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• W2044208802 abstract "Article1 July 2002free access Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE Gretel Buchwald Gretel Buchwald Max-Planck-Institut für Molekulare Physiologie, Abt. Strukturelle Biologie, Otto-Hahn-Str. 11, D-44227 Dortmund, Germany Search for more papers by this author Andrea Friebel Andrea Friebel Max von Pettenkofer-Insitut, Pettenkoferstr. 9a, D-80336 München, Germany Search for more papers by this author Jorge E. Galán Jorge E. Galán Section of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT, 06536 USA Search for more papers by this author Wolf-Dietrich Hardt Wolf-Dietrich Hardt Institute of Microbiology, ETHZ/LFV B20, Schmelzberg Str. 7, 8092 Zürich, Switzerland Search for more papers by this author Alfred Wittinghofer Corresponding Author Alfred Wittinghofer Max-Planck-Institut für Molekulare Physiologie, Abt. Strukturelle Biologie, Otto-Hahn-Str. 11, D-44227 Dortmund, Germany Search for more papers by this author Klaus Scheffzek Corresponding Author Klaus Scheffzek European Molecular Biology Laboratory (EMBL), Structural and Computational Biology Programme, Meyerhofstr. 1, D-69117 Heidelberg, Germany Search for more papers by this author Gretel Buchwald Gretel Buchwald Max-Planck-Institut für Molekulare Physiologie, Abt. Strukturelle Biologie, Otto-Hahn-Str. 11, D-44227 Dortmund, Germany Search for more papers by this author Andrea Friebel Andrea Friebel Max von Pettenkofer-Insitut, Pettenkoferstr. 9a, D-80336 München, Germany Search for more papers by this author Jorge E. Galán Jorge E. Galán Section of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT, 06536 USA Search for more papers by this author Wolf-Dietrich Hardt Wolf-Dietrich Hardt Institute of Microbiology, ETHZ/LFV B20, Schmelzberg Str. 7, 8092 Zürich, Switzerland Search for more papers by this author Alfred Wittinghofer Corresponding Author Alfred Wittinghofer Max-Planck-Institut für Molekulare Physiologie, Abt. Strukturelle Biologie, Otto-Hahn-Str. 11, D-44227 Dortmund, Germany Search for more papers by this author Klaus Scheffzek Corresponding Author Klaus Scheffzek European Molecular Biology Laboratory (EMBL), Structural and Computational Biology Programme, Meyerhofstr. 1, D-69117 Heidelberg, Germany Search for more papers by this author Author Information Gretel Buchwald1, Andrea Friebel2, Jorge E. Galán3, Wolf-Dietrich Hardt4, Alfred Wittinghofer 1 and Klaus Scheffzek 5 1Max-Planck-Institut für Molekulare Physiologie, Abt. Strukturelle Biologie, Otto-Hahn-Str. 11, D-44227 Dortmund, Germany 2Max von Pettenkofer-Insitut, Pettenkoferstr. 9a, D-80336 München, Germany 3Section of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT, 06536 USA 4Institute of Microbiology, ETHZ/LFV B20, Schmelzberg Str. 7, 8092 Zürich, Switzerland 5European Molecular Biology Laboratory (EMBL), Structural and Computational Biology Programme, Meyerhofstr. 1, D-69117 Heidelberg, Germany *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2002)21:3286-3295https://doi.org/10.1093/emboj/cdf329 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The bacterial enteropathogen Salmonella typhimurium employs a type III secretion system to inject bacterial toxins into the host cell cytosol. These toxins transiently activate Rho family GTP-binding protein-dependent signaling cascades to induce cytoskeletal rearrangements. One of these translocated Salmonella toxins, SopE, can activate Cdc42 in a Dbl-like fashion despite its lack of sequence similarity to Dbl-like proteins, the Rho-specific eukaryotic guanine nucleotide exchange factors. To elucidate the mechanism of SopE-mediated guanine nucleotide exchange, we have analyzed the structure of the complex between a catalytic fragment of SopE and Cdc42. SopE binds to and locks the switch I and switch II regions of Cdc42 in a conformation that promotes guanine nucleotide release. This conformation is strikingly similar to that of Rac1 in complex with the eukaryotic Dbl-like exchange factor Tiam1. However, the catalytic domain of SopE has an entirely different architecture from that of Tiam1 and interacts with the switch regions via different amino acids. Therefore, SopE represents