Abhishek Upadhyay

Huan-Lin Wu

Christopher Williams

Terry Field

Edouard E Galyov

Jean Mh Van Den Elsen

Stefan Bagby

Chr Istopher Williams

Jean M H Van Den Elsen

Guanine nucleotide exchange

Keywords: mallei, Burkholderia mallei, B. pseudomallei, Burkholderia pseudomallei, CPK, creatine phosphokinase, DH, Dbl homology, heteronuclear single quantum coherence, IPAP, in-phase anti-phase, NMR, nuclear magnetic resonance, NOE, nuclear Overhauser effect, NOESY, nuclear Overhauser effect spectroscopy, PH, pleckstrin homology, RDC, residual dipolar coupling, rmsd, root mean square deviation, TTS, type III secretion, TTSS, type III secretion system bacterial pathogen, guanine nucleotide exchange factor, protein-protein interaction, protein structure, Rho GTPase, type III secretion

teaching and research institutions in France or abroad, or from public or private research centers.

INTRODUCTION

Burkholderia pseudomallei (B. pseudomallei) is the aetiological agent of melioidosis, a severe emerging infection of humans and animals that is endemic in south-east Asia and tropical Australia and that has the potential to spread worldwide [START_REF] Dance | Melioidosis[END_REF][START_REF] White | Melioidosis[END_REF][START_REF] Peacock | Melioidosis[END_REF]. Melioidosis has a range of clinical manifestations, including rapidly fatal septicaemia, pneumonia, skin and soft tissue abscesses, and osteomyelitis or septic arthritis. Infection is usually via contaminated soil, dust or water [START_REF] Brett | Pathogenesis of and immunity to melioidosis[END_REF][START_REF] Currie | Endemic melioidosis in tropical northern Australia: a 10-year prospective study and review of the literature[END_REF][START_REF] Currie | Melioidosis: acute and chronic disease, relapse and re-activation[END_REF]. Asymptomatic infection is common in areas where the infection is endemic and progression to disease depends on the condition of the host [START_REF] Currie | Endemic melioidosis in tropical northern Australia: a 10-year prospective study and review of the literature[END_REF]. Between the fatal and asymptomatic extremes, the infection may be chronic or run a relapsing course.

Latency and relapse are common even in patients treated with appropriate antibiotics [START_REF] Chaowagul | Relapse in melioidosisincidence and risk factors[END_REF]. B. pseudomallei is closely related to Burkholderia mallei (B. mallei), the pathogen that causes glanders, a disease of horses and other solipeds. B. mallei can also affect humans and is often fatal if left untreated [START_REF] Wilkinson | Glanders: medicine and veterinary medicine in common pursuit of a contagious disease[END_REF]. Due to the severity of the infection, aerosol infectivity and worldwide availability, both B. pseudomallei and B. mallei are considered to be potential bioweapons [START_REF] Rotz | Public health assessment of potential biological terrorism agents[END_REF]. There is currently no vaccine against B. pseudomallei [10].

The molecular mechanisms of B. pseudomallei pathogenesis are not completely understood [START_REF] Stevens | Exploitation of host cells by Burkholderia pseudomallei[END_REF]. B. pseudomallei has a 7.3 Mb genome, unusually large for a prokaryote, comprising two chromosomes with sixteen genomic islands possibly acquired through very recent lateral transfer [START_REF] Holden | Genornic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei[END_REF]. The B. pseudomallei genome contains at least three loci encoding putative type III secretion systems [START_REF] Rainbow | Distribution of type III secretion gene clusters in Burkholderia pseudomallei, B. thailandensis and B. mallei[END_REF]. One of these, Bsa, is homologous to the inv/spa/prg type III secretion system (TTSS) of Salmonella typhimurium [START_REF] Rainbow | Distribution of type III secretion gene clusters in Burkholderia pseudomallei, B. thailandensis and B. mallei[END_REF][START_REF] Attree | A second type III secretion system in Burkholderia pseudomallei: Who is the real culprit?[END_REF][START_REF] Stevens | An inv/mxi-spa-like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen[END_REF]. Type III secretion systems resemble molecular syringes for the injection of multiple bacterial effector proteins into the host cell cytoplasm that modify host cell physiology to the benefit of the pathogen [START_REF] Johnson | The type III needle and the damage done[END_REF][START_REF] Galàn | Protein delivery into eukaryotic cells by type III secretion machines[END_REF]. TTSSs are central to the virulence of many Gram negative pathogens, including Salmonella, Shigella, Yersinia, enteropathogenic E. coli and the four major genera of plant pathogenic bacteria [18,[START_REF] Pallen | Bioinformatics, genomics and evolution of non-flagellar type-III secretion systems: a Darwinian perpective[END_REF]. BopE, encoded within the Bsa locus, is secreted via the Bsa TTSS and influences invasion of HeLa cells probably via its function as a guanine nucleotide exchange factor (GEF) for Rho GTPases that regulate the actin network [START_REF] Stevens | A Burkholderia pseudomallei type III secreted protein, BopE, facilitates bacterial invasion of epithelial cells and exhibits guanine nucleotide exchange factor activity[END_REF]. BopE shares sequence homology with the Salmonella translocated effector proteins SopE [START_REF] Wood | SopE, a secreted protein of Salmonella dublin, is translocated into the target eukaryotic cell via a Sip-dependent mechanism and promotes bacterial entry[END_REF][START_REF] Hardt | S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells[END_REF] and SopE2 [START_REF] Bakshi | Identification of SopE2, a Salmonella secreted protein which is highly homologous to SopE and involved in bacterial invasion of epithelial cells[END_REF][START_REF] Stender | Identification of SopE2 from Salmonella typhimurium, a conserved guanine nucleotide exchange factor for cdc42 of the host cell[END_REF] (supplemental Figure S1) which play an important role in Salmonella invasion of nonphagocytic intestinal epithelial cells. SopE is a potent guanine nucleotide exchange factor for the mammalian Rho GTPases Cdc42 and Rac1 in vitro and in vivo whereas SopE2 efficiently activates Cdc42 but not Rac1 [START_REF] Friebel | SopE and SopE2 from Salmonella typhimurium activate different sets of Rho GTPases of the host cell[END_REF]. The structures of SopE [START_REF] Buchwald | Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE[END_REF] and SopE2 [START_REF] Williams | Solution structure, backbone dynamics, and interaction with Cdc42 of Salmonella guanine nucleotide exchange factor SopE2[END_REF] We have previously shown that BopE is monomeric in aqueous solution, adopts a single conformation that is predominantly -helical, is stable over a wide range of pH and able to refold independently [START_REF] Upadhyay | Biophysical characterization of the catalytic domain of guanine nucleotide exchange factor BopE from Burkholderia pseudomallei[END_REF]. Now, as part of an examination of the structural and mechanistic relationships between BopE and its counterparts SopE and SopE2 from Salmonella, we report here the three-dimensional structure in solution of the catalytic domain of BopE (BopE residues 78-261 where 261 is the C-terminal residue of the full length protein) and NMR and kinetic analyses of the interaction of BopE 78-261 with the Rho GTPase Cdc42.

