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Affinity-purified VgaA-LC in complex with 70S ribosomes from Staphylococcus aureus

ABSTRACT:

SUBMITTER: Caillan Thomas Crowe-McAuliffe

PROVIDER: EMPIAR-10683 | biostudies-other |

REPOSITORIES: biostudies-other

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Publications

Structural basis of ABCF-mediated resistance to pleuromutilin, lincosamide, and streptogramin A antibiotics in Gram-positive pathogens.

Crowe-McAuliffe Caillan C Murina Victoriia V Turnbull Kathryn Jane KJ Kasari Marje M Mohamad Merianne M Polte Christine C Takada Hiraku H Vaitkevicius Karolis K Johansson Jörgen J Ignatova Zoya Z Atkinson Gemma C GC O'Neill Alex J AJ Hauryliuk Vasili V Wilson Daniel N DN

Nature communications 20210611 1

Target protection proteins confer resistance to the host organism by directly binding to the antibiotic target. One class of such proteins are the antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F-subtype (ARE-ABCFs), which are widely distributed throughout Gram-positive bacteria and bind the ribosome to alleviate translational inhibition from antibiotics that target the large ribosomal subunit. Here, we present single-particle cryo-EM structures of ARE-ABCF-ribosome compl ...[more]

PMID: 34117249

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Project description:During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of a stress signaling network termed the stringent response. RsgA, RbgA, Era, and HflX are four ribosome-associated GTPases (RA-GTPases) that bind to (p)ppGpp in Staphylococcus aureus. These enzymes are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) ribosome-associated states. Entry into the OFF state occurs upon hydrolysis of GTP, with GTPase activity increasing substantially upon ribosome association. When bound to (p)ppGpp, GTPase activity is inhibited, reducing 70S ribosome assembly and growth. Here, we determine how (p)ppGpp impacts RA-GTPase-ribosome interactions. We show that RA-GTPases preferentially bind to 5'-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a regulatory mechanism within the cell to shut down ribosome biogenesis during stress. Stopped-flow fluorescence and association assays reveal that when bound to (p)ppGpp, the association of RA-GTPases to ribosomal subunits is destabilized, both in vitro and within bacterial cells. Consistently, structural analysis of the ppGpp-bound RA-GTPase RsgA reveals an OFF-state conformation similar to the GDP-bound state, with the G2/switch I loop adopting a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated ribosome assembly and growth control. IMPORTANCE The stringent response is a bacterial signaling network that utilizes the nucleotides pppGpp and ppGpp to reprogram cells in order to survive nutritional stresses. However, much about how these important nucleotides control cellular reprogramming is unknown. Our previous work revealed that (p)ppGpp can bind to and inhibit the enzymatic activity of four ribosome-associated GTPases (RA-GTPases), enzymes that facilitate maturation of the 50S and 30S ribosomal subunits. Here, we examine how this occurs mechanistically and demonstrate that this interaction prevents the accommodation of RA-GTPases on ribosomal subunits both in vitro and within bacterial cells, with the ppGpp-bound state structurally mimicking the inactive GDP-bound conformation of the enzyme. We additionally reveal that these GTPase enzymes have a greater affinity for OFF-state-inducing nucleotides, which is a mechanism likely to control ribosome assembly during growth. With this, we further our understanding of how ribosome function is controlled by (p)ppGpp, enabling bacterial survival during stress.

Project description:Bacterial and eukaryotic hibernation factors prevent translation by physically blocking the decoding center of ribosomes, a phenomenon called ribosome hibernation that often occurs in response to nutrient deprivation. The human pathogen Staphylococcus aureus lacking the sole hibernation factor HPF undergoes massive ribosome degradation via an unknown pathway. Using genetic and biochemical approaches, we find that inactivating the 3'-to-5' exonuclease RNase R suppresses ribosome degradation in the Δhpf mutant. In vitro cell-free degradation assays confirm that 30S and 70S ribosomes isolated from the Δhpf mutant are extremely susceptible to RNase R, in stark contrast to nucleolytic resistance of the HPF-bound 70S and 100S complexes isolated from the wild type. In the absence of HPF, specific S. aureus 16S rRNA helices are sensitive to nucleolytic cleavage. These RNase hot spots are distinct from that found in the Escherichia coli ribosomes. S. aureus RNase R is associated with ribosomes, but unlike the E. coli counterpart, it is not regulated by general stressors and acetylation. The results not only highlight key differences between the evolutionarily conserved RNase R homologs but also provide direct evidence that HPF preserves ribosome integrity beyond its role in translational avoidance, thereby poising the hibernating ribosomes for rapid resumption of translation. IMPORTANCE Ribosome hibernation is pivotal for the rapid recovery of translation after quiescence in both bacteria and eukaryotes. Ribosome hibernation factors sterically occlude the entry of mRNA and tRNA and are thought to primarily maintain ribosomes in a translation-repressive state, thereby providing a pool of readily recyclable 70S or 80S complexes upon dissociation of the hibernation factors. Ribosomes in Staphylococcus aureus cells lacking the sole hibernation factor HPF are extremely unstable. Here, we show that HPF binding inhibits ribosome degradation by the evolutionarily conserved exoribonuclease RNase R. The data not only uncover a direct protective role of HPF in ribosome stability but also reinforce the versatility of RNase R in RNA processing, decay, and ribosome quality control.

Dataset Information

Affinity-purified VgaA-LC in complex with 70S ribosomes from Staphylococcus aureus

Publications

Structural basis of ABCF-mediated resistance to pleuromutilin, lincosamide, and streptogramin A antibiotics in Gram-positive pathogens.

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