Crystal Structure of Human RhoA in a Dominantly Active Form Complexed with a GTP Analogue*

Kentaro IharaDagger , Sachiko Muraguchi, Masato KatoDagger , Toshiyuki Shimizu, Masahiro Shirakawa, Shinya Kuroda§, Kozo Kaibuchi§, and Toshio Hakoshimapar

From the Divisions of Structural Biology and § Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-01, Japan, and the  Inheritance and Variation Group, PRESTO, Japan Science and Technology, Kyoto 619-02, Japan

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The 2.4-Å resolution crystal structure of a dominantly active form of the small guanosine triphosphatase (GTPase) RhoA, RhoAV14, complexed with the nonhydrolyzable GTP analogue, guanosine 5'-3-O-(thio)triphosphate (GTPgamma S), reveals a fold similar to RhoA-GDP, which has been recently reported (Wei, Y., Zhang, Y., Derewenda, U., Liu, X., Minor, W., Nakamoto, R. K., Somlyo, A. V., Somlyo, A. P., and Derewenda, Z. S. (1997) Nat. Struct. Biol. 4, 699-703), but shows large conformational differences localized in switch I and switch II. These changes produce hydrophobic patches on the molecular surface of switch I, which has been suggested to be involved in its effector binding. Compared with H-Ras and other GTPases bound to GTP or GTP analogues, the significant conformational differences are located in regions involving switches I and II and part of the antiparallel beta -sheet between switches I and II. Key residues that produce these conformational differences were identified. In addition to these differences, RhoA contains four insertion or deletion sites with an extra helical subdomain that seems to be characteristic of members of the Rho family, including Rac1, but with several variations in details. These sites also display large displacements from those of H-Ras. The ADP-ribosylation residue, Asn41, by C3-like exoenzymes stacks on the indole ring of Trp58 with a hydrogen bond to the main chain of Glu40. The recognition of the guanosine moiety of GTPgamma S by the GTPase contains water-mediated hydrogen bonds, which seem to be common in the Rho family. These structural differences provide an insight into specific interaction sites with the effectors, as well as with modulators such as guanine nucleotide exchange factor (GEF) and guanine nucleotide dissociation inhibitor (GDI).

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Rho is a small GTPase1 that was first purified from mammalian tissue membrane (1) and cytosol (2) fractions and was identified as the gene product of the ras homologue gene, rho (3). Rho has three mammalian isoforms, RhoA, RhoB, and RhoC, that exhibit high sequence homology with 83% identities (4). Rho cycles between GTP-bound and GDP-bound forms in a similar manner as Ras and other small GTPases. The level of the active GTP-bound form is regulated by its own GDI, GEF, and GAP. The interconversion between the GTP-bound and GDP-bound forms allows Rho to act as a molecular switch that regulates intercellular signaling pathways. Rho is implicated in the cytoskeletal responses to extracellular signals including lysophosphatidic acid and certain growth factors, which result in the formation of stress fibers and focal adhesion (5-7). Recent isolation and characterization of putative target proteins for Rho from the bovine brain (8, 9) have led to a possible mechanism by which Rho regulates cytokinesis, cell motility, or smooth muscle contraction (10, 11). These proteins contain the MBS made up of myosin phosphatase and a novel serine/threonine kinase, Rho-kinase, that has been shown to phosphorylate MBS to inactivate myosin phosphatase and also to phosphorylate the MLC. Accumulation of phosphorylated MLC induces a conformational change in myosin II that increases its interaction with actin and enables the formation of myosin filaments (12). Rho-kinase is identical to ROK from the rat brain (13) and p160ROCK from human megakaryocytic leukemia cells (14), which are members of a growing family of serine/threonine protein kinases that include myotonic dystrophy kinase. Other target proteins for Rho contain PKN (15, 16), Rhophilin (16), Rhotekin (17), and Citron (18). Further signaling pathways for actin polymerization have appeared to involve PtdIns 4-phosphate 5-kinase (19) and p140mDia (20), as downstream effectors.

Rho has two related small GTPases, Rac and Cdc42, that are also involved in regulating the organization of the actin cytoskeleton, whereas the cell morphological effects induced by these GTPases are clearly different in appearance. Rac regulates lamellipodium formation and membrane ruffling, and Cdc42 regulates filopodium formation. Rac is also known to be involved in the activation of NADPH oxidase in phagocytes. Rac has two mammalian isoforms, Rac1 and Rac2, that exhibit a high sequence homology with 90% identities. Rac and Cdc42 also share a significant homology with 68% identities and, actually, bind to some common target proteins for activation. Rho, however, exhibits a relatively low similarity to those GTPases, an approximately 45% identity with both Rac and Cdc42. These differences in similarity are thought to be essential for the activation of several downstream target proteins of each small GTPase, although we do not yet understand the molecular basis of the specificities. Based on these differences, RhoA, RhoB, and RhoC are hereafter referred to as the RhoA subfamily, and Rac1, Rac2, and Cdc42 as the Rac1 subfamily. The Rho-binding domains of the target proteins consist of less than 100 residues and have been classified into at least two motifs (9, 21). The class 1 of the Rho-binding motif is characterized as a polybasic region followed by a leucine-zipper-like motif and is found in PKN, Rhophilin, Rhotekin, and MBS. Rho-kinase and Citron make up another class of the Rho-binding motif, the class 2, that has a putative coiled-coil motif located at the C terminus of the segment that is similar to myosin rod. It is of considerable interest that these sequences of the Rho-binding domains have no similarity to the binding domain of an activated Cdc42Hs-associated kinase (ACK) (22), a p21(Cdc42/Rac1)-activated protein kinase (PAK) (23), or the Ras-binding domain of Raf-1 (24).