the first example of a non-Dbl-like protein capable of inducing guanine nucleotide exchange in Rho family proteins. Introduction Ras-related GTP-binding proteins of the Rho family (e.g. RhoA/B, Cdc42 and Rac1/2) are key regulators of vital cellular functions including cytoskeletal rearrangements, cell shape, motility, cytokinesis, gene expression, cell aggregation and cell–cell adhesion (Hall, 1998). They exert their function by cycling between a biologically inactive GDP-bound and an active GTP-bound conformation (Bourne et al., 1990, 1991). As spontaneous guanine nucleotide dissociation is very slow, activation is dependent on guanine nucleotide exchange factors (GEFs), which enhance nucleotide exchange rates by inducing structural changes in the nucleotide-binding site (Vetter and Wittinghofer, 2001). All cellular Rho family-specific GEFs identified so far share a common module, the Dbl homology (DH) domain. This domain forms the catalytically active part of these molecules responsible for binding to the switch I and switch II regions of the Rho component (Cerione and Zheng, 1996; Cherfils and Chardin, 1999). These regions in GTP-binding proteins define major conformational differences between the active and inactive states (Milburn et al., 1990). The bacterial enteropathogen Salmonella typhimurium is one of the leading causes of gastroenteritis in developed countries. During its evolution as a pathogen, S.typhimurium has acquired a variety of mechanisms to colonize, replicate and survive within its animal hosts (Groisman and Ochman, 1996). Essential for S.typhimurium virulence is a specialized protein secretion and delivery system termed type III that is encoded within the SPI-1 pathogenicity island of this bacterium (Galán and Curtiss, 1989; Watson et al., 1998; reviewed by Wallis and Galyov, 2000). This system mediates the transfer of several bacterial proteins that have the capacity to modulate signal transduction into the host cell cytosol. The translocated toxins trigger responses including cell death in macrophages, nuclear responses resulting in pro-inflammatory cytokine production, and actin cytoskeletal rearrangements leading to bacterial internalization by normally non-phagocytic cells such as epithelial cells (reviewed by Galán, 1999). In fact, S.typhimurium serves as an important model to study the cell biology of bacterial host cell invasion (Galán and Zhou, 2000). Recent work has demonstrated that S.typhimurium invasion of host cells is triggered mainly by three translocated effector proteins (Mirold et al., 2001; Zhou et al., 2001): SopE, SopE2 (see below) and SopB, a protein with inositol phosphatase activity (Hong and Miller, 1998; Norris et al., 1998). Cdc42 is an essential component of the host cellular signaling cascades triggered by the translocated S.typhimurium effector proteins during invasion (Chen et al., 1996). The effector proteins SopE and SopE2 (70% amino acid sequence identity; Wood et al., 1996; Hardt et al., 1998b; Bakshi et al., 2000; Stender et al., 2000) bind directly to and activate host cellular Cdc42 by catalyzing guanine nucleotide exchange (Hardt et al., 1998a; Rudolph et al., 1999a; Stender et al., 2000). In fact, transient expression of SopE or SopE2 in cultured cells is sufficient to activate Cdc42, induce actin cytoskeletal reorganization and mediate internalization of non-invasive S.typhimurium mutants (Hardt et al., 1998b; Stender et al., 2000). Biochemical analyses have demonstrated that, unlike many other bacterial toxins, SopE and SopE2 activate host cellular Rho family proteins by transient interaction and not by chemical modification. Even though SopE and SopE2 do not share any recognizable sequence similarity to any known proteins from bacteria or eukaryotes, particularly the DH family proteins, they act as genuine GEFs (Rudolph et al., 1999a; Stender et al., 2000). Their ability to form nucleotide-free binary complexes with Rho proteins and to stimulate the rate of nucleotide exchange by several orders of magnitude are strikingly similar to those of genuine eukaryotic GEFs such as RCC1, a GEF for Ran (Klebe et al., 1995), or Cdc25, a GEF for Ras (Lenzen et al., 1998). Preliminary experiments