title: Structural and biochemical analysis ofEscherichia coliObgE, 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 fromEscherichia coli(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.
authors.affiliation: From the Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels.|the VIB-VUB Center for Structural Biology, 1050 Brussels.
authors.forename: Sotirios
authors.initials: S
authors.lastname: Gkekas
authors.affiliation: From the Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels.|the VIB-VUB Center for Structural Biology, 1050 Brussels.
authors.forename: Ranjan Kumar
authors.initials: RK
authors.lastname: Singh
authors.affiliation: From the Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels.|the VIB-VUB Center for Structural Biology, 1050 Brussels.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Shkumatov
authors.affiliation: From the Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels.|the VIB-VUB Center for Structural Biology, 1050 Brussels.
authors.forename: Joris
authors.initials: J
authors.lastname: Messens
authors.affiliation: the Centre of Microbial and Plant Genetics, KU Leuven, University of Leuven, 3001 Leuven, and.|the Department of Life Science Technologies, Smart Systems and Emerging Technologies Unit, IMEC, 3001 Leuven, Belgium.
authors.forename: Maarten
authors.initials: M
authors.lastname: Fauvart
authors.affiliation: the Centre of Microbial and Plant Genetics, KU Leuven, University of Leuven, 3001 Leuven, and.
authors.forename: Natalie
authors.initials: N
authors.lastname: Verstraeten
authors.affiliation: the Centre of Microbial and Plant Genetics, KU Leuven, University of Leuven, 3001 Leuven, and.
authors.forename: Jan
authors.initials: J
authors.lastname: Michiels
authors.affiliation: From the Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, wim.versees@vib-vub.be.|the VIB-VUB Center for Structural Biology, 1050 Brussels.
authors.forename: Wim
authors.initials: W
authors.lastname: Versées
mesh_terms: D002414:Cations, Monovalent / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011188:Potassium / Q000737:chemistry* / Q000378:metabolism; D000072417:Protein Domains; D055503:Protein Multimerization
chemical_list: D002414:Cations, Monovalent; D029968:Escherichia coli Proteins; C491572:ObgE protein, E coli; D020559:Monomeric GTP-Binding Proteins; D011188:Potassium
keywords: GTPase; X-ray crystallography; X-ray scattering; biophysics; crystal structure; enzyme kinetics; enzyme structure