Rho and the related small GTPases are the most common targets for bacterial toxins and are of major importance for the entry of bacteria into mammalian host cells. It is well known that various bacterial toxins can modify Rho by ADP-ribosylation, -glucosylation, and -deamidation. These toxins are classified into three families, C3-like exoenzymes such as Clostridium botulinum C3 ADP-ribosyltransferase, large clostridial cytotoxins such as Clostridium difficile toxins A, and Rho-activating toxins such as Escherichia coli CNFs (25). The C3-like exoenzymes act on members of the RhoA subfamily, but most of the large clostridial cytotoxins inactivate all members of the Rho family. The Rho-activating toxins activate members of the RhoA subfamily and Cdc42. No activity for Ras, Rap, and Ran has been reported for the bacterial toxins of these three families, but Clostridium sordelli HT, one of the large clostridial cytotoxins, is known to inactivate Ras and Rap. There is no interpretation for these emerging differences in the specificity of the small GTPases. Hence, it becomes essential to examine the three-dimensional structures of Rho to understand how their interactions with the target proteins control the various signaling processes and how the modifications by bacterial toxins change the activities of their target GTPases for bacterial invasion. We report here the crystal structure of recombinant human RhoA, which is dominantly activated with substitution of Gly14 by valine (RhoAV14), complexed with GTP analogue, GTPgamma S, and we compare it with the structures of H-Ras and other related GTPases.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Preparation and Crystallization of RhoAV14-- The cloning, expression, and purification of the dominantly active form of recombinant human RhoAV14 complexed with GTPgamma S and Mg2+ were carried out according to the methods described previously (8, 9, 11, 15). Detail procedures will be described elsewhere. The resulting active sample, used in this study, is verified with MALDI-TOF MS (JMS-ELITE, PerSeptive Inc.) and N-terminal analysis (M492, Applied Biosystems). The protein is truncated at Ala181 and has one additional serine residue at the N terminus. Crystals were obtained at 4 °C by the hanging-drop vapor diffusion method from solutions containing 10 mg/ml GTPgamma S-RhoAV14, 50 mM Tris-HCl buffer, pH 8.5, 10% PEG8000, 7.5% 1,4-dioxane equilibrated against 100 mM concentration of the same buffer containing 20% PEG8000 and 15% 1,4-dioxane. Plate-like crystals (Form A) grew within a few days and were found to diffract up to 2.4 Å resolution. The crystals belong to space group P21212 (a = 62.02 Å, b = 74.78 Å, c = 50.52 Å), with one molecule in the asymmetric unit. Hexagonal crystals (Form B) also were obtained from solutions containing 10 mg/ml GTPgamma S-RhoAV14, 50 mM sodium acetate buffer, pH 4.6, 10% 2-propanol equilibrated against 100 mM of the same buffer containing 20% 2-propanol. Crystals had hexagonal or trigonal lattice parameters with a rather long c axis (a = b = 60.80 Å, c = 214.56 Å) and diffracted at 3.0 Å.

Data Collection and Structure Determination-- The structural analysis was performed using Form A. Intensity data were collected at 10 °C using an R-AXIS IIc imaging plate detector with CuKalpha x-rays generated by a rotating anode RU-300H (RIGAKU, Japan). The diffraction data were processed with PROCESS (RIGAKU). A summary of the data processing statistics are given in Table I. The initial phases were calculated by molecular replacement with the program AMoRe (26) using a search model based on the structure of human H-Ras (Protein Data Bank code 5P21, Brookhaven National Laboratory), with which RhoA shares a 27.5% identity. Several searches with a polyalanine model using different ranges of intensity data and integration radii resulted in a unique solution. Rigid body refinements of the searched model were performed with X-PLOR (27). The model obtained was divided into the secondary structure elements, and again, rigid body refinements were performed, followed by solvent flattening/histogram matching with the program DM (28). Four regions of insertions and deletions were inspected on the resulting 2Fo-Fc map that was generated with the program O (29). The structure was built and refined through alternating cycles using the programs O and X-PLOR, respectively.

Structure Refinement-- Three regions were poorly defined in the resulting map. The first is at the loop and beta -strand residues, Asp28-Gly50, which contain the switch I region connected to strand B2, and the second is at the residues of the switch II region. All residues in these regions were rebuilt on their omit maps. Structures of these parts were found to have large displacements from those of H-Ras (see text). The last is at the inserted residues, Glu125-Glu137, which is specific for members of the Rho-family. After several cycles of refinements incorporating solvent water molecules located at regions other than the inserted residues, we defined the residues forming a short 310-helix connected to an alpha -helix. The GTPgamma S molecule and the Mg2+ ion were identified unequivocally by their appearance in 2Fo-Fc maps. The gamma -sulfur atom of GTPgamma S was also identified by its appearance in Fo-Fc maps and its standard sulfur-phosphorus bond distance (1.9 Å), which is longer than the nonbridging oxygen-phosphorus bond (1.5 Å). Three N-terminal residues have uninterpretable densities implying complex disorder. The structure consists of one RhoAV14 molecule of 178 residues, one GTPgamma S, one Mg2+ ion, and 38 water molecules. The side chains of Arg5, Glu54, Arg68, and His126 are poorly defined in the current structure. There are no residues in disallowed regions as defined in PROCHECK (30). A summary of the refinement statistics is given in Table I. The structure was inspected using the program QUANTA (Molecular Simulators Inc).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Data collection and refinement statistics

