Biochemical Analysis of SopE from Salmonella typhimurium, a Highly Efficient Guanosine Nucleotide Exchange Factor for RhoGTPases

doi:10.1074/jbc.274.43.30501

Biochemical Analysis of SopE from Salmonella typhimurium, a Highly Efficient Guanosine Nucleotide Exchange Factor for RhoGTPases*

From the Max von Pettenkofer-Institut, Ludwig Maximilians Universität, Pettenkoferstrasse 9a, 80336 München, Germany, the ^§Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany, and the ^¶Freie Universität Berlin, Institut für Chemie-Biochemie, Thielallee 63, 14195 Berlin, Germany

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 10⁵-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 α-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.

Footnotes

↵* This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Bundesministerium für Bildung und Forschung (to W.-D. H.) and by the Fonds der Chemischen Industrie (to C. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ These authors contributed equally to the experimental work.
↵‖ To whom correspondence should be addressed. Tel.: 89-5160-5263; Fax: 89-5160-5223; E-mail: hardt@m3401.mpk.med.uni-muenchen.de.
↵2 M. G. Rudolph, unpublished data.
↵3 M. G. Rudolph, unpublished data.
↵4 W.-D. Hardt and J. E. Galán, unpublished data.
Abbreviations:

GEF

guanine nucleotide exchange factor

GST

glutathione S-transferase

HPLC

high pressure liquid chromatography

mant

O-(N-methylanthraniloyl)

mGDP

mant-GDP
- Received July 6, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.