EXPERIMENTAL Biophysical and biological characterisation, NMR sample generation and NMR spectroscopy of recombinant BopE 78-261

The methods used to obtain BopE 78-261 NMR samples and to derive backbone and side chain resonance assignment, plus biophysical characteristics of BopE 78-261 , have been described previously [START_REF] Upadhyay | Biophysical characterization of the catalytic domain of guanine nucleotide exchange factor BopE from Burkholderia pseudomallei[END_REF][START_REF] Wu | Assignment of the 1 H, 13 C and 15 N resonances of the catalytic domain of guanine nucleotide exchange factor BopE from Burkholderia pseudomallei[END_REF]. The 1 H, 13 C and 15 N chemical shifts of BopE 78-261 are in the BioMagResBank database (http://www.bmrb.wisc.edu) under accession number BMRB-5974. Biological activity of exactly the same BopE 78-261 construct as used here has been demonstrated previously: BopE 78-261 was shown to have guanine nucleotide exchange activity towards Cdc42 and Rac1 in vitro [START_REF] Stevens | A Burkholderia pseudomallei type III secreted protein, BopE, facilitates bacterial invasion of epithelial cells and exhibits guanine nucleotide exchange factor activity[END_REF].

All NMR data were acquired at 25ºC on a Varian Unity INOVA spectrometer operating at a nominal proton frequency of 600 MHz, using a triple resonance 5 mm probe equipped with z-axis pulsed field gradients. NMR data were processed using the NMRPipe/NMRDraw software suite [START_REF] Delaglio | NMRPipe: a multidimensional spectral processing system based on Unix pipes[END_REF] and analysed using the SPARKY assignment program [START_REF] Goddard | University of California[END_REF]. NOE distance restraints were obtained by analysis of 1 H-1 H 2D NOESY [36] (100 ms and 175 ms mixing times), 15 N-NOESY HSQC [START_REF] Zhang | Backbone 1 H and 15 N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed-field gradient NMR techniques[END_REF] (50 ms, 100 ms, and 150 ms mixing times) and simultaneous 3D 15 N/ 13 C-edited NOESY [38] (100 ms mixing time) spectra. Backbone 1 D NH residual dipolar coupling (RDC) restraints were measured for BopE 78-261 aligned with respect to the magnetic field using a stretched polyacrylamide gel; gels were made using an apparatus based on that described previously [START_REF] Chou | A simple apparatus for generating stretched polyacrylamide gels, yielding uniform alignment of proteins and detergent micelles[END_REF]. RDCs were measured using IPAP-HSQC [START_REF] Ottiger | Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra[END_REF].

Structure calculation

Each NOE was assigned to one of four restraint distances based on the peak intensity:

1.8-2.8 Å, 1.8-3.3 Å, 1.8-5.0 Å and 1.8-6.0 Å, corresponding to strong, medium, weak and very weak NOEs. Distances involving methyl groups, aromatic ring protons, and nonstereospecifically assigned methylene protons were represented as a ( r -6 ) -1/6 sum [START_REF] Nilges | A calculation strategy for the structure determination of symmetrical dimers by 1 H NMR[END_REF]. 0. protons. Backbone dihedral angles and were predicted from 13 C , 13 C , 13 C', 1 H and backbone 15 N chemical shifts using TALOS [START_REF] Cornilescu | Protein backbone angle restraints from searching a database for chemical shift and sequence homology[END_REF]. The dihedral angles were restrained to TALOS-predicted values ± 30 o for -helices and ± 40 o for -strands and dihedral angles were restrained to TALOS-predicted values ± 50 o . Hydrogen bond restraints were obtained from hydrogen-deuterium exchange experiments: uniformly 15 N-labelled BopE 78-261 in NMR buffer was lyophilised and resuspended in 99.96% D 2 O. A series of 1 H-15 N HSQC spectra was then recorded to determine amide protons protected from exchange with the solvent. For hydrogen bond distance constraints, the NH-O distance was assigned lower and upper distance bounds of 1.5 and 2.5 Å, and the N-O distance assigned lower and upper distance bounds of 2.5 and 3.5 Å.

Structures were calculated using the Python interface of Xplor-NIH 2.16.0 [START_REF] Schwieters | The Xplor-NIH NMR molecular structure determination package[END_REF]44], using simulated annealing starting from random extended structures. Default values were used for all force constants and molecular parameters. The ensemble of NMR structures was analysed for violated restraints using the VMD-Xplor visualisation package [45]. The structure determination was carried out iteratively whereby consistently violated restraints were reassigned, wherever possible, using existing structures or removed until a consistent set of constraints was obtained with few violations in the ensemble. The ensemble of structures was further refined with Xplor-NIH standard refinement protocols using the final set of restraints. The quality of the structures was assessed using PROCHECK-NMR [START_REF] Laskowski | Aqua and Procheck-NMR: programs for checking the quality of protein structures solved by NMR[END_REF].

