Cryo-EM structures of C. glutamicum pre- and posttranslocational 70S ribosome in complex with Kasugamycin

Abstract text (incl. figure legends and references)

The Actinobacteria Corynebacterium glutamicum is the workhorse in modern biotechnology, e.g. in industrial biosynthesis of amino acids and heterologous protein production. The detailed architecture of the C. glutamicum translation machinery, the 70S ribosome, is hitherto unknown, impeding its structure-function analysis, and limiting drug optimization. The majority of aminoglycoside antibiotic producers are Actinobacteria, still C. glutamicum is sensitive to the antibiotic Kasugamycin (KSG). Our study aims to understand the impact KSG has on the 70S ribosome of C. glutamicum (Cg70S) and how the simple mutations ΔbS22 and ΔKsgA contribute to resistance. Therefore, we present the three-dimensional structures of the wildtype, ΔbS22 and ΔKsgA ribosome solved to high resolution (<3 Å) with cryo-electron microscopy. By exploiting the potential of single particle analysis in the structural analysis of dynamic processes, particles were classified in 3D heterogeneous refinement according to their t-RNA position in the ribosome. We captured Cg70S ribosomes in the pre- and posttranslocational state in absence and presence of KSG. For both states, we were able to localize the KSG-binding pocket within the mRNA path and in close proximity to the P-tRNA binding site. In direct comparison of the molecular models derived from the WT, ΔbS22 and ΔKsgA EM density maps, it became clear that the deletions do not significantly alter the molecular structure of the KSG binding pocket. Interestingly however, depending on the absence or presence of KSG in wildtype- or ΔbS22-Cg70S the particle ratio between the two distinct states differs significantly, suggesting a role of bS22 in the stabilization of the pretranslocational state. ΔKsgA results in undermethylation of the 16s rRNA, but the structure of the KSG binding pocket is preserved. Here, the alteration of the protein content of the small ribosomal subunit, visible in the EM density maps, seems to be the key to resistance by influencing the presence of bS22 and other small ribosomal subunit proteins and thereby modifying ribosomal dynamics. The here presented cryo-EM structures of the C. glutamicum ribosome provide the structural framework for understanding antibiotic KSG resistance.