Structural and biochemical analysis of Escherichia coli ObgE, a central regulator of bacterial persistence

Abstract

The Obg protein family belongs to the TRAFAC (translation factor) class of P-loop GTPases and is conserved from bacteria to eukaryotes. Essential roles in many different cellular processes have been suggested for the Obg protein from (ObgE), and we recently showed that it is a central regulator of bacterial persistence. Here, we report the first crystal structure of ObgE at 1.85-Å resolution in the GDP-bound state, showing the characteristic N-terminal domain and a central G domain that are common to all Obg proteins. ObgE also contains an intrinsically disordered C-terminal domain, and we show here that this domain specifically contributed to GTP binding, whereas it did not influence GDP binding or GTP hydrolysis. Biophysical analysis, using small angle X-ray scattering and multi-angle light scattering experiments, revealed that ObgE is a monomer in solution, regardless of the bound nucleotide. In contrast to recent suggestions, our biochemical analyses further indicate that ObgE is neither activated by K ions nor by homodimerization. However, the ObgE GTPase activity was stimulated upon binding to the ribosome, confirming the ribosome-dependent GTPase activity of the Obg family. Combined, our data represent an important step toward further unraveling the detailed molecular mechanism of ObgE, which might pave the way to further studies into how this GTPase regulates bacterial physiology, including persistence.

Structural and functional investigation of the Small Ribosomal Subunit Biogenesis GTPase A (RsgA) from Pseudomonas aeruginosa

“…The affinity towards nucleotide obtained for Pa RsgA is also higher than those reported for other GTPases involved in bacterial ribosome maturation . Interestingly a submicromolar affinity for GDP and GTP (K D = 0.18 μ m for GDP and 0.37 μ m for GTP) have been recently obtained for ObgE from E. coli . The high affinity of Pa RsgA for nucleotides together with the slow dissociation rate of GDP opens the question whether nucleotide exchange occurs spontaneously due to the higher concentration of GTP in the cell or if a guanine exchange factor to substitute the GDP with GTP is required in vivo to sustain its catalytic activity in a physiological context.…”

GTP Binding Is Necessary for the Activation of a Toxic Mutant Isoform of the Essential GTPase ObgE

“…The N-terminal Obg domain is widely conserved and is essential for Obg function [2,8]. It consists of six left-handed type II helices and an eight-stranded β-barrel that contacts the G domain [8][9][10]. The Obg domain most likely mediates protein-protein interactions with effector molecules [8,11].…”

“…The Obg domain most likely mediates protein-protein interactions with effector molecules [8,11]. In Escherichia coli Obg (ObgE), this domain spans from amino acid residue 1 until 157 [9]. Nucleotide binding and hydrolysis is performed by the G domain.…”

“…Nucleotide binding and hydrolysis is performed by the G domain. This domain consists of a six-stranded β-sheet and five α-helices and undergoes conformational changes in response to nucleotide binding [1,2,[8][9][10]. These conformational changes are thought to be transmitted to the N terminus through a flexible linker region between the G and N domains [8,12].…”

“…These conformational changes are thought to be transmitted to the N terminus through a flexible linker region between the G and N domains [8,12]. In E. coli, the G domain contains amino acid residues 158 to 340 [9]. The final 50 residues of ObgE make up the C-terminal domain [9].…”