Translational Regulation via L11: Molecular Switches on the Ribosome Turned On and Off by Thiostrepton and Micrococcin

Abstract

The thiopeptide class of antibiotics targets the GTPase-associated center (GAC) of the ribosome to inhibit translation factor function. Using X-ray crystallography, we have determined the binding sites of thiostrepton (Thio), nosiheptide (Nosi), and micrococcin (Micro), on the Deinococcus radiodurans large ribosomal subunit. The thiopeptides, by binding within a cleft located between the ribosomal protein L11 and helices 43 and 44 of the 23S rRNA, overlap with the position of domain V of EF-G, thus explaining how this class of drugs perturbs translation factor binding to the ribosome. The presence of Micro leads to additional density for the C-terminal domain (CTD) of L7, adjacent to and interacting with L11. The results suggest that L11 acts as a molecular switch to control L7 binding and plays a pivotal role in positioning one L7-CTD monomer on the G' subdomain of EF-G to regulate EF-G turnover during protein synthesis.

Archaeal ribosomal stalk protein interacts with translation factors in a nucleotide-independent manner via its conserved C terminus

“…The individual sites for interaction with IF2, EF-Tu, EF-G, and RF3 have been mapped to the α4-loop-α5 region of purified L12 by NMR (19), and the results are consistent with the functional findings from site-directed mutagenesis (8,33). The binding of the C-terminal domain of L12 to EF-G was also detected in the ribosome•EF-G complex by cryo-EM (14,15) and in recent crystal structure data (16). Although crystal structure data are not available for the C-terminal portion of the archaeal/eukaryotic ribosomal stalk protein, some structural features have been deduced by NMR analysis of peptides that comprise the 13 C-terminal amino acids from human and Leishmania brazililiensis P1/P2 (34).…”

“…Kinetic experiments under saturating nucleotide conditions indicated that EF-G•GTP and EF-G•GDP bind to the ribosome with similar affinities (30). It can be inferred from current evidence that a conformational change in EF-G including the switch I region (31) occurs after GTP hydrolysis on the ribosome and induces the rearrangement of the ribosomal factorbinding center, which is composed of the sarcin/ricin loop and the L11 (E. coli terminology) binding region, including the H43/H44 domain, of the 23S/28S rRNA (15)(16)(17)32). This structural rearrangement of the factor-binding center, which depends on GTP hydrolysis, seems to be responsible for a change in the affinity of binding of the factor to the ribosome and recycling of the factor.…”

Structure of BipA in GTP form bound to the ratcheted ribosome

“…The "GTPase-associated center" (GAC) is responsible for recruiting translational factors and stimulating their GTPase activity. In addition to the SRL, a key component of the GAC, the other two crucial regions are the ribosomal uL10-bL12 stalk (comprised of uL10 protein and 4-6 copies of bL12) and its base comprising the uL11 region (uL11 protein and 23S rRNA helices H43 and H44) (37). Both the uL10-bL12 stalk and the uL11 region are extremely mobile elements of the ribosome.…”

Targeting Antibiotic Resistance

“…Thiopeptides are inhibitors of protein synthesis, but their mode of action differs depending on the size of the macrocycle. Thiostrepton A ( 15 ),79 the archetypal thiopeptide, and micrococcin P1 possess 26‐membered macrocyclic cores and are known to bind to the GTPase‐associated region of the ribosome/L11 protein complex 80. Thiopeptides with 29‐membered macrocyclic cores such as GE2270 A ( 16 ), on the other hand, interact with GTP‐bound bacterial EF‐Tu, preventing the formation of the ternary complex with aa‐tRNA 81…”