NMR titration of Cdc42 7 against BopE 78-261

Binding of unlabelled human Cdc42 7 to 15 N-labelled BopE 78-261 was monitored by recording 1 H- 15 N HSQC spectra as a function of BopE 78-261 :Cdc42 7 ratio. Cdc42 7 is Cdc42 lacking seven C-terminal amino acids; it was shown previously that C-terminal truncation of Cdc42 does not interfere with SopE GEF activity [START_REF] Rudolph | Biochemical analysis of SopE from Salmonella typhimurium, a highly efficient guanosine nucleotide exchange factor for Rho GTPases[END_REF]. Cdc42 7 was purified from E. coli BL21(DE3) as previously described [START_REF] Rudolph | Biochemical analysis of SopE from Salmonella typhimurium, a highly efficient guanosine nucleotide exchange factor for Rho GTPases[END_REF]. The NMR titration was performed as previously described [START_REF] Williams | Solution structure, backbone dynamics, and interaction with Cdc42 of Salmonella guanine nucleotide exchange factor SopE2[END_REF][START_REF] Mcalister | NMR analysis of interacting soluble forms of the cell-cell recognition molecules CD2 and CD48[END_REF]. Briefly, two initial NMR samples were prepared in 0.5 ml NMR buffer (20 mM sodium phosphate pH 5.5, 50 mM NaCl) with 10% D 2 O. Sample A contained 0.5 mM 15 N-labelled BopE 78-261 (molar ratio of 1.0:0.0 BopE 78-261 :Cdc42 7) and sample B contained 0.5 mM 15 N-labelled BopE 78-261 and 1.34 mM Cdc42 7 (molar ratio of 1.0:2.7 BopE 78-261 :Cdc42 7). The buffer composition of both samples was identical as both samples were extensively exchanged into the same batch of sample buffer. Throughout the titration the concentration of BopE 78-261 was maintained at a constant 0.5 mM and the Cdc42 7 concentration was varied to give a series of BopE 78-261 :Cdc42 7 molar ratios from 1.0:0.0 to 1.0:2.7. A 1 H-15 N HSQC spectrum was acquired at each titration point with 512 complex 1 H points and 192 complex 15 N points with 32 scans per increment and spectral the aliquots to the other NMR tube (i.e., from tube A to tube B and vice versa). This procedure was repeated until a series of twelve 1 H- 15 N HSQC experiments at BopE 78- 261 :Cdc42 7 molar ratios between 1.0:0.0 and 1.0:2.7 was completed.

Generation and characterisation of BopE mutants

BopE 78-261 double mutants N224P/R230Q (mutant 1), N216P/L226P (mutant 2) and R207E/N216P (mutant 3) were made using the following pairs of primers (shown as 5'-3'): TCGCCCACGCTCGTCGAGTTCCAGCAGACGGT (N224PR230Q for) and CTGCTGGAACTCGACGAGCGTGGGCGAACGCTC (N224PR230Q rev);

CGCCCGCGTTGCCGGCCGAGCGTTCGAACACGCCCGTCGAGT (N216PL226P for)

and ACGGGCGTGTTCGAACGCTCGGCCGGCAACGCGGGCGCGACGA (N216PL226P rev); TGCGGAGCAGCAGGCGATCGATCTCGTCGCGCCCGCGTTGCC (R207EN216P for) and CGCGGGCGCGACGAGATCGATCGCCTGCTGCTCCGCATAC (R207EN216P rev). The mutants were constructed by overlapping PCR. The two overlapping primers (for and rev) were used in PCR with upstream and downstream primers to amplify two parts of the gene (upstream-rev and for-downstream, respectively). The resulting DNA fragments were purified, mixed and used as a template for a third PCR with upstream and downstream primers to amplify the mutated gene. The resulting DNA fragment in each case was digested with EcoR1 and BamH1 and cloned into pGEX4T1 (GE Healthcare). The cloned DNA was then sequenced. The mutant proteins were expressed and purified in the same way as wild type BopE 78-261 [START_REF] Upadhyay | Biophysical characterization of the catalytic domain of guanine nucleotide exchange factor BopE from Burkholderia pseudomallei[END_REF].

Filter binding assays

Cdc42 7 was loaded at 25ºC for 10 minutes with ) and 98 backbone 1 D NH RDC restraints (Table 1). The ensemble of twenty final simulated annealing structures, selected from forty calculations on the basis of lowest energy, and the average structure are shown in Figure 1. Over the regular secondary structure elements, the ensemble of structures has a backbone rmsd from the mean of 0.65 Å and an rmsd of 1.13 Å for all non-hydrogen atoms. A Ramachandran plot of the structures with Procheck-NMR [START_REF] Laskowski | Aqua and Procheck-NMR: programs for checking the quality of protein structures solved by NMR[END_REF] indicates that 96.7% of the residues (excluding Gly and Pro residues) lie in the most favoured or additionally allowed regions. The few non-glycine residues to fall into the generously allowed regions and disallowed regions correspond to residues located at the termini or loop regions where the NMR restraint density is low. GlyAlaGlyThr 174 catalytic motif between helices 3 and 4, and a loop between helices 5 and 6 (Figure 1). 2).

Three-dimensional structure of BopE

The interactions between the two three-helix bundles of BopE 78-261 comprise an intricate network of charge and hydrophobic interactions. Among the residues involved are BopE residue Pro 204 (corresponding to Ala 199 in SopE and SopE2) promotes these inter-bundle interactions by disrupting helix 5 into two parts termed 5' and 5''. As a consequence, 5' is positioned to bridge the 1 4 5 and 2 3 6 bundles and its residues are able to interact with residues in 2 and 6 of the 2 3 6 bundle (Figure 3). connecting element is a three-residue turn rather than the seven-residue loop observed in SopE2 69-240 and SopE 78-240 (Figure 3 and supplemental Figure S1). We reason that this protrusion of the polypeptide chain in the 5-6 loop at the apex of the , not observed in BopE 78-261 due to the key amino acid differences described here, counteracts extensive interbundle interaction in SopE 78-240 and SopE2 69-240 .

NMR investigation of the interaction between BopE 78-261 and Cdc42

In order to probe BopE 78-261 binding to Cdc42 in solution, twelve 2D 1 H- 15 N HQSC experiments on mixtures of varying ratios of uniformly 15 Due to peak overlap the degree and rate of broadening could not be quantified for a quarter of the approximately 175 backbone NH peaks. At a Cdc42 7:BopE 78-261 ratio of 1.0:1.0, many peaks were broadened to below 20% of their original height with the greatest concentrations of less rapidly broadened peaks found at the terminal regions, particularly the C-terminal region (Figure 4). The highest concentration of particularly rapidly broadened peaks (to noise level at a Cdc42 7:BopE 78-261 ratio of 1.0:1.0) occurred in helix 2; the equivalent SopE helix is involved in the interface between SopE 78-240 and Cdc42 in the SopE 78-240 -Cdc42 crystal structure [START_REF] Buchwald | Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE[END_REF].