indicate that its catalytic activity is much higher than that of genuine eukaryotic Rho-GEFs of the DH family (A.Wittinghofer, unpublished). For this reason, it was of great interest to study the structural basis of SopE-mediated guanine nucleotide exchange. Here we have analyzed by X-ray crystallography at 2.3 Å resolution the interaction between the catalytic fragment of SopE and its host cellular target, Cdc42. We found that the structure of SopE is entirely different from that of Dbl-like RhoGEFs. Yet, the interaction of SopE locks the switch I and switch II regions of Cdc42 in the same conformation as that observed in the equivalent regions of Rac1 when bound to its celluar GEF Tiam1 (Worthylake et al., 2000), or in the Cdc42–Dbs complex (Rossman et al., 2002). These data provide the first insight into how proteins lacking any similarity to the Dbl-like protein family can act as efficient GEFs for Rho family GTP-binding proteins. Results and discussion Structure determination and overview For crystallographic analysis, we used C-terminally truncated Cdc42 (amino acids 1–178) and the catalytic domain of SopE (SopEC, residues 78–240) (SopE from now onwards). It was shown previously that C-terminal truncation of Cdc42 does not interfere with SopE-mediated GEF activity (Rudolph et al., 1999a). The components of the complex were expressed in Escherichia coli and purified from cell lysates according to standard protocols. After mixing the constituents with a 2-fold molar excess of Cdc42, the SopE–Cdc42 complex was purified by size exclusion chromatography, crystallized, and the structure determined as described in Materials and methods. The current structural model of the asymmetric unit consists of two SopE–Cdc42 complexes, with most of the structure being well defined in its electron density. Altogether, six sulfate ions and 160 solvent molecules were added on the basis of Fo − Fc electron density maps. A summary of the crystallographic data analysis is given in Table I. Figure 1A shows a representative segment of the experimental electron density map. Figure 1.The GEF domain of SopE. (A) Segment of the experimental electron density map, contoured at 1.5σ showing a section of the complex interface. (B) Diagram of the structure of SopE, with helices as cylinders and the catalytic loop highlighted in red. (C) Sequences of the GEF domains of SopE (S.typhimurium, accession No. O52623; S.dublin, accession No. O06949; S.typhi, accession No. Q9RPM6) and SopE2 (accession No. Q9KIZ2), with the assignment of secondary structure elements according to the program DSSP (Kabsch and Sander, 1993). Selected residues in the interface (see Figure 2) are marked with filled circles. The catalytic loop involved in close contact with Cdc42 is highlighted. (D) Diagram of the structure of the DH domain of Tiam1, with helices as cylinders, as seen in the Rac1–Tiam1 complex (Worthylake et al., 2000), in the same orientation relative to the GTP-binding protein as SopE in (B). The functionally important lysine residue (see Figure 3) is included. Download figure Download PowerPoint Table 1. Summary of crystallographic analysis Native CH3HgCl K3UO2F5 Data collectiona Resolution (Å) 20−2.3 25−2.3 20−2.5 Highest shell (Å) 2.4−2.3 2.4−2.3 2.6−2.5 Unique reflections 40 507 (4737) 76 154 (9239) 59 109 (6611) Redundancy 8.7 (7.6) 2.6 (2.6) 1.9 (1.9) I/σ 15.2 (5.5) 12.48 (5.18) 7.49 (2.37) Rsym (%)b 11.2 (32.8) 5.6 (20.2) 7.2 (29.4) RF (%)c – 28.9 13.8 Completeness (%) 99.9 (100.0) 97.5 (98.6) 99.3 (99.8) MIRAS analysis Resolution (Å) – 20−2.6 20−2.6 Soaking Concentration/time 0.1 mM/13 h 5 mM/108 h No. of sites 4 3 Rcullisd 0.75 0.86 Anomalous Rcullis 0.89 0.99 Phasing powere 1.29 0.78 Anomalous phasing power 7.15 1.85 Overall figure of merit 0.52 Refinement Resolution (Å) 20−2.3 Reflections 40 297 Rwork (%)f 22.7 Rfree (%)g 25.8 No. of atoms Protein 5308 Sulfate 6 Solvent 160 R.m.s. deviations Bonds (Å) 0.006 Angles (°) 1.25 a Numbers in parentheses give values for the highest resolution bin. b Rsym = Σ|Ihi − Ih|/ΣIhi, where Ihi is the scaled intensity of the ith symmetry-related observation of reflection h and Ih is the mean value. c RF = 2Σh|FPH − FP|/Σh|FPH + FP|, where FPH and FP are the