Interplay between an ATP-binding cassette F protein and the ribosome from Mycobacterium tuberculosis

Abstract

EttA, energy-dependent translational throttle A, is a ribosomal factor that gates ribosome entry into the translation elongation cycle. A detailed understanding of its mechanism of action is limited due to the lack of high-resolution structures along its ATPase cycle. Here we present the cryo-electron microscopy (cryo-EM) structures of EttA from Mycobacterium tuberculosis (Mtb), referred to as MtbEttA, in complex with the Mtb 70S ribosome initiation complex (70SIC) at the pre-hydrolysis (ADPNP) and transition (ADP-VO4) states, and the crystal structure of MtbEttA alone in the post-hydrolysis (ADP) state. We observe that MtbEttA binds the E-site of the Mtb 70SIC, remodeling the P-site tRNA and the ribosomal intersubunit bridge B7a during the ribosomal ratcheting. In return, the rotation of the 30S causes conformational changes in MtbEttA, forcing the two nucleotide-binding sites (NBSs) to alternate to engage each ADPNP in the pre-hydrolysis states, followed by complete engagements of both ADP-VO4 molecules in the ATP-hydrolysis transition states. In the post-hydrolysis state, the conserved ATP-hydrolysis motifs of MtbEttA dissociate from both ADP molecules, leaving two nucleotide-binding domains (NBDs) in an open conformation. These structures reveal a dynamic interplay between MtbEttA and the Mtb ribosome, providing insights into the mechanism of translational regulation by EttA-like proteins.

CRISPRi chemical genetics and comparative genomics identify genes mediating drug potency in Mycobacterium tuberculosis

“…3b,c ). rv2477c is an orthologue of the Escherichia coli gene ettA (~58% amino acid identity), a ribosome-associated ABC-F protein that regulates the translation elongation cycle 49 – 51 . Due to its sequence similarity, we will refer to rv2477c as ettA .…”

Unraveling the mechanisms of intrinsic drug resistance in Mycobacterium tuberculosis

“…For example, several ABC proteins have been annotated as efflux pumps even though these proteins lack transmembrane helices ( Duan et al., 2019 ; Laws et al., 2022 ). Two such previously annotated efflux pumps, Rv1473 and EttA, turn out to indeed influence drug activity in Mtb but have nothing to do with efflux and rather are ATP-dependent regulators of the ribosome ( Sharkey et al., 2016 ; Cui et al., 2022 ; Li et al., 2022 ). Furthermore, genetic studies of efflux pumps should be validated with biochemical approaches to unambiguously demonstrate drug efflux activity.…”

Genome-encoded ABCF factors implicated in intrinsic antibiotic resistance in Gram-positive bacteria: VmlR2, Ard1 and CplR

“…The F subfamily of ABC ATPases includes both ribosome-associated antibiotic resistance (ARE) ABCFs (Ero et al, 2019; Wilson et al ., 2020), as well as multiple housekeeping factors that assist protein synthesis and ribosome assembly (Boël et al, 2014; Chen et al, 2014; Cui et al, 2022; Murina et al, 2019). ARE-ABCF factors mediate antibiotic resistance in human pathogens (exemplified by staphylococcal VgaA (Gentry et al, 2008) and closely related Listeria monocytogenes VgaL/Lmo0919 (Crowe-McAuliffe et al, 2021; Dar et al, 2016)), in environmental bacteria (exemplified by B. subtilis VmlR (Ohki et al, 2005)), and also confer self-resistance in antibiotic producers (exemplified by Streptomyces lincolnensis LmrC (Koberska et al, 2021)).…”

“…PhO-resisting PoxtA and OptrA have even shorter ARDs, which act by perturbing the positioning of the CCA-end of the P-site tRNA, rather than directly distorting the PTC (Crowe-McAuliffe et al ., 2022). Finally, the P-site tRNA-contacting PtIM elements of the housekeeping ABCFs are in general shorter than the ARD elements of ARE-ABCFs (Boël et al ., 2014; Chen et al ., 2014; Cui et al ., 2022).…”