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Abstract
Even though the Obg protein is essential for bacterial viability, the cellular functions of this universally conserved GTPase remain enigmatic. Moreover, the influence of GTP and GDP binding on the activity of this protein is largely unknown. Previously, we identified a mutant isoform of ObgE (the Obg protein of Escherichia coli) that triggers cell death. In this research we explore the biochemical requirements for the toxic effect of this mutant ObgE* isoform, using cell death as a readily accessible read-out for protein activity. Both the absence of the N-terminal domain and a decreased GTP binding affinity neutralize ObgE*-mediated toxicity. Moreover, a deletion in the region that connects the N-terminal domain to the G domain likewise abolishes toxicity. Taken together, these data indicate that GTP binding by ObgE* triggers a conformational change that is transmitted to the N-terminal domain to confer toxicity. We therefore conclude that ObgE*–GTP, but not ObgE*–GDP, is the active form of ObgE* that is detrimental to cell viability. Based on these data, we speculate that also for wild-type ObgE, GTP binding triggers conformational changes that affect the N-terminal domain and thereby control ObgE function.
Highlights
Obg proteins belong to the TRAFAC class of P-loop GTPases that typically switch between a GTP- and a GDP-bound state by GTP hydrolysis or nucleotide exchange [1,2]
In this study we present evidence that implies that ObgE*-mediated cell death is triggered by the GTP-bound version of the protein
Since the G domain itself is insufficient to cause toxicity, we postulate that GTP binding by ObgE* leads to changes in the N-terminal domain that trigger cell death
Summary
Obg proteins belong to the TRAFAC (translation factor) class of P-loop GTPases that typically switch between a GTP- and a GDP-bound state by GTP hydrolysis or nucleotide exchange [1,2]. 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]. 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]. The final 50 residues of ObgE make up the C-terminal domain [9] This domain is not well conserved among different species and is thought to mediate species-specific Obg functions [2]. In E. coli, the C-terminus of ObgE is intrinsically disordered [9]
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