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Overall Structure-- The major features of the fold, consisting of a six-stranded beta -sheet surrounded by helices connected with loops, are basically conserved as found in H-Ras (31, 32) and other related small GTPases (33-36) (Fig. 1). The beta -sheet is formed by the anti-parallel association of two extended beta -strands (B2 and B3) and the parallel association of five extended beta -strands (B3, B1, B4-B6). RhoAV14 contains five alpha -helices (A1, A3, A3', A4, and A5) and three 310-helices (H1-H3). There are three insertion and one deletion sites, which are common in the members of the Rho family, as can be seen from the sequence and secondary structure element alignment of RhoA and H-Ras (Fig. 2). The 13-residue insertion (Asp124-Gln136) is located at the loop between strand B5 and helix A4. Excluding the deletion and insertion residues, the Calpha -carbon atoms of RhoAV14 and the corresponding dominantly activated H-RasV12, which is complexed with GTP (37), superimpose with a root mean square (r.m.s.) deviation of 1.68 Å for 163 common Calpha -carbon atoms. This superposition yields an r.m.s. deviation of 0.87 Å for the atoms of the guanine nucleotide. Segments that involve major differences are located at the switch I and II regions and part of the antiparallel beta -sheet, consisting of the C-terminal half of strand B2 and the N-terminal half of strand B3, in addition to the insertion and deletion sites described above (Fig. 3A). Recently, Wei et al. have reported the crystal structure of RhoA bound to GDP (38). Compared with this RhoA-GDP structure, the significant conformational changes were found to be localized in the switch I and II regions (Fig. 3B), as described for H-Ras (31, 39). Excluding these regions, the Calpha -carbon atoms of RhoAV14-GTPgamma S and RhoA-GDP superimpose with a r.m.s. deviation of 0.48 Å. The nonhydrolyzable nucleotide GTPgamma S binds to the protein with a Mg2+ ion that has a typical octahedral coordination sphere (Fig. 4).


View larger version (64K):
[in this window]
[in a new window]
 
Fig. 1.   Structure of RhoAV14-GTPgamma S. Shown is a ribbon representation of RhoAV14 complexed with GTPgamma S (yellow) and Mg2+ (a gray ball) with beta -strands (red), alpha -helices (green), and 310-helices (blue). Three water molecules (pink balls) are also illustrated. One water molecule (Wat1) participates in the guanine-base recognition of GTPgamma S, the second (Wat2) participates in the binding of the ribose, and the last (Wat3) is a putative nucleolytic water molecule. The secondary structure elements, Mg2+ ion, water, and GTP molecules are labeled as well as the N and C termini.


View larger version (97K):
[in this window]
[in a new window]
 
Fig. 2.   Sequence alignment of human RhoA with the related human GTPases. Conserved residues are highlighted in yellow for the RhoA subfamily (RhoA, RhoB, and RhoC), in red for the Rac1 subfamily (Rac1, Rac2, and Cdc42), in blue for the Rho family, in light green for the Ras family (K-Ras, N-Ras, and H-Ras) and in gray for all members. The secondary structure elements of RhoAV14-GTPgamma S and H-RasV12-GTP complexes are indicated at the top and below the aligned sequences, respectively. The alpha -helices (A1-A5) are in green, the extended beta  (B1-B6) are in green, and the 310-helices (H1-H3) are in blue. Three functional regions of RhoA are also indicated with marks for the residues that participate in interactions with GTPgamma S (circles) and the Mg2+ ion (rectangles). The dominantly active mutation of glycine to valine is indicated by a star. The sequences are taken from SwissProt. The accession numbers are (RhoA), (RhoB), (RhoC), (Rac1), (Rac2), (Cdc42), (K-Ras), (N-Ras), and (H-Ras).


View larger version (94K):
[in this window]
[in a new window]
 
Fig. 3.   Structural comparison of RhoAV14-GTPgamma S with H-RasV12-GTP and RhoA-GDP. A, superposition of Calpha -carbon atom tracings of RhoAV14 bound to GTPgamma S (magenta) and H-RasV12 bound to GTP (green) (Protein Data Bank code 521P). Segments displaying large displacements are highlighted in red for RhoAV14 and yellow for H-RasV12. These include switch I (residues 27-36 for RhoA), the anti-parallel beta -sheet formed by strands B2 and B3 (residues 43-53), and switch II (residues 59-78). The 13-residue insertion (residues 122-137), forming helix A3', and the segments helix A3 (residues 90-106), L7 (residues 107-110), and L9 (residues 151-154), which have insertion or deletion to produce large displacements, are in blue for RhoAV14. B, superposition of Calpha -carbon atom tracings of RhoAV14-GTPgamma S (magenta) and RhoAN25 bound to GDP (green) (38) with segments displaying large displacements in red and yellow, respectively. These include switch I and the C-terminal flanking region (residues 28-44) and the N-terminal region of switch II (residues 62-69). Val14 of RhoAV14 is highlighted in blue.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4.   GTPgamma S bound to RhoAV14. A cartoon of GTPgamma S binding to RhoAV14 with Mg2+ and water molecules. All dashed lines correspond to hydrogen bonding interactions (distance less than 3.5 Å), and the corresponding distances (Å) are indicated. The residues whose main chains participate in the hydrogen bonding are represented by rectangles, and the residues whose side chains participate in the hydrogen bonding are represented by ovals. The coordination bonds to the Mg2+ ion are indicated by arrows. The possible hydrogen bond between Gln63 and Wat-3 has a longer distance (3.8 Å). The hydrogen bonds observed in the current structure but not in H-Ras are highlighted in red.

Insertion Regions-- The N-terminal segment (Glu125-Lys133) of the 13-residue insertion forms an alpha -helix designated as A3', which is followed by an extended loop. A short 310-helix, designated as H3 (Fig. 1), is induced at the segment flanking the N terminus of this insertion, with large displacements of Arg122 and Asn123 from those of H-RasV12 (3.0 Å and 6.1 Å, respectively). Compared with RhoA-GDP, however, no significant conformational change exists in the 13-residue insertion and its N-terminal flanking regions. This folding seems to be basically similar to that of Rac1 complexed with GMP-PNP (36) but shows many differences in details. It is notable that the sequences of this region of members in the RhoA subfamily is rather different from those of the Rac1 subfamily. Among the key residues in stabilization of helices H3 and A3' (Fig. 5A), Arg122 and Asp124 are conserved in the Rho family but Arg128, Glu137, and Lys140 are variant in the Rac1 subfamily. No water molecule is found to be involved in the structural stabilization of the RhoA insertion region, though Rac1 forms a water-mediated hydrogen bond between the main chains. The conserved residues Leu131 and Pro138 of helix A3' form a hydrophobic patch with Thr127 and Pro89, which are also conserved or conservatively replaced in the Rho family. Rac1 adds Ile126 (Arg128 of RhoA) to the hydrophobic patch.