(B) Appearance of multiple cross peaks per BopE 78-261 backbone NH

The second major observation upon increasing the Cdc42 7:BopE 78-261 ratio was the appearance of a peak or peaks in addition to the original backbone NH peak for approximately seventy of the 175 backbone NH peaks; single extra peaks accounted for about 75% of these seventy cases. In fifty-six instances these additional peaks could be assigned to Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2007 The Authors Journal compilation © 2007 Biochemical Society was predominant. Clusters of residues exhibiting multiple backbone NH peaks are located in the 1-2 loop and adjacent parts of 1 and 2, the -hairpin and loops adjacent to thehairpin including the putative 171 GAGT 174 catalytic motif, and around the 5-6 loop (Figure 5). There is also a sequence of such residues in 6.

(

Guanine nucleotide exchange activity of BopE 78-261 and BopE 78-261 mutants

In order to investigate further the requirement for a conformational change in BopE for catalysis of nucleotide exchange in Rho GTPases, three BopE 78-261 double mutants were made. These were N224P/R230Q (mutant 1), N216P/L226P (mutant 2) and R207E/N216P

(mutant 3). The mutations were selected according to their potential for changing BopE 78-261 from its relatively closed conformation to a more open conformation closer to those observed for SopE 78-240 in its complex with Cdc42 [START_REF] Buchwald | Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE[END_REF] and unbound SopE2 69-240 [START_REF] Williams | Solution structure, backbone dynamics, and interaction with Cdc42 of Salmonella guanine nucleotide exchange factor SopE2[END_REF], as follows:

N224P to induce a SopE/E2-like bulge in the 5-6 loop; R230Q to further disrupt the 5-6

interaction; N216P and L226P to induce a SopE/E2-like bulge in the 5-6 loop; R207E to disrupt the 5-6 interaction and N216P to induce a SopE/E2-like bulge in the 5-6 loop.

Like the wild type recombinant BopE 78-261 , the mutants were cloned and expressed as GST fusions. Mutant 1 expressed relatively poorly in E. coli but could be purified, mutant 2 expressed at low levels but disappeared during purification (perhaps this mutant is misfolded and therefore rapidly degraded) and mutant 3 expressed well and could be purified. In filter binding assays [49] with bovine serum albumin as negative control, the order of nucleotide exchange catalytic efficiency was: BopE 78-261 N224P/R230Q (mutant 1) > SopE2 69-240 > wild type BopE 78-261 >> BopE 78-261 R207E/N216P (mutant 3); in fact, mutant 3 showed essentially no catalytic activity (Figure 6). The reason for the lack of nucleotide exchange activity in mutant 3 is unclear but the R207E/N216P double mutation obviously induces changes that disrupt rather than enhance BopE function. The N224P/R230Q double mutation in BopE 78- 261 , on the other hand, produces a much more effective guanine nucleotide exchange factor than wild type BopE 78-261 and a better guanine nucleotide exchange factor than even SopE2 69- 240 (Figure 6), itself a better guanine nucleotide exchange factor for Cdc42 than SopE 78-240 [START_REF] Friebel | SopE and SopE2 from Salmonella typhimurium activate different sets of Rho GTPases of the host cell[END_REF]. This result, showing that mutations designed to abrogate important inter-bundle A number of putative type III-secreted effector proteins have been identified by analysis of the B. pseudomallei genome sequence [START_REF] Stevens | An inv/mxi-spa-like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen[END_REF]. One of these proteins, BopE, is a homologue of the potent guanine nucleotide exchange factors SopE [START_REF] Wood | SopE, a secreted protein of Salmonella dublin, is translocated into the target eukaryotic cell via a Sip-dependent mechanism and promotes bacterial entry[END_REF][START_REF] Hardt | S. Typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells[END_REF] and SopE2 [START_REF] Bakshi | Identification of SopE2, a Salmonella secreted protein which is highly homologous to SopE and involved in bacterial invasion of epithelial cells[END_REF][START_REF] Stender | Identification of SopE2 from Salmonella typhimurium, a conserved guanine nucleotide exchange factor for cdc42 of the host cell[END_REF] from Salmonella enterica (supplemental Figure S1). SopE and SopE2 catalyse nucleotide exchange in mammalian Rho GTPases, contributing to disruption of the host cell membrane and invasion of the host cell [START_REF] Galàn | Protein delivery into eukaryotic cells by type III secretion machines[END_REF][START_REF] Wood | SopE, a secreted protein of Salmonella dublin, is translocated into the target eukaryotic cell via a Sip-dependent mechanism and promotes bacterial entry[END_REF][START_REF] Bakshi | Identification of SopE2, a Salmonella secreted protein which is highly homologous to SopE and involved in bacterial invasion of epithelial cells[END_REF][START_REF] Friebel | SopE and SopE2 from Salmonella typhimurium activate different sets of Rho GTPases of the host cell[END_REF][START_REF] Hardt | S. Typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells[END_REF][START_REF] Friebel | Purification and biochemical activity of Salmonella exchange factor SopE[END_REF]. BopE likewise acts as a GEF for the Rho GTPases Cdc42 and Rac1 in vitro and may play a role in the invasion of non-phagocytic epithelial cells [START_REF] Stevens | A Burkholderia pseudomallei type III secreted protein, BopE, facilitates bacterial invasion of epithelial cells and exhibits guanine nucleotide exchange factor activity[END_REF]. This work shows that BopE and SopE/SopE2 catalytic domains adopt similar three-dimensional folds comprising two three-helix bundles but also shows that BopE has a more compact conformation, involving significant inter-bundle interaction, than its

  are entirely different from those of the best characterised eukaryotic GEFs which comprise a catalytic Dbl homology (DH) domain and an adjacent pleckstrin homology (PH) domain [28-30], although there are similarities in the catalytic mechanisms [31]. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 6 Dec 2007 as manuscript BJ20071546 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071546 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  2 Å was added to the upper bounds for strong and medium NOE restraints involving amide S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 6 Dec 2007 as manuscript BJ20071546 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071546 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 6 Dec 2007 as manuscript BJ20071546 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071546 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society widths of 8000 Hz in 1 H and 2000 Hz in 15 N. The initial NMR samples represented the end points of the titration. Intermediate values of BopE 78-261 :Cdc42 7 were obtained by simultaneously taking equal aliquots from both sample A and sample B and then transferring

