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

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.

Identification of Substrates of Cytoplasmic Peptidyl-Prolyl Cis/Trans Isomerases and Their Collective Essentiality in Escherichia Coli

“…Similarly, various components of rRNA modification, the RNA helicase DeaD, ribosome assembly factors like BipA and ObgE GTPases were identified as binding partners of various PPIs. BipA and ObgE are known to impact the ribosome assembly [ 33 , 34 ]. Concerning ribosomal proteins such as S2 and S3, they not only aggregate in Δ6 ppi bacteria, but were also identified as co-eluting partner proteins with PpiB, PpiC, FklB, and FkpB.…”

Vibrio vulnificus induces the death of a major bacterial species in the mouse gut via cyclo-Phe-Pro

“…The thermodynamic parameters upon titration of rObgE (40 μM, nucleotide-free) with a solution of 400 μM cFP were measured at 25 °C in a buffer consisting of 20 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM MgCl 2 , and 1 mM β-mercaptoethanol. The nucleotide-free rObgE was prepared by treating with calf intestine alkaline phosphatase (Promega) and passing a gel filtration AKTA-FPLC system (Superdex 200, GE Healthcare) at a flow rate of 0.3 ml/min in a buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, and 5 mM MgCl 2 as previously described [ 60 ]. rObgE in the eluted fractions (1 ml) was concentrated using Amicon® Ultra-15 10K (Millipore) and its buffer was exchanged with that used for an ITC assay.…”