View larger version (85K):
[in this window]
[in a new window]
 


View larger version (64K):
[in this window]
[in a new window]
 
Fig. 5.   Various parts of RhoAV14-GTPgamma S. A, the 13-residue insertion subdomain of RhoAV14. The carbon, nitrogen, oxygen, and sulfur atoms are in white, blue, red, and yellow, respectively. The Calpha -carbon atom tracing is in brown. The hydrogen bonds involving the side chains are indicated by thin white lines. B, interactions in switches I and II and P-loop regions of RhoAV14 (white) bound to GTPgamma S (magenta). The Calpha -carbon atom tracings of the corresponding regions of RhoA-GDP (green), together with the side chains of switch I, are superimposed. C, part of RhoAV14 switch I that displays large displacements from the corresponding part of H-RasV12. The corresponding part of switch I and the guanosine moiety of H-RasV12-GTP complex are shown with green lines. The guanosine (the carbons in brown) of GTPgamma S bound to RhoAV14 is also shown with residues in the guanine-binding site. The two water molecules are indicated by red balls. D, superposition of Calpha -carbon atom tracings of switch II of RhoAV14 (white) and H-RasV12 (green). The side chains that stabilize the unique conformation of the RhoAV14 switch II are added as well as the functionally important residues. For clarity, the hydrogen bond between the side chain of Arg70 and the main chain of Ala61 (see text) is not shown in this figure. E, rearrangement of the side-chain packing of the guanine recognition site for accommodation of a water molecule (Wat-1) of the RhoAV14-GTPgamma S complex. The corresponding part of H-RasV12-GTP is superimposed with the carbon in green. F, modification sites by bacterial toxins located at the C terminus of switch I and part of beta -sheet B2 (white) -B3 (brown). The stacking interaction between the side-chain carbonyl group of Asn41 and the indole ring of Trp58 is indicated by a broken line. The target residue, Thr37, for glucosylation by large closteidal cytotoxins is located at the N terminus of switch I. The Mg2+ ion and the triphosphate group of GTPgamma S is shown with a ball-and-stick model.

The outer surface of helix A3' is covered with charged residues whose side chains form hydrogen bonds and/or ion pairs, Glu125-Arg129 and Glu130-Lys133 pairs. These residues are conserved or conservatively substituted in the RhoA subfamily, but are replaced by other amino acid residues in the Rac1 subfamily. It is interesting that, in the Rac1 subfamily, Glu125 and Arg129 are substituted by lysine and glutamic acid, respectively, and Glu130 and Lys133 are substituted by lysine/arginine and glutamic acid, respectively. Therefore, these pairs of acidic and basic residues of Rac1 could form hydrogen bonds or ion pairs as observed in the current structure, though most of the exposed side chains of the residues of Rac1 corresponding to the residues 125-135 of RhoA are highly mobile.

Compared with H-Ras, helix A3 has a one-residue insertion at the center and two Pro residues (Pro96 and Pro101), which cause a disruption of the normal hydrogen-bonding pattern of an alpha -helix, whereas H-Ras has no Pro residue on this helix. These differences induce a relatively large discrepancy (2.05 Å at Pro96) of helix A3 from that of H-RasV12 (Fig. 3A). Both Pro96 and Pro101 face the solvent region so as to induce pronounced kinks that serve to maximize the contacts with strands B1 and B4.

Phosphate-binding Loop-- The G14V mutation of RhoA and the G12V mutation of H-Ras exhibit less than one-tenth the GTPase activity of the wild-type GTPases. Crystal structures of H-RasV12 complexed with GDP (31, 39) and GTP (37) show that the mutation causes no significant conformational change at the phosphate-binding region, though there are large differences in mobility and conformation predominantly localized in the switch II region (see below). Similar results were obtained in RhoA. The r.m.s. deviation of the 12GXG(V)XXGKT/S19 motifs between RhoAV14-GTPgamma S and RhoA-GDP is small (0.31 Å) and that between RhoAV14-GTPgamma S and H-RasV12-GTP is relatively small (0.84 Å). However, the bulky side chain of Val14 contacts with Gly62 and Gln63, and these contacts cause a displacement (0.71 Å) of the Calpha -carbon atom of Val14 from the corresponding atom of the wild-type RhoA (Fig. 5B).

Switch I-- The switch I region in RhoAV14-GTPgamma S is well ordered in the crystal structure as well as that of RhoA-GDP. In contrast, in Rac1-GMP-PNP, residues 32-36 of switch I (34-38 in RhoA) are disordered. Dramatic changes in the conformations of switch I and its C-terminal flanking regions, as compared with RhoA-GDP, occurs with the largest displacements (5.4 Å and 6.4 Å) at Pro36 and Phe39, respectively (Fig. 5B). Similar conformational changes whether bound to GTP or GDP were reported for H-Ras (31, 40), the Galpha subunits of the trimeric GTPases such as transducin-alpha (41), thereby playing a key role as a molecular switch in signal transduction. Tyr34 and Pro36 of RhoAV14 flip their side chains toward the nucleotide so as to shield the triphosphate group from the solvent region. The phenolic ring of Tyr34 stacks on the Pro36 and also contacts with Ala15 and Val14 to close the entrance of the phosphate-binding pocket. These conformational changes are accompanied by the flipping out of hydrophobic residues, Val35, Val38, and Phe39, toward the solvent region. It should be noted that Val38 and Phe39 form a hydrophobic patch on the molecular surface together with Tyr66 and Leu69 of switch II. These contacts play a pivotal role to induce the stable conformation of switch II (see below). Switch I of RhoAV14 displays a significantly different conformation from that of H-RasV12-GTP. Large displacements of the residues of switch I begin from Asp28 and end at Pro36 with the largest displacement (4.1 Å) at Glu32 (Figs. 5C). These displacements, which result in differences in recognition of the ribose of the guanine nucleotide, seem to be caused by Pro31, which restricts the main chain torsion angles. Since Pro31 is well conserved in the Rho family but is replaced by other residues in Ras (Val), Rab (Val), and Ran (Asp), the displacements could be a common structural feature of members of the Rho family. While large displacements were observed in switch I as described, no significant difference in the position and orientation of Thr37, which coordinates to the Mg2+ ion, is seen between RhoAV14 and H-RasV12.