derivative and native structure amplitudes. d Generalized Rcullis as implemented in CNS (Brünger et al., 1998). e As defined in CNS (Brünger et al., 1998). f Rwork = Σh|Fo − Fc|/ΣhFo, where Fo and Fc are the observed and calculated structure factor amplitudes for reflection h, respectively. g Value of Rfree calculated for 10% randomly chosen reflections not included in the refinement. SopE SopE is composed of six α-helices arranged in two three-helix bundles. The two bundles measure ∼40 Å in length and 30 Å in width and are arranged in a V-shaped fashion (Figure 1B). We observed only one small two-stranded β-sheet, which is followed by a peptide segment (residues 165–172) connecting the two sides of the ‘V’. This segment harbors a 166GAGA169 motif, which is proposed to represent the catalytic core of SopE (see Figure 1C). Analysis of the surface charge distribution with the program GRASP (Nicholls et al., 1991) did not show preferentially acidic or basic character. A homology search with the program DALI (Holm and Sander, 1993) did not identify similarity with the Rho family-specific DH domains from the eukaryotic RhoGEFs Sos, Trio, βPIX or Tiam1 (Aghazadeh et al., 1998; Liu et al., 1998; Soisson et al., 1998; Worthylake et al., 2000). The DH domain is also an α-helical protein, but the location and topological arrangement of helices are quite different, as shown in Figure 1D. SopE also has no similarity to other nucleotide exchange factors for Ras-related GTP-binding proteins (Boriack-Sjodin et al., 1998; Cherfils et al., 1998; Mossessova et al., 1998; Renault et al., 1998). Structural relationships were found with proteins containing three-helix bundle ‘motifs’, with ribosome recycling factor (Selmer et al., 1999; Kim et al., 2000) and the helical segment of human guanylate-binding protein showing the highest scores of 5.8 and 5.7, respectively (Prakash et al., 2000). These similarities do not reflect common functional aspects, but rather confirm the three-helix bundle as a structural element of a large number of proteins. The complex with Cdc42 The overall structure of Cdc42 is similar to that found in crystal structures of other Rho family proteins (e.g. Hirshberg et al., 1997; Wei et al., 1997). Conformational changes are restricted to the switch regions of Cdc42 that interact directly with SopE (discussed below). The switch I and switch II regions of Ras-related GTP-binding proteins are involved in nucleotide binding and define the major conformational differences between GTP- and GDP-bound forms (Milburn et al., 1990) (see below). Presumably as a consequence of the crystallization conditions (see Materials and methods), sulfate ions are found in various favorable binding sites, the functionally most important of which is the phosphate-binding (P) loop (Saraste et al., 1990). This sulfate ion is located in the position which would be occupied by the β-phosphate moiety when nucleotide is present. Like the β-phosphate, it is stabilized by interactions with main chain amide groups derived from the P-loop motif (GxxxxKS/T) and the side chain amino group of Lys16. A similar situation is found in the complex between Rac1 and the RhoGEF Tiam1 (Worthylake et al., 2000) (see below), in the Ran–RCC1 complex (Renault et al., 2001) or in guanosine-bound Ras (Scheffzek et al., 1994). The complex interface The complex interface is formed primarily by helices α2, α4 and α5 from SopE, including flanking regions that communicate mainly with the switch regions of Cdc42 (Figure 2A and B). The interface buries ∼2800 Å of accessible surface area, which is similar to that of the Rac1–Tiam1 (Worthylake et al., 2000) and Dbs–Cdc42 (Rossman et al., 2002) complexes and somewhat higher than those of other Rho regulatory complexes (Rittinger et al., 1997; Hoffman et al., 2000; Scheffzek et al., 2000). As in other G-protein–GEF complexes (Kawashima et al., 1996; Wang et al., 1997; Boriack-Sjodin et al., 1998; Goldberg, 1998), the base-binding pocket is not occluded by the GEF component and is accessible from the solvent. SopE does not interact with P-loop residues, which is consistent with the observation that Cdc42G12V is an efficient substrate (Rudolph et al., 1999a). Figure 