  [ 3 H]GDP in reaction buffer containing 30 mM HEPES, 100 mM KCl, 0.1 mM EDTA, pH 7.5, 1 g of CPK (Sigma), 0.5 mM DTT. MgCl 2 was added to a final concentration of 2.8 mM and the mixture was incubated for another 2 minutes. Exchange reactions were started by adding the respective guanine nucleotide exchange factor and unlabelled GDP to the reaction mixture containing Cdc42 7 and [ 3 H]GDP. BSA (Sigma) was used as a negative control and SopE2 69-240 was used as a positive control. Aliquots were withdrawn and the reaction stopped by quenching in ice cold wash buffer, containing 30 mM HEPES, 100 mM KCl, 0.1 mM EDTA, 5 mM MgCl 2 , pH 7.5 followed by analysis by nitrocellulose filter binding assay [49]. Filters were washed twice with wash buffer, containing 30mM HEPES, 100 mM KCl, 0.1 mM EDTA, 5 S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 6 Dec 2007 as manuscript BJ20071546 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071546 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society mM MgCl 2 , pH 7.5, dried, and radioactivity bound to the filters was analysed by scintillation counting in a Tri-Carb Liquid Scintillation Counter 1600 TR (Packard, Meriden). RESULTS AND DISCUSSION Structure determination of BopE 78-261 A semi-automated procedure for iterative NOE assignment was used to generate the structure of BopE 78-261 . The final structures were generated using 2452 NOE-derived distance restraints (comprising 784 intra-residue, 1151 sequential and medium range and 517 long range NOEs where long range means between amino acids five or more apart in the sequence), 192 hydrogen bond restraints, 255 and dihedral angle restraints (132 and 123

  five arginine residues at sequence positions 100, 182, 200, 207 and 230 that are almost unique to BopE: SopE and SopE2 do not possess arginines in any of the corresponding positions (supplemental Figure S1) but the putative bacterial GEF family member CopE from Chromobacterium violaceum (accession AAQ57975) has arginines corresponding to BopE Arg 200 and Arg 207 . Three of the BopE arginines, Arg 200 , Arg 207 and Arg 230 , form part of the association between helices 5 and 6 while Arg 182 and Glu 125 are suitably located to link helices 4 and 2 at the putative Cdc42-binding face (based on the SopE 78-240 -Cdc42 complex structure[START_REF] Buchwald | Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE[END_REF]) of BopE through a potential salt bridge. Arg 100 (in helix 1) occupies a hydrophobic pocket between helices 2 and 5.

  In contrast to BopE Pro 204 , three SopE/E2 prolines appear to impede inter-bundle interaction and therefore contribute to the more open conformation adopted by SopE2 69-240 in solution relative to BopE 78-261 . Near to the apex of the formed by the two three-helix bundles, the loop connecting 5 and 6 in SopE2 69-240 and SopE 78-240 bulges (Figure3), S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 6 Dec 2007 as manuscript BJ20071546 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071546 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society presumably due to the presence of Pro 211 , Pro 219 and Pro 221 . Due to the lack of prolines at positions corresponding to 219 (Asn 224 in BopE) and 221 (Leu 226 in BopE), BopE 78-261 helix 6 begins earlier in the amino acid sequence than SopE/E2 6 and the BopE 78-261 5-6

  N-labelled BopE 78-261 and unlabelled human Cdc42 7 were performed. Two main types of behaviour were observed for peaks in BopE 78-261 HSQC spectra upon increasing the ratio of Cdc42 7 to BopE 78-261 : general broadening of peaks characterised by intensity loss throughout the spectrum; and for more than one third of residues the appearance of one or more additional peaks per backbone amide NH, indicating that BopE samples more than one conformation upon interaction with Cdc42 with slow exchange between the conformations.(A) BopE 78-261 cross peak broadening with increasing Cdc42 7 concentrationAlmost all of the backbone NH peaks in 1 H-15 N HQSC spectra of BopE 78-261 broadened as a function of increasing Cdc42 7 concentration (Figure4) until, at the highest Cdc42 7:BopE 78-261 ratio of 2.7:1, there was a subset of fifteen peaks that remained relatively intense (fourteen of which can be assigned as Thr 78 , Gly 79 , Asp 80 , Glu 109 , Phe 110 , Gly 160 , Glu 251 , Lys 252 , Ala 254 , Thr 255 , Asn 256 , Ala 257 , Gly 260 , Ala 261 and hence comprise amino acids in presumably relatively flexible parts of the protein near the N-and C-termini plus the 1-2 and pre--hairpin loops) plus a subset of readily detectable peaks (some of which can be assigned as Ala 81 , Lys 82 , Gln 83 , Ala 84 (all near N-terminus), Asp 162 , Gly 165 , Val 166 ( -hairpin), Gly 190 ( 4-5 loop), Glu 221 ( 6) and Ser 248 (unstructured C-terminal region)) and about forty further peaks that were still detectable just above the noise level. The remaining backbone NH peaks (in excess of one hundred) were broadened into the noise. Most asparagine and glutamine side chain NH 2 cross peaks were still present at the highest Cdc42 7:BopE 78-261 ratio of 2.7:1.0. The rate of backbone NH peak broadening was reasonably uniform across the sequence, suggesting that the major contributors to broadening are molecular weight increase upon complexation (a 1:1 BopE 78-261 :Cdc42 7 complex is just over double the molecular S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 6 Dec 2007 as manuscript BJ20071546 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071546 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society weight of BopE 78-261 ), shape change upon complexation with potential for non-linear increase in effective rotational correlation time, and exchange between free and bound BopE 78-261 .