Strands B2 and B3-- The switch I loop is connected to the anti-parallel beta -sheet of strands B2 and B3, which is followed by switch II. This two-stranded sheet is located at the edge of the six-stranded beta -sheet and is sitting on helices A1 and A5 to form a hydrophobic core. Compared with H-RasV12-GTP, a large displacement of these strands expands between a stretch from Ala44 to Val53, which moves toward helices A1 and A5, with the largest shift being 2.9 Å at Asp45. It is notable that the sequence of this region is highly conserved in the RhoA subfamily. This displacement seems to be caused mainly by differences in hydrophobic interactions between these strands and helices A1 and A5. Strands B2 and B3 are connected by a reverse turn of type II formed by 48VDGK51. H-RasV12 and H-Ras also have a type II reverse turn at this position with the corresponding segment, 46IDGE49. Since the third residue of this type of reverse turn should have a Gly residue to avoid the steric clash with the main-chain carbonyl group of the second residue, mutations of this residue to any other residue destroy the reverse turn, which results in large conformational changes of the strands or displacement of the beta -sheet. This may possibly explain why mutations of Gly48 in H-Ras inhibits effector function (42) even though this residue is distal from the effector-binding site encompassing switch I and the N-terminal half of strand B2, as observed in the crystal of Rap1A-Raf1 complexes (35).

Switch II-- The segment between strands B3 and B4 contains the switch II region that has a key residue Gln63 (Gln61 of H-Ras) of GTPase activities. Mutation of Gln61 of H-Ras to almost any other amino acid blocks intrinsic and GAP-stimulated GTPase activity (43). In the crystal structures of cellular H-Ras complexed with either GDP (31), GMP-PCP (39) or GMP-PNP (32), the highly conserved 57DTAGQE62 motif of switch II exhibits flexibility to adapt to alternative conformations although this motif of oncogenic H-RasV12 complexed with GTP has only one major conformation (37). In RhoA-GDP, residues 63-65 are also disordered. The electron density of this region of RhoAV14 is well defined and has a single conformation at the present resolution. RhoAV14 has two 310-helices, H1 (64EDY66) and H2 (70RPL72), which are separated by a short loop of three residues (67DRL69). The sequence of this region is well conserved in the Rho family but is different from those regions in the Ras family (Fig. 2). A similar conformation is also seen in the crystal structure of Rac1-GMP-PNP. In contrast, H-RasV12 has a 310-helix at the position corresponding to the short loop of RhoAV14 with an alpha -helix of five residues corresponding to residues 71-75 of RhoAV14. These differences induce a large displacement of Glu64 (3.8 Å), together with re-orientations of the side chains of Gln63, Glu64 and Asp65 from the corresponding residues of H-RasV12 (Fig. 3A). Lys98 and Glu102, both of which are located at helix A3, play crucial roles in the conformation of the segment by forming multiple hydrogen bonds to switch II (Fig. 5D). These two residues are conserved in members of the Rho family but are replaced in H-Ras. Moreover, the segment from helices H1 to H2 makes hydrophobic contacts strands B2 and B3. In this hydrophobic core, H-RasV12 has an additional residue Tyr71, which is replaced by a small residue (Ser73) in RhoA. This difference causes a movement of the helix H2 toward strand B2. These differences seem to be one of the main reasons why the conformations of the segment are so different between RhoAV14 and H-RasV12.

Magnesium Ion Binding-- The strong GTP/GDP-binding and the GTPase activity of small GTPases have been shown to be absolutely dependent on the presence of divalent ions. The Mg2+ ion of the present structure is located at a position similar to those in H-RasV12-GTP and in H-Ras-GMP-PNP, as well as that in RhoA-GDP. The displacement of the ion from the corresponding position in the GDP-bound form is 1.04 Å. The Mg2+ ion plays a key role in bringing together the functional regions of the phosphate-binding, switches I and II, as observed in H-Ras. Actually, the stereochemistry of Mg2+ coordination is identical to that in H-Ras-GMP-PNP. In contrast, the stereochemistry of Mg2+ coordination in RhoA-GDP is different from the current form but also is different from that in H-Ras-GDP.

Guanosine Nucleotide Binding-- The glycosyl conformation of GTPgamma S is anti with the C2'-endo sugar pucker. The guanine base is trapped in a hydrophobic pocket, in a manner similar to H-RasV12-GTP, to be recognized by several interactions with the conserved residues of the 116GXKXDL121 and 160SAK162 motifs (Figs. 5C). A major difference in base recognition is the water-mediated hydrogen bonds to the N7 and O6 atoms of the guanine base. The water molecule (Wat-1) is completely buried inside the hydrophobic binding pocket with a hydrogen bond to Gly17. The space for the accommodation of this water molecule is mainly produced by a rearrangement of the side-chain packing of the pocket, involving Leu21, Asn117, and Cys159 (Fig. 5E). In H-Ras, Cys159 of RhoA is replaced by a Thr residue. In addition to the base recognition, the 2'-hydroxyl group of the ribose also has a water-mediated hydrogen bond to switch I, although in H-RasV12-GTP, the hydroxyl group of the ribose forms direct hydrogen bonds with the main chains corresponding to Pro31 and Glu32 of RhoA. As mentioned above, these differences in the recognition of the ribose are caused by the large displacements of switch I. Similar water-mediated hydrogen bonds in base and sugar recognition have also been found in RhoA-GDP and in Rac1-GMP-PNP.