2.The Cdc42–SopE complex. (A) Ribbon representation of the complex with SopE in blue and Cdc42 in yellow. The switch regions are shown in green. The catalytic loop inserted between the switch regions is highlighted in red. (B) Schematic representation of the Cdc42–SopE interaction with important interactions indicated. Hydrophobic contacts are in green, polar interactions in red. Arrowheads mark side chain and straight ends indicate main chain contributions. (C) Close up view of the interface region with the switch regions of GDP-bound Cdc42 (in light gray) included. Side chains of residues considered important for the interaction are shown. Dashed segments indicate disordered regions, modeled stereochemically. The gray ball near Ala59 is the Mg2+ ion from the Cdc42–GDP structure. (D) Stereo view of the nucleotide-binding region, showing the specific rearrangements of the switch regions associated with SopE–Cdc42 interaction, as discussed in the text. (E) Multiple turnover kinetics of nucleotide exchange on Cdc42·mantGDP mediated by GST–SopEG168A. Release of mantGDP was followed by fluorescence spectrometry in buffer S (50 mM NaCl, 50 mM Tris–HCl pH 7.6, 5 mM MgCl2) in the presence of 1 mM GDP. The assays were performed with 1 μM Cdc42·mantGDP and 250 nM GST, 10 mM EDTA, 25 nM GST–SopEG168A (gray line) or 25 nM GST–SopE (excitation wavelength 366 nm, emission wavelength 440 nm, step size 1 nm, bandpass 4 nm). Download figure Download PowerPoint In order to explore the catalytic mechanism of SopE-mediated guanine nucleotide exchange, we have superimposed the structure of the nucleotide-free SopE–Cdc42 complex (Figure 2A) with the structure of GDP-loaded Cdc42 (Rudolph et al., 1999b). The core of the G-domains is virtually identical in both structures, but major conformational changes are observed in the switch I/II regions of Cdc42 (Figure 2C). The most notable feature of SopE contributing to these conformational changes is the catalytic 166GAGA169-loop of SopE, which is inserted between the switch regions of Cdc42 and appears to exert a push-and-pull-type movement whereby switch I is pushed aside and switch II is pulled towards the loop (Figure 2C). Insertion of the 166GAGA169-loop of SopE induces two consecutive peptide flips in the C-terminal half of switch I, rotating Phe37cdc42 by ∼90° and Tyr40cdc42 by almost 180° (Figure 2D). The resulting conformation is stabilized by a salt bridge between Asp38cdc42 and Lys198sopE along with a hydrogen bond between the hydroxyl group of Tyr40cdc42 and the main chain carbonyl group of Gln194sopE. In addition, the hydrophobic core burying the aromatic side chains of Phe37cdc42 and Tyr40cdc42 appears to contribute to stabilization (Figure 2). The major contribution of the catalytic 166GAGA169-loop to the proposed mechanism of SopE-mediated nucleotide release is supported by site-directed mutagenesis of Gly168sopE to alanine. The mutant protein, under the conditions employed, does not stimulate nucleotide exchange on Cdc42 as measured by a fluorescence assay where Cdc42-bound fluorescent mant-nucleotide is exchanged against a large excess of unlabeled nucleotide (Figure 2E). The displacement of switch I seems to be stabilized by interactions of Asp124sopE with the hydroxyl group of Tyr32cdc42, as well as with the main chain amide groups of Thr35cdc42 and Val36cdc42. The side chains of these residues also participate in the interface. Presumably as a consequence of the observed interactions, the N-terminal portion of switch I becomes disordered (residues 26–31) and extruded from the nucleotide-binding pocket (Figure 2B and C). Switch II is displaced towards the catalytic loop and its C-terminal end is involved in interactions with the area around helices α2 and α4 of SopE, forming an extensive interface (Figure 2B and C). Hydrophobic contributions come from Tyr64cdc42, and a number of polar contacts involving Tyr64cdc42, Asp65cdc42, Arg66cdc42 and Ser71cdc42 apparently stabilize the interface in this area (Figure 2B and C). The most extensive contacts are formed between the catalytic loop of SopE and the N-terminal part of switch II, involving residues Ala59cdc42, Gly60cdc42 and Gln61cdc42. They form a network of polar