Truncation of the C-terminal domain from CgtA of Mycobacterium smegmatis reduces its ribosome binding property but increases its GTPase activity

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis, presents a significant public health concern due to the emergence and prevalence of multi-drug-resistant strains. On this note, this study explores the potential of CgtA as a therapeutic drug target, derived from the bacterium Mycobacterium smegmatis, a well-established model organism used globally for TB research. CgtA is a GTPase protein essential for the survival of many bacterial species, and is involved in several essential processes; although the exact pathways of its association with these processes are still unknown. CgtA exhibits a three-domain structure, comprising of the conserved Obg and GTPase domain, followed by a highly variable C-Terminal Domain (CTD) of unknown function. This study explores the interaction and activity of CgtA from M. smegmatis (CgtA_ms) with ribosome. Through comparative ribosome binding analyses and Surface Plasmon Resonance studies, we demonstrate that the CTD of CgtA_ms facilitates binding of CgtA_ms to both 70S ribosomes and 50S ribosomal subunits. Interestingly, the 50S ribosome-associated GTPase activity of wt CgtA_ms increased by 2.6-fold, while that of CTD-truncated CgtA_ms increased by 3.5-fold., suggesting CTD acts as a negative regulator of ribosome-associated GTPase activity of CgtA_ms. These observations suggest that the CTD-CgtA_ms have a dual role; as a positive factor for ribosome binding, and as a negative factor for GTPase activity. Given the high amino acid sequence similarity between CgtA from M. smegmatis and M. tuberculosis, these findings may facilitate the investigation of CgtA as a potential therapeutic target for TB.