Triphosphate Binding-- GTP and GDP bind to small GTPases with dissociation constants on the order of nanomolar. This strong binding affinity is well demonstrated in the current structure. The triphosphate moiety of GTPgamma S has 21 direct and 9 water-mediated hydrogen bonds to the protein, together with 2 magnesium coordinations. These involve six residues of the phosphate-binding loop, four residues of switch I, three residues of switch II, and four residues of base-recognition motifs. It is notable that most of these residues of the phosphate-binding loop interact with the triphosphate through their main-chains, especially the amino groups. This is the reason why the amino acid sequence of the 12GXGXXGKT/S19 motif contains many variant residues. The residues whose side chains participate in the interactions with the triphosphate are invariant Lys18, Tyr34, and Asp59. The conformation of the triphosphate exhibits similarity to that of GDP bound to RhoA, with relatively small displacements of the alpha - and beta -phosphates from those of GDP, 0.75 Å and 0.67 Å, respectively. Two oxygen atoms of the gamma -phosphate make contact with the protein by several hydrogen bonds, together with the coordination to the Mg2+ ion buried inside the pocket formed by switches I and II and the phosphate-bonding loops. These heavy interactions allow the gamma -phosphate to orient the gamma -sulfur atom toward Val14, Tyr34, and Pro36. Similar configurations of the gamma -thiophosphate were also observed in the crystal structures of transducin-alpha (44) and Gialpha 1 (45) complexed with GTPgamma S. In all these crystals complexed with GTPgamma S, the gamma -sulfur atom is the closest atom to the side-chain amide group of Gln63 (Gln200 of transducin-alpha and Gln204 of Gialpha 1) among the gamma -thiophosphate atoms.

Putative Nucleophilic Water Molecule-- We identified one water molecule (Wat-3) that is close enough to the gamma -phosphate to perform an in-line nucleophilic attack. The water molecule is located at a position 10° off from this line at a distance of 3.6 Å from the phosphorus atom and forms a hydrogen bond (3.3 Å) to the gamma -sulfur atom, although the distance to the gamma -oxygen atoms of the phosphate group is too long to form a hydrogen bond (3.6 Å for both). Similar water molecules have been located in analogous positions close to the gamma -phosphate in the crystal structures of transducin-alpha and Gialpha 1 complexed with GTPgamma S, as well as of H-Ras, Rac1, and EF-Tu (46) complexed with GMP-PNP, although no water molecule corresponding to Wat-3 has been found in RhoA-GDP. Gln63 positions the side-chain oxygen atom at a distance of 3.8 Å from the hydrolytic water and the side-chain nitrogen atom at a distance of 3.8 Å from the side-chain carboxyl group of Asp65.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Modification Sites by Bacterial Toxins-- C. botulinum C3 ADP-ribosyltransferase transfers an ADP-ribose moiety of NAD to Asn41 of Rho (47). The side-chain of Asn41, which is located at strand B2, forms a hydrogen bond (3.1 Å) to the main-chain carbonyl group of Glu40. This hydrogen bond allows Asn41 to interact with the indole ring of Trp58 of strand B3 (Fig. 5F). The distances between the nearest atoms of the indole ring and the carbonyl oxygen atom of the side chain of Asn41 range from 3.3 to 3.5 Å, which indicates the existence of a stacking interaction between them. Because the indole ring is a strong electron-donor, this interaction may help to enhance the nucleophilic properties of the side-chain nitrogen atom of Asn41. It should be noted that the hydrophobic side chains of Val38, Phe39, and Val43 are exposed to the solvent region around Asn41, together with Trp58. This unusual feature of the molecular surface may be related to the interaction with C3-like exoenzymes. Asn41 orients the side chain away from the switch I loop. This is consistent with the fact that the ADP-ribosylation on Rho affected neither the GTPgamma S binding nor its intrinsic GTPase activity. Furthermore, the ADP-ribosylation on Rho did not affect its interaction with rhoGAP (48). Recent data using Swiss 3T3 cells indicates that the ADP-ribosylation of Rho enhances its binding to PtdIns 4-phosphate 5-kinase and acts as a dominantly negative inhibitor (19, 49). This also suggests that the ADP-ribosylation does not impair the intrinsic properties of the switch I conformation though PtdIns 4-phosphate 5-kinase could bind to the GDP-bound form, and therefore, the binding may be different from those of other effectors that do not bind to the GDP-bound form. Rac and Cdc42 are not subjected to ADP-ribosylation (50). This may be related to the global conformation of the anti-parallel beta -sheet formed by strands B2 and B3 since the sequence of this region of RhoA subfamily is conserved but is different from that in the Rac1 subfamily. On the molecular surface around Asn41, Val43 is replaced by Ser/Ala and Glu40 is replaced by Asp in Rac and Cdc42.

Recent biochemical data have shown that Thr37 is glucosylated by the major virulence factors of C. difficile, toxin A and B (51). The glucosylated RhoA induces the disaggregation of actin filaments. It also appears that GDP-bound RhoA is a superior substrate for Toxin B to GTP-bound RhoA. This is consistent with the crystal structures: in RhoA-GDP, the side chain of Thr37 does not participate in either Mg2+ ion or phosphate binding, whereas it participates in both in the current structure. Since Thr37 orients the side chain inside the loop, its glucosylation must accompany a structural deformation of the loop. This structural change could extend to strand B2. Actually, it has been shown that the glucosylation of Thr37 inhibits ADP-ribosylation by C3-like exoenzymes (52).

CNFs from E. coli and dermonecrotic toxins (DNTs) from Bordetella species induce the massive reorganization of the actin cytoskeleton and inhibit cell division, leading multinucleated cells. Recently, CNF1 has been shown to cause the deamidation of Gln63 of RhoA, resulting in a dominantly active form, RhoAE63 (53-54). CNF1 acts preferentially with RhoA but also inhibits the GAP-stimulated GTPase activity of Cdc42 and of Rac at high concentrations. These actions of CNF1 may be related with the unique conformation and/or conformational properties of switch II. The differences in the CNF1 activity on RhoA, Cdc42 and Rac probably indicate that this toxin may interact with these small GTPases through segments other than switch I, though it remains unclear.

