EspM2 is a RhoA guanine nucleotide exchange factor

Abstract

We investigated how the type III secretion system WxxxE effectors EspM2 of enterohaemorrhagic Escherichia coli, which triggers stress fibre formation, and SifA of Salmonella enterica serovar Typhimurium, which is involved in intracellular survival, modulate Rho GTPases. We identified a direct interaction between EspM2 or SifA and nucleotide-free RhoA. Nuclear Magnetic Resonance Spectroscopy revealed that EspM2 has a similar fold to SifA and the guanine nucleotide exchange factor (GEF) effector SopE. EspM2 induced nucleotide exchange in RhoA but not in Rac1 or H-Ras, while SifA induced nucleotide exchange in none of them. Mutating W70 of the WxxxE motif or L118 and I127 residues, which surround the catalytic loop, affected the stability of EspM2. Substitution of Q124, located within the catalytic loop of EspM2, with alanine, greatly attenuated the RhoA GEF activity in vitro and the ability of EspM2 to induce stress fibres upon ectopic expression. These results suggest that binding of SifA to RhoA does not trigger nucleotide exchange while EspM2 is a unique Rho GTPase GEF.

Fig. 1

RhoA binds directly to EspM2 and SifA. A. Surface plasmon resonance showing concentration-dependent binding of RhoA to EspM2^29–196. Concentrations of RhoA varying from 0.05 µM to 50 µM were flowed at 50 µl min⁻¹ (duration indicated by black bar) over a CM5 sensor chip with EspM2^29–196 covalently bound to the surface. Control substituted signals are shown. B. Inhibition of EspM2^29–196–RhoA interaction by GDP or GTP. For RhoA (4 µM) flowing over an EspM2^29–196-bound surface in buffer containing GDP/GTP, the response maxima relative to response maxima in the absence of nucleotide are plotted with respect to GDP/GTP concentration. C. Binding of EspM2^29–196 to RhoA and Rac1. Averaged response maxima for three representative concentrations compare the strength of EspM2 binding with the two GTPases. D. Surface plasmon resonance demonstrates RhoA concentration dependence for binding SifA. Concentrations of SifA varying from 0.5 µM to 6 µM were flowed at 50 µl min⁻¹ over a CM5 sensor chip with RhoA covalently bound to the surface.

Fig. 2

EspM2 is a RhoA GEF. A. EspM2^29–196 mediates loading of mant-GTP into RhoA. mant-GTP (0.5 µM) was incubated with 2 µM RhoA in presence of 250 mM EDTA (squares), or 0.05–50 µM EspM2^29–196 (circles) or in presence of buffer only (triangles). The insertion of the mant-GTP into the nucleotide binding pocket of RhoA in presence of EspM2^29–196 detected by an increase in the fluorescent emission was found to be concentration dependent. B. EspM2^29–196 weakly induces loading of mant-GTP into Cdc42. mant-GTP (0.5 µM) was incubated with 2 µM Cdc42 in presence of 250 mM EDTA (squares), or 0.05–50 µM EspM2^29–196 (circles) or in presence of buffer only (triangles). Slow loading of mant-GTP into Cdc42 was only detected when high concentrations of EspM2^29–196 were added. C. EspM2^29–196 does not induce nucleotide exchange for Rac1. mant-GTP (0.5 µM) was incubated with 2 µM Rac1 in presence of 250 mM EDTA (squares), or 0.05–50 µM EspM2^29–196 (circles) or in presence of buffer only (triangles). No efficient loading of man-GTP into Rac1 was observed after incubation with up to 50 µM EspM2^29–196. D. Incubation of 50 µM EspM2^29–196 in the exchange buffer alone did not change the fluorescence intensity. E. SifA does not induce nucleotide exchange in RhoA, Cdc42, Rac1 or H-Ras. mant-GTP (0.5 µM) was incubated with 2 µM RhoA (blue circles), Cdc42 (pink circles), Rac1 (green circles) or H-Ras (orange circles) in presence of 5 µM SifA. No loading of man-GTP into any of the small GTPases tested was observed in these conditions. Results shown are the average of three independent experiments.

Fig. 3

Model of EspM2^29–196 and the EspM2^29–196–RhoA complex. A. ¹H ¹⁵N TROSY-HSQC titration of RhoA against EspM2^29–196. Black peaks show no addition of RhoA while red peaks show fourfold molar excess of RhoA. Chemical shift changes were deemed significant if the peak intensity was reduced by more than 80% (i.e. D145). B. Homology model of EspM2^29–196 created with SWISS-MODEL (Arnold et al., 2006). Data from the RhoA NMR titration (red) and alanine mutations (blue) have been mapped onto the model. Those residues with chemical shift changes in the NMR titrations and which have also been mutated to alanines are coloured cyan. C. Model of the EspM2^29–196–RhoA complex created by superimposing the EspM2^29–196 model and the crystal structure of RhoA (pdb:1xcg; Derewenda et al., 2004) onto the crystal structure of the SopE–Cdc42 complex (pdb:1gzs; Buchwald et al., 2002). All residues identified by the RhoA titration appear at the interface except for those within the C-terminal helix. In this model the intimate contacts with EspM2 are through the switch regions I and II of RhoA.

Fig. 4

Activity of EspM2^29–196 mutants. A. Multiple sequence alignment of the putative catalytic loop and flanking regions of the WxxxE proteins: EspM2 of EHEC O157:H7 Sakai, IpgB1 and IpgB2 of Shigella flexneri, EspT of Citrobacter rodentium, Map of EPEC E2348/69 and SifA and SifB of S. Typhimurium. The loop region of SopE was added for comparison, with the catalytic region highlighted. Similar residues are highlighted in grey. A stretch of residues in the putative catalytic loop of SifA that is different from the other WxxxE effectors is highlighted. EspM2 residues selected for mutagenesis are indicated by a star. B. Swiss 3T3 cells were transfected with pRK5 encoding myc-tagged EspM2^29–196, EspM2^29–196 W70A and EspM2^29–196 I127A. Actin was stained with Oregon green phalloidin and the myc tag was detected with monoclonal antibody. C. Quantification of stress fibres formation in cell transfected with different EspM2^29–196 mutants. Results are displayed as mean ± SEM. D. SPR comparison of RhoA binding to wild-type EspM2 and EspM2 Q124A. No significant difference in binding was detected over a range of RhoA concentrations. Shown is the averaged response of three repeats. E. EspM2^29–196 Q124A is impaired in loading mant-GTP into RhoA. mant-GTP (0.5 µM) was incubated with 2 µM RhoA in presence of 1 µM EspM2^29–196 (blue circles) or 1 µM (green triangles), 5 µM (orange triangles) and 50 µM (red triangles) EspM2^29–196 Q124A.