main chain and van der Waals interactions with Ser165sopE, Gly166sopE, Gly168sopE and Ala169sopE. The most dramatic effect on the conformation of switch II is a peptide flip involving Ala59cdc42, whereby its methyl group points into and thus blocks the Mg2+-binding site (Figure 2C). Strikingly, a similar conformation of Ala59cdc42 has been observed in GDP-bound RhoA in the absence of Mg2+ (Shimizu et al., 2000) and also in other G-protein–GEF complexes (Boriack-Sjodin et al., 1998; Worthylake et al., 2000). The conformation of Ala59cdc42 is stabilized by a main chain–main chain interaction with Ala169sopE. Implications for nucleotide release We have shown earlier that SopE increases the guanine nucleotide exchange on Rho proteins 105-fold above the intrinsic reaction rate (Rudolph et al., 1999a), which is similar to the catalytic rates of GEFs for Ras-related proteins (Klebe et al., 1995; Lenzen et al., 1998; Hutchinson and Eccleston, 2000). The structure of the nucleotide-free SopE–Cdc42 complex provides important insights to explain the accelerated guanine nucleotide release stimulated by the bacterial exchange factor. Reorientation of switch I causes extrusion of residues potentially important for binding of the nucleotide, especially of the guanine base. The rearrangements in switch I include the disordering of its N-terminal segment, as inferred from ill-defined electron density in this region (Figure 2C). This leads to extrusion of Phe28cdc42 from the nucleotide-binding pocket, where it usually provides a major contribution to the binding of GDP/GTP. Similarly to Ras, the mutation of this amino acid to leucine leads to 140-fold increased nucleotide dissociation rates (Reinstein et al., 1991; Lin et al., 1997), highlighting the importance of this hydrophobic interaction for nucleotide affinity. In the nucleotide-free Ras–Sos complex, the switch I region of Ras is flipped out of its position close to the nucleotide by a long helical extension on Sos, and it has been argued that this is the key feature driving guanine nucleotide release (Boriack-Sjodin et al., 1998). Here we also find that most of the switch I region is pushed aside due to the action of the catalytic loop of SopE, arguing that reorientation of residues such as Glu31cdc42 in addition to Phe28cdc42 might contribute to accelerated nucleotide release. In the Rac1–GppNHp, but not in the Cdc42–GDP complex, Glu31rac1 is coordinated to the 3′-oxygen of the ribose and this residue is conserved in Rho family proteins. Therefore, we speculate that the specific reorientation of Phe28cdc42 and Glu31cdc42 and/or the general rearrangement of the effector loop, which runs alongside the nucleotide and partially shields it from the solvent, might both contribute to the accelerated nucleotide release. In switch II, a peptide flip positions the methyl group of Ala59cdc42 within 2 Å of the Mg2+-binding site (Figure 2C and D). Since Mg2+ is important for high-affinity binding of GDP/GTP from Ras-like GTP-binding proteins (Vetter and Wittinghofer, 2001) and excess Mg2+ decreases dissociation of GDP from Cdc42 by 140-fold (Zhang et al., 2000), the SopE-induced displacement of Mg2+ might be an important step in guanine nucleotide release. The conformation of switch II in SopE-bound Cdc42 is strikingly similar to that of switch II regions in Mg2+-free GDP-bound RhoA (Shimizu et al., 2000) and Tiam1-bound Rac1 (Worthylake et al., 2000), with Ala59rac1 pointing into the Mg2+-binding site. It is possible that part of the switch II region is stabilized in a Mg2+-free conformation and that the GEF activity of SopE is due at least partially to its effect on removal of the metal ion. The role of the methyl group of Ala59 in the GEF reaction has been investigated for the Ras–Sos system by site-directed mutagenesis (Hall et al., 2001). These studies showed that this residue might not be important for nucleotide release in the Ras–Sos reaction. In order to assess the role of the almost invariant Ala59 for the RhoGEF mechanism in more detail, however, site-directed mutagenesis combined with fast kinetic analysis would be necessary. Reorientation of Thr35 in switch I, in turn, is unlikely to have an effect on nucleotide