GTP/GDP Switching and Effector Binding-- The fundamental mechanism of the molecular switch, which involves the significant conformational changes in switch I and II regions, in signal transduction seems to be common in small GTPases and Galpha subunits of trimeric GTPases, as described for H-Ras (31), transducin-alpha (41), and Gialpha 1 (55) and Rap2A (56). However, the present RhoAV14 structure reveals large conformational deviations from H-RasV12 in the regions containing switches I and II. The structures of two other small GTPases, ADP-ribosylation factor (Arf) (33) and Ran (34), also showed significant conformational variations in the switch regions, whose structures are also different from those of the present RhoAV14: switch II of Arf forms a long beta -strand, and Ran has a completely different orientation of switch II that contains a short beta -strand. All these results indicate that small GTPases from different families may have a similar fold but with significant variations in the switch regions.

There are several biochemical data indicating that the switch I region of RhoA is involved in its effector binding. It has been suggested from analyses of the chimeric proteins of Rho and Ras that the switch I region (residues 32-42) is essential for the induction of actin stress fiber formation (57). Either Cdc42 or Rac shows no significant binding to the target proteins of RhoA, such as Rho-kinase and the others described above. At switch I and its franking regions of RhoAV14, residues that are exposed to the solvent region are well conserved in the RhoA subfamily but are replaced in the Rac1 subfamily. It is of interest that most of the side chains of these residues protrude onto the same molecular surface (Fig. 6). The double mutant Rap1A (58), which mimics Ras, binds to the Ras-binding domain of c-Raf1 through several residues that are located at the same side of the corresponding molecular surface of RhoAV14. Among them, residues whose side chains form the specific hydrogen bonds to the Ras-binding domain are located at the N-terminal half of strand B2. It is notable that most of these residues are replaced by non-conservative, mainly hydrophobic, residues (Val33, Val35, Phe39-Asn41, and Val43) in RhoAV14-GTPgamma S to form hydrophobic patches on the molecular surface, as described.


View larger version (76K):
[in this window]
[in a new window]
 
Fig. 6.   Molecular surface of RhoAV14. Residues whose mutations abolish the interaction with GEF are in yellow. Asn41 is also highlighted in green. Switches I and II are shown in red and blue, respectively. This surface also contains most of the residues corresponding to the effector-binding residues as seen in the complex between the Ras-binding domain of Raf1 and a double mutant Rap1A (E30D/K31E), which mimics Ras.

In addition to switch I, the second effector site is suggested in the C-terminal two-thirds of the molecule (57). However, little is currently known about the possible second effector site of RhoA. Recent mutagenesis experiments have indicated that the 13-residue insertion region of Rac1 participates in the interaction with p67phox but not in the interaction with PAK, and a combinational use of the multiple effector-binding sites has therefore been proposed (59). Since there are several structural differences in the 13-residue insertion regions between RhoAV14-GTPgamma S and Rac1-GMP-PNP, it may be possible that RhoA also utilizes the insertion region in the specific binding with its own effector proteins, but this remains to be seen in future experiments. It should be noted that the 13-residue insertion region has no significant displacement from that in RhoA-GDP and, therefore, has no switching function between GTP-bound and GDP-bound forms. It is well known that the Galpha subunits of trimeric GTPases contain four insertion regions if compared with small GTPases. The 13-residue insertion region of the members of the Rho family corresponds to the third insertion region that forms an additional helix at the N-terminal portion of the segment (44), although no homology has been detected between RhoA and each of Galpha subunits and no possible function has been assigned to this insertion region.

GEF and GDS Binding-- GEFs for small GTPases of the Rho family have been identified as Dbl-containing proteins that contain a region with a sequence homology to the dbl oncogene product (60). While most of these Dbl-containing proteins can act on multiple members of the Rho family in vitro, some have a limited specificity for one type of the GTPases in vivo. Among them, Lbc shows selectivity for Rho (61) but Tiam-1 for Rac (62) and Cdc24 for Cdc42 (63). Analysis of RhoA/Cdc42Hs chimeric proteins has suggested that residues of switch I and switch II are involved in the specific interaction with Lbc (64). Based on mutation analyses, Lys27, Tyr34, Thr37, and Phe39 in switch I and Asp76 in switch II have been identified as Lbc-sensitive residues. These residues are located at nearly the same side of the molecule, which may form a surface of interaction for Lbc. It is of interest that this surface is almost the same as that for a tentative effector binding (Fig. 6). The side chains of all these residues are highly projected toward the solvent region but Thr37 is buried inside the switch I loop. This might be one reason why the mutation of T37A has an affinity for Lbc comparable with the wild-type RhoA although most of the other mutants failed to associate with Lbc. In contrast, extensive mutations in switch II have been reported to have no significant change in their sensitivity to Lbc. Most of these residues are found on the other side of the molecule or inside the protein. It is notable that the ADP-ribosylated residue by C3-like exoenzymes, Asn41, is also located at this surface and may inhibit the binding to Lbc. There is another protein having guanine nucleotide exchange activity, smgGDS, that has no homology to the Dbl-containing proteins but has some homology to Cdc25 of yeast (65, 66). smgGDS shows wider specificity than Dbl homologues and acts on Ki-Ras, Rap in addition to the Rho family members. Rho seems to interact with smgGDS through its molecular surface containing Asn41 since ADP-ribosylation of Rho by C3-like exoenzymes is reduced in the presence of smgGDS (67).

