin-ricin loop (SRL) of 23S rRNA as one of the most pivotal rRNA elements for trGTPase functioning (8)(9)(10)(11)(12). The SRL represents one of the most highly conserved sequences of ribosomal RNA, and its tip has been shown to closely approach the bound GTP molecule in the respective G domains of trGTPases (9,12).…”

mentioning

confidence: 99%

Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

Koch

Flür

Kreutz

et al. 2015

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.