affinity since it is coordinated to Mg2+ only in the triphosphate form, and its mutation to alanine in Ras reduces Mg2+ affinity in that complex only 4-fold (Spoerner et al., 2001). Studies on Ras have shown that the β-phosphate contributes significantly to binding affinity of the nucleotide, since GMP and guanosine bind with five orders of magnitude weaker affinity than GTP or GDP (John et al., 1990). Since removal of Mg2+ or Phe28cdc42 would only partially account for the 105-fold stimulation of nucleotide release from Cdc42 by SopE, it is possible that SopE, like other GEFs, interferes with binding of the phosphate moiety of the nucleotide. Structures of Ras, Arf and EF-Tu complexed with their cognate GEFs indeed show GEF side chains pointing into phosphate-binding regions (Kawashima et al., 1996; Wang et al., 1997; Boriack-Sjodin et al., 1998; Goldberg, 1998). However, no such residues were found in our structure or in other GEF–G-protein complexes where a sulfate ion is bound in the P-loop (Worthylake et al., 2000; Renault et al., 2001). On the basis of the Ran–RCC1 complex (Renault et al., 2001), it has been suggested that the sulfate-bound structure in the crystal represents an intermediate along the kinetic pathway of nucleotide exchange, and that rearrangement of the nucleotide-binding site after release of the anion may produce conformational changes that lead to the nucleotide-free structures observed in Ras–Sos, Arf–Sec7 and EF-Tu–EF-Ts (Kawashima et al., 1996; Boriack-Sjodin et al., 1998; Goldberg, 1998). The SopE-induced conformational rearrangements of switch I and switch II shown here disrupt several favorable interactions necessary for tight guanine nucleotide binding and induce nucleotide release. Since the nucleotide-binding pocket is almost freely accessible from the solvent, unrestricted rebinding of nucleotide and formation of a low-affinity trimeric complex would be possible. In the course of the complete GEF reaction, concomitant restructuring of the switch regions would not be compatible with the SopE binding and would thus lead to dissociation of SopE and formation of a high-affinity nucleotide complex. Comparison with the eukaryotic Rac1–Tiam1 complex and evolutionary relationship In order to compare the catalytic mechanism of SopE-mediated guanine nucleotide exchange with that of eukaryotic RhoGEFs, we have superimposed the structure of the Cdc42–SopE complex with that of the Rac1–Tiam1 complex (Worthylake et al., 2000). Alignment of both complexes on the G-domain cores revealed that the switch regions of the Rho proteins have a remarkably similar local arrangement (Figure 3) such as the flip of Phe28cdc42/rac1 and Ala59cdc42/rac1 as well as displacement of Phe37cdc42/rac1. Only the local arrangement around Tyr40cdc42/rac1 differs significantly between the two structures (Figure 3). In the Rac1–Tiam1 complex, the side chain of Tyr40rac1 is rotated by 180° relative to its orientation in the Cdc42–SopE complex. Nevertheless, SopE and the eukaryotic GEF Tiam1 induce very similar overall rearrangements in the switch I and switch II regions of the Rho component to accelerate nucleotide release. Figure 3.Similar GEF mechanisms by SopE and Tiam1 using distinct structural elements. The Cdc42–SopE and Rac1–Tiam1 complexes have been aligned on the G-domain shown in gray. The P loop and switch I/II regions are shown for the individual molecules. Helices α2 and α4 from SopE are in blue, and helix α8 from Tiam1 in red. The view shows the similar location of the catalytic loop from SopE and the important Lys1195 from Tiam1. Download figure Download PowerPoint In sharp contrast, however, the GEF components of both protein complexes are completely different (Figure 1B and D). While both GEFs have a high α-helical content, they have different tertiary folds and both use different amino acids to interact with their Rho targe" @default.
• W2044208802 created "2016-06-24" @default.
• W2044208802 creator A5028091742 @default.
• W2044208802 date "2002-07-01" @default.
• W2044208802 modified "2023-10-10" @default.
• W2044208802 title "Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE" @default.
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