GDI Binding-- In addition to inhibiting nucleotide dissociation, GDIs mediate partitioning their cognate small GTPases between the membrane and the cytosol (68). RhoGDI inhibits the guanine nucleotide exchange of all members of the Rho family. Recent structural studies have suggested that rhoGDI binds to the cognate GTPases via an immunoglobulin-like domain that has a hydrophobic pocket for binding to the C-terminal isoprenyl group (69, 70). Although this immunoglobulin-like domain has little effect on the rate of nucleotide dissociation from the GTPases, it has been suggested that this binding directs the flexible N-terminal arm of rhoGDI to GTPases, resulting in the inhibition of nucleotide exchange. It is of interest to question how the N-terminal arm interacts with GTPases because the C terminus having the isoprenyl group is located on the molecular surface of the GTPases opposite to the nucleotide binding surface. It has been reported that GDI effectively prevents ADP-ribosylation by C3-like exoenzymes and the nucleotide-exchange activity of smgGDS (67). Furthermore, the nucleotide-exchange activity of Dbl also was remarkably reduced (66). Taken together, these results suggest that the N-terminal arm of rhoGDI may interact with GTPases on the molecular surface, which has residues interact with GEF and GDS as well as C3-like exoenzymes (Fig. 6). This hypothesis provides a framework for analyzing the interactions of rhoGDI, GEF, and GDS with RhoA.

Effects of the gamma -Sulfur Atom and the G14V Mutation on the GTPase Activity-- The GTPgamma S molecule exhibits its resistance to hydrolysis, which is conferred by the gamma -thiophosphorothioate. In the present structure, the gamma -thiophosphate turns the sulfur-phosphorus bond toward Gln63 and positions the gamma -sulfur atom to come into contact with the putative nucleophilic water molecule. Therefore, the bulky sulfur atom, which has a van der Waals radius (1.8 Å) much larger than that of the oxygen atom (1.4 Å), sterically shields the phosphorus atom from the close approach of the nucleophilic water molecule and could interfere with the stabilization of the transition state by Gln63. The gamma -sulfur atom could also interfere with the stabilization of the transition state by the Arg residue from GAP (71). Similar mechanisms for the resistance of the GTPgamma S molecule to hydrolysis, which is conferred by the gamma -thiophosphorothioate, are possible for transducin-alpha and Gialpha 1. Based on the crystal structure of transducin-alpha complexed with GTPgamma S, it has also been pointed out that Arg174, which is a key residue stabilizing the transition state, prevents the thiophosphate from reaching the transition state, due to a steric clash between the firmly anchored guanidino group and the sulfur atom (44).

It has been suggested that the role of the key Gln residue (Gln63 of RhoA) is to stabilize the transition state by direct hydrogen bonds doubly bonded to the gamma -phosphate and the putative nucleophilic water molecule. The transition state should induce conformational changes around the active site of the current structure since the present conformation of Gln63 directs the side-chain carbonyl group toward the nucleolytic water molecules (3.8 Å) but also positions the side-chain amide group away from the phosphate. The gamma -sulfur atom of the phosphate is closest to the side-chain amide group of Gln63, as described above, but the distance between them is more than 5 Å. The contacts of the branched side chain of Val14 with the N terminus of switch II seem to push Gln63 away from the gamma -phosphate group and reduce the conformational flexibility of the side chain of Gln63. Actually, the Cgamma carbon atoms of Val14 and Gln63 have a contact of 3.6 Å. Any rotation around the side-chain torsions of Gln63 could not bring the side-chain amide group to a position close enough to interact with the gamma -sulfur atom because of the steric hindrance of the bulky side chain of Val14. We postulate that these steric effects are a possible means of inhibiting GTP hydrolysis by the dominantly active mutation V14 of RhoA. Thus, the mechanism of dominant activation by the G14V mutation of RhoA seems to be similar to that of G12V of H-Ras even though the conformations of switches I and II are quite different.

    ACKNOWLEDGEMENTS

We thank Drs. S. Takayama, F.-S. Che, and T. Katsuragi for technical assistance and helpful discussions. We acknowledge Dr. Z. S. Derewenda for providing the coordinates of RhoA-GDP.

    FOOTNOTES

* This work was supported in part by Grants in Aid for Scientific Research on Priority Areas (06276104) and Biometallics (09235220) (to T. H.) and for Cancer Research (to K. K.) from the Ministry of Education, Science and Culture of Japan.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.

The atomic coordinates and structure factors (code 1A2B) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

Dagger Supported by a research fellowship from the Japan Society for the Promotion of Science.

par To whom correspondence should be addressed. Tel.: 81-0743-72-5570; Fax: 81-0743-72-5579; E-mail: hakosima{at}bs.aist-nara.ac.jp.

1 The abbreviations used are: GTPase, guanosine triphosphatase; GDI, guanine nucleotide dissociation inhibitor; GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; MBS, myosin-binding subunit; MLC, myosin light-chain; PKN, protein kinase N; PtdIns 4-phosphate 5-kinase, phosphatidylinositol 4-phosphate 5-kinase; PAK, p21(Cdc42/Rac1)-activated protein kinase; CNF, cytotoxic necrotizing factor; GTPgamma S, guanosine 5'-3-O-(thio)-triphosphate; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy; PEG8000, polyethylene glycol-8000; r.m.s., root-mean square; GMP-PNP, guanosine-5'-(beta gamma -imino)triphosphate; GMP-PCP, guanosine-5'-(beta gamma -methylene)triphosphate; Arf, ADP-ribosylation factor; smgGDS, small GTP-binding protein guanine nucleotide dissociation stimulator.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Yamamoto, K., Kondo, J., Hishida, T., Teranishi, Y., and Takai, Y. (1988) J. Biol. Chem. 263, 9926-9932[Abstract/Free Full Text]
  2. Morii, N., Sekine, A., Ohashi, Y., Nakao, K., Imura, H., Fujiwara, M., and Narumiya, S. (1988) J. Biol. Chem. 263, 12420-12426[Abstract/Free Full Text]
  3. Madaule, P., and Axel, R. (1985) Cell 41, 31-40[Medline] [Order article via Infotrieve]
  4. Simon, M. (1996) Trends Biochem. Sci. 21, 178-181[CrossRef][Medline] [Order article via