title: Biochemical analysis of SopE from Salmonella typhimurium, a highly efficient guanosine nucleotide exchange factor for RhoGTPases.
abstract: RhoGTPases are key regulators of eukaryotic cell physiology. The bacterial enteropathogen Salmonella typhimurium modulates host cell physiology by translocating specific toxins into the cytoplasm of host cells that induce responses such as apoptotic cell death in macrophages, the production of proinflammatory cytokines, the rearrangement of the host cell actin cytoskeleton (membrane ruffling), and bacterial entry into host cells. One of the translocated toxins is SopE, which has been shown to bind to RhoGTPases of the host cell and to activate RhoGTPase signaling. SopE is sufficient to induce profuse membrane ruffling in Cos cells and to facilitate efficient bacterial internalization. We show here that SopE belongs to a novel class of bacterial toxins that modulate RhoGTPase function by transient interaction. Surface plasmon resonance measurements revealed that the kinetics of formation and dissociation of the SopE.CDC42 complex are in the same order of magnitude as those described for complex formation of GTPases of the Ras superfamily with their cognate guanine nucleotide exchange factors (GEFs). In the presence of excess GDP, dissociation of the SopE.CDC42 complex was accelerated more than 1000-fold. SopE-mediated guanine nucleotide exchange was very efficient (e.g. exchange rates almost 10(5)-fold above the level of the uncatalyzed reaction; substrate affinity), and the kinetic constants were similar to those described for guanine nucleotide exchange mediated by CDC25 or RCC1. Far-UV CD spectroscopy revealed that SopE has a high content of alpha-helical structure, a feature also found in Dbl homology domains, Sec7-like domains, and the Ras-GEF domain of Sos. Despite the lack of any obvious sequence similarity, our data suggest that SopE may closely mimic eukaryotic GEFs.
authors.affiliation: Max von Pettenkofer-Institut, Ludwig Maximilians Universität, Pettenkoferstrasse 9a, 80336 München, Germany.
authors.forename: M G
authors.initials: MG
authors.lastname: Rudolph
authors.forename: C
authors.initials: C
authors.lastname: Weise
authors.forename: S
authors.initials: S
authors.lastname: Mirold
authors.forename: B
authors.initials: B
authors.lastname: Hillenbrand
authors.forename: B
authors.initials: B
authors.lastname: Bader
authors.forename: A
authors.initials: A
authors.lastname: Wittinghofer
authors.forename: W D
authors.initials: WD
authors.lastname: Hardt
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D017433:Protein Structure, Secondary; D012486:Salmonella typhimurium / Q000378:metabolism*; D013379:Substrate Specificity; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism; D020666:ras-GRF1 / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; C103987:SopE protein, Salmonella; D020666:ras-GRF1; D006153:Guanosine Diphosphate; D020558:GTP Phosphohydrolases; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D018631:ras Proteins