a

  particular amino acid by proximity to the corresponding original backbone NH peak. At least two of the sixteen Asn and Gln side chain NH 2 groups also displayed a second pair of peaks in the presence of Cdc42 7. In the vast majority of cases with one or more extra peaks, upon increasing the Cdc42 7:BopE 78-261 ratio the Cdc42 7-induced extra peaks increased in height or sometimes reached a plateau as the original backbone NHpeaks decreased in height. The chemical shift difference between the original backbone NH peak and Cdc42 7-induced additional peak(s) at a Cdc42 7:BopE 78-261 ratio of 1.0:1.0 was calculated according to the formula ave = [( HN 2 + ( N 2 /25))/2] 1/2 , where HN and N correspond to the chemical shift difference in the amide 1 H and 15 N chemical shifts between the original NH peak and the Cdc42 7-induced extra peak(s); the ave values are shown in Figure 5A. In the cases where more than one Cdc42 7-induced extra peak could be assigned to a specific amino acid, the value plotted is the average of the ave values. For sixty-seven residues, only one backbone NH peak was observed throughout the titration; the approximate sequence positions of these residues are highlighted in Figure 5A. For the remaining forty or so backbone NH peaks, overlap hindered the observation of peak behaviour during the titration. The presence of the Cdc42 7-induced additional peaks for residues in several parts of BopE 78-261 indicates that BopE 78-261 samples more than one conformation in the presence of Cdc42 7 with the Cdc42 7-induced conformations in slow exchange with the initial Cdc42 7-free conformation. The fact that in about 75% of cases with more than one NH peak the additional peak was a single peak indicates that one Cdc42 7-induced conformation S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 6 Dec 2007 as manuscript BJ20071546 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071546

( 2 -

 2 3 loop) was involved in the Cdc42-induced slow conformational exchange but the behaviour of Asp 107 could not be monitored due to peak overlap. The significance of the positions of slowly exchanging residues in BopE 78-261 is discussed in the next section. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 6 Dec 2007 as manuscript BJ20071546 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071546 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society Implications of BopE 78-261 tertiary structure and BopE 78-261 -Cdc42 NMR titration for BopE interaction with Rho GTPasesThe question arises as to whether the conformational difference between the catalytic domain of BopE and those of SopE and SopE2 has implications for interaction with Rho GTPases. Analysis of the interface between SopE 78-240 and Cdc42 in the SopE 78-240 -Cdc42 complex crystal structure[START_REF] Buchwald | Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE[END_REF] reveals that the interaction can be broken down into two major components: a groove on SopE 78-240 accommodates a ridge on Cdc42 formed by residues 35 to 41 (switch region I) and the gap between the two three-helix bundles of SopE 78-240 accommodates Cdc42 residues Val 36 and Asp 38 (supplemental Figure2). The latter interaction in particular indicates that, in its closed conformation, BopE 78-261 would experience steric clashes with Cdc42. The resulting implication is that BopE catalytic domain must undergo a change from its closed conformation to a more open conformation like those of SopE and SopE2 catalytic domains in order to carry out its guanine nucleotide exchange function. A requirement for such a large scale conformational change is consistent with, and may at least partially explain, the observed differences in turnover number for guanine nucleotide exchange between BopE 78-261 and its Salmonella counterparts: a k cat of 0.48 s -1 was measured for BopE 78-261 -induced guanine nucleotide exchange in Rac1 (a similar rate was measured for Cdc42)[START_REF] Stevens | A Burkholderia pseudomallei type III secreted protein, BopE, facilitates bacterial invasion of epithelial cells and exhibits guanine nucleotide exchange factor activity[END_REF], whereas the k cat values for guanine nucleotide exchange in Cdc42 are 5 ± 1 s -1 and 19 ± 3 s -1 for SopE 78-240 and SopE2 69-240 respectively[START_REF] Friebel | SopE and SopE2 from Salmonella typhimurium activate different sets of Rho GTPases of the host cell[END_REF].It might then be asked whether BopE catalytic domain exists in equilibrium in solution between closed and open forms, or does it undergo a conformational change upon interaction with target protein? These two possibilities are not necessarily mutually exclusive -there may be an equilibrium in solution for unbound BopE catalytic domain but one that lies strongly towards the closed conformation. The results of the BopE 78-261 -Cdc42 7 titration are consistent with a significant conformational change in BopE 78-261 upon binding to Cdc42: when superimposed on the structure of BopE 78-261 (Figure5B), it is apparent that many of the amino acids that sampled one or more Cdc42 7-induced conformations during the BopE 78-261 -Cdc42 7 titration are located in potential hinge areas for a closed-to-open conformational change involving relative reorientation of the two three-helix bundles of BopE 78-261 . These hinge areas include the 1-2 loop and adjacent residues in 1 and 2, residues in the region between 3 and 4 that includes the -hairpin and 171 GAGT 174 putative catalytic motif, and residues in and around the 5-6 turn. Residues in the central part of 2 also show slow exchange between initial and Cdc42 7-induced conformations, consistent with a change in conformation and/or position of the 3-4 loop C-terminal to the -hairpin that associates with this part of 2 in Cdc42-free BopE 78-261 (Figure1). It is also striking that few of the amino acids with multiple NH peaks are located in areas that would be involved in any intra-S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 6 Dec 2007 as manuscript BJ20071546 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071546 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society bundle conformational changes, suggesting that the three-helix bundles themselves remain largely unchanged. The considerably greater magnitude of the Cdc42 7-induced chemical shift differences between free and Cdc42 7-bound states of BopE 78-261 compared to the magnitude of the chemical shift changes observed in the SopE2 69-240 -Cdc42 7 titration underpin the conclusion that BopE 78-261 undergoes greater structural change than SopE2 69-240 upon binding of the Rho GTPase.

  interactions and thereby induce a more open conformation in BopE 78-261 can substantially improve nucleotide exchange catalytic efficiency, adds further strong experimental support to that from NMR titration for the hypothesis that BopE GEF domain undergoes Rho GTPaseinduced change from a closed to an open conformation. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 6 Dec 2007 as manuscript BJ20071546 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071546 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society CONCLUSIONS The molecular mechanisms of B. pseudomallei pathogenesis are not well understood.

S t a g e 2

 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 6 Dec 2007 as manuscript BJ20071546 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071546 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure 1

 1 Figure 1 Structure of the BopE GEF domain (residues 78-261) (A) Backbone (N, C and C') trace of the twenty lowest energy structures coloured as a continuum from blue at N-terminus to red at C-terminus. (B) Ribbon diagram of the average structure coloured as in part A. The -helices and -hairpin are labelled. The 171 GAGT 174 putative catalytic motif lies between the -hairpin and 4.

Figure 2

 2 Figure 2 Comparison of the structures of SopE, SopE2 and BopE catalytic GEF domains Representations of the crystal structure of Cdc42-bound SopE 78-240 (green; PDB code 1GZS), solution structure of SopE2 69-240 (cyan; PDB codes 1R6E and 1R9K) and solution structure of BopE 78-261 (purple; PDB codes 2JOK and 2JOL)demonstrating the similarities and differences in the SopE 78-240 , SopE2 69-240 and BopE 78-261 structures. All three structures consist of two three-helix bundles with a connecting -hairpin that is followed by a loop that contains the 166 GAGA 169 (SopE/E2) / 171 GAGT 174 (BopE) catalytic motif.

Figure 3

 3 Figure 3 SopE 78-240 and BopE 78-261 conformation comparison In order to highlight the major secondary structure and conformational differences between SopE 78-240 (green) and BopE 78-261 (purple), the -helices of the two structures are shown and the locations of relevant proline residues are highlighted. Note the contrast between the protuberance of the 5-6 loop in SopE 78-240 due to Pro 211 , Pro 219 and Pro 221 and the compactness of the corresponding turn in BopE 78- 261 . Also note the disruption of BopE 78-261 helix 5 by Pro 204 into two parts, labelled 5' and 5'', that permits 5' in particular to interact with the 2 3 6 bundle. Both characteristics arise from the presence or absence of proline residues and result in the greater compactness of BopE 78-261 relative to SopE 78-240 and SopE2 69-240 . The viewpoint for this figure is approximately 180º different from that used for Figures 1 and 2.

Figure 4

 4 Figure 4 Reduction in BopE 78-261 backbone NH peak height as a function of BopE residue number upon titration with Cdc42 7 The percentage reduction in peak height is shown at a BopE 78-261 :Cdc42 7 molar ratio of 1.0:1.0. Only one BopE 78-261 residue, Cys-131, showed no reduction in peak S t a g e 2 ( a ) P O S T -P R I N T

Figure 6 S

 6 Figure 6 S t a g e 2 ( a ) P O S T -P R I N T

78-261 and comparison with Salmonella SopE 78-240 and SopE2 69-240 BopE

has been identified

[START_REF] Stevens | An inv/mxi-spa-like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen[END_REF]

as a homologue of the Salmonella effector proteins SopE and SopE2 (supplemental Figure

S1

). Overall, BopE has approximately 16% and 17% sequence identity with SopE and SopE2. Within the catalytic domain (comparing residues 78-240 of SopE and SopE2 with residues 78-240 of BopE), the sequence identity/similarity with SopE and SopE2 is approximately 25%/40% and 24%/39%. BopE 78-261 consists of six major -helices termed 1 to 6 arranged in two threehelix bundles, 1 4 5 and 2 3 6.

The three-helix bundles are connected by a loop between helices 1 and 2, a -hairpin (residues 162 to 168) followed by a loop that contains the putative (by comparison with SopE which has a 166 GlyAlaGlyAla 169 catalytic motif) 171

  As an illustration of the more extensive association between the bundles in BopE 78-261 , the buried surface areas between the three-helix bundles are 1693 Å 2 in SopE 78-240 , 1849 Å 2 in SopE2 69-240 and 2148 Å 2 in BopE 78-261 . Also, we have assigned 56 inter-bundle NOEs in BopE 78-261 compared with 20 such NOEs in our previous SopE vs BopE). (Note that the buried surface area and rmsd figures plus visual inspection (Figure2) show that SopE2 69-240 is somewhat intermediate as it has a slightly more closed conformation than SopE 78-240 ; it must be emphasised however that the only available SopE 78-240 structure is from the complex with Cdc42 so it is possible that unbound SopE 78-240 also has a more closed SopE2 69-240 -like conformation). When individual three-

	structure determination of SopE2 69-240 [27]. The greater conservation of bundle structure
	relative to bundle-bundle orientation is quantitatively illustrated by rmsd values for
	superimposed C traces and by comparison of inter-helical angles. When the catalytic
	domains are superimposed, the rmsd values are 2.5 Å (SopE vs SopE2), 3.9 Å (SopE2 vs
	BopE) and 5.0 Å (

The BopE 78-261 fold is similar to that of its Salmonella counterparts SopE 78-240 and SopE2 69-240 but is more closed and compact with substantial interaction between the two S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 6 Dec 2007 as manuscript BJ20071546 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071546 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society three-helix bundles (Figures

2 and 3

). helix bundles are superimposed, the corresponding values are 2.3Å, 2.9Å and 2.3Å for the 1 4 5 bundle and 1.6Å, 2.8Å and 2.8Å for the 2 3 6 bundle. Calculation of inter-helical angles shows that the angles between helices in different bundles tend to differ considerably between BopE 78-261 and the two Salmonella GEFs (Table

  -induced chemical shift changes fell into two groups, one of which showed very good agreement with the SopE 78-240 residues involved in important intermolecular interactions in the SopE 78-240 -Cdc42 crystal structure[START_REF] Buchwald | Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE[END_REF]: this group included SopE2 69-240 residues Gln 109 ( 2), Asp 124 ( 2), Gly 165 (adjacent to catalytic motif), Gly 166 , Gly 168 , Ala 169 (all catalytic motif), Val 174 ( 4), Gln 194 ( 5) and Lys 198[START_REF] Currie | Endemic melioidosis in tropical northern Australia: a 10-year prospective study and review of the literature[END_REF]. The second group of perturbed SopE2 69-240 residues comprised several scattered internal residues and isolated residues on the opposite side of the molecule to the binding interface. In contrast to SopE2 69- 240 , slow exchange between unbound and Cdc42 7-bound conformations of BopE 78-261 was observed during the BopE 78-261 -Cdc42 7 titration. The chemical shift differences between these states of BopE 78-261 were, in general, four to five or more times the magnitude of the Cdc42 7-induced chemical shift changes observed in the SopE2 69-240 -Cdc42 7 titration. The BopE equivalents (BopE residues Asp 128 , Gly 171 , Gly 173 , Thr 174 and Thr 179 ) of five of the Cdc42-perturbed SopE2 69-240 residues (SopE2 69-240 residues Asp 124 , Gly 166 , Gly 168 , Ala 169 and Val 174 ) listed above were involved in the Cdc42-induced slow conformational exchange whereas Ser 170 , Tyr 199 and Gln 203 , the BopE equivalents of SopE residues Gly 165 , Gln 194 and Lys 198 , were not. The behaviour of Gln 113 (BopE equivalent of SopE Gln 109 ) during the titration could not be monitored due to peak overlap. Of the BopE equivalents of a further two SopE 78-240 residues that interact with Cdc42 in the SopE 78-240 -Cdc42 crystal structure but that were not significantly perturbed in the SopE2 69-240 -Cdc42 7 NMR titration[START_REF] Williams | Solution structure, backbone dynamics, and interaction with Cdc42 of Salmonella guanine nucleotide exchange factor SopE2[END_REF], Ala135

	C) Comparison of BopE 78-261 -Cdc42 and SopE2 69-240 -Cdc42 titration results
	Very similar NMR titrations, both using Cdc42 7 and the same protocol, have now
	been carried out to study the BopE 78-261 -Cdc42 (this work) and SopE2 69-240 -Cdc42 [27]
	interactions. BopE 78-261 and SopE2 69-240 both experienced widespread backbone NH peak
	broadening upon increasing the ratio of Cdc42 7 to BopE 78-261 /SopE2 69-240 . The broadening
	was, if anything, more rapid in the SopE2 69-240 -Cdc42 7 titration. The SopE2 69-240 NH peaks
	that underwent Cdc42 7

Table 1 Structural statistics on NMR-derived structures of BopE GEF domain

 1 Salmonella homologues. The most open conformation of the three is for Cdc42-bound SopE 78-240 , with unbound SopE2 69-240 slightly more closed. It is worth noting, however, that SopE residues involved in contacting Cdc42 in the SopE 78-240 -Cdc42 complex crystal structure [26] are largely conserved or conservatively substituted in BopE (supplemental Figure S1). SopE residues (Asp 103 , Gln 109 , Asp 124 and Gly 168 ) shown by mutation to be functionally important [31] are, moreover, conserved in BopE. It seems likely, therefore, that despite its more closed conformation, BopE ultimately utilises the same mechanism as SopE and other Rho GEFs [52] in catalysing guanine nucleotide exchange in Rho GTPases. This would require that BopE change from closed to open conformations in the presence of Rho GTPase target proteins. Such a conformational change is evidenced here by the results of a BopE 78-261 -Cdc42 NMR titration and measurements of nucleotide exchange catalytic efficiency comparing wild type and mutant BopE GEF domain. Phosphorylation of BopEwould not seem to be required for any conformational change as we and others have shown that BopE 78-261 purified from E. coli exhibits guanine nucleotide exchange factor activity[START_REF] Stevens | A Burkholderia pseudomallei type III secreted protein, BopE, facilitates bacterial invasion of epithelial cells and exhibits guanine nucleotide exchange factor activity[END_REF].Finally, given the sequence and conformational differences between BopE and SopE/E2 catalytic domains, it is possible that there are as yet unknown differences in specificity among the members of this family of bacterial guanine nucleotide exchange factors, with the potential for modulation of the activities of small G-proteins in addition to Cdc42 and Rac1.

	Total number of NOE restraints	2452
	intraresidue	784
	sequential/med. range (i to i+1 4)	1151
	long range	517
	Number of dihedral angle restraints	255
	Number of hydrogen bond restraints	192
	Number of backbone 1 D NH RDC restraints Rmsd for backbone atoms a	98 0.65Å
	Rmsd for non-hydrogen atoms a	1.15Å
	Average numbers of NOE violations
	>0.3Å (per structure)	5
	>0.5Å (per structure)	1
	Average number of dihedral angle violations >5 o (per structure)	0
		Average structure	Ensemble
	Ramachandran plot regions b
	Most favoured (%)	88.8	80.8
	Additional allowed (%)	8.7	15.9
	Generously allowed (%)	1.9	2.6
	Disallowed (%)	0.6	0.7
	a The rmsd from the mean structure calculated over residues 83-99, 110-133, 143-156, 177-
	189, 205-217, and 220-246.

S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 6 Dec 2007 as manuscript BJ20071546 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071546 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society b Calculated with PROCHECK-NMR [46]. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 6 Dec 2007 as manuscript BJ20071546 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071546 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 2 Comparison of helix crossing angles in the solution structures of BopE 78-261 and SopE 69-240 and the crystal structure of Cdc42-bound SopE 78-240

 2 The BopE 78-261 helix crossing angle minus the SopE2 69-240 helix crossing angle. e The BopE 78-261 helix crossing angle minus the SopE 78-240 helix crossing angle. f The SopE2 69-240 helix crossing angle minus the SopE 78-240 helix crossing angle.

	Crossing angle (deg) a

a Calculated with Interhlx (K. Yap, University of Toronto; (http://nmr.uhnres.utoronto.ca/ikura/interhlx). b SopE2 GEF domain NMR structure (PDB entry 1R9K). c SopE GEF domain crystal structure (PDB entry 1GZS). d

THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071546 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

This work was supported at Bath by The Wellcome Trust (grant 060998) and at IAH by BBSRC. The Wellcome Trust is acknowledged for purchase of the 600 MHz NMR spectrometer (grant 051902) used in this study. C.W. was supported by a PhD studentship from EPSRC. We thank Julian Eaton (Bath) for constructive criticism of the manuscript and help with rmsd calculations, Lewis Kay (Toronto) for some of the pulse sequences used here, Charles Schwieters for help with the Python interface of Xplor-NIH, Kyoko Yap for the programme Interhlx, Graham Pavitt's group (Manchester) for instruction on filter binding assays and Mareike Posner (Bath) for advice on enzyme assays.

height. For the remainder of residues that appear with 0% reduction on this plot, peak height could not be quantified due to peak overlap. Note that prolines, which do not give rise to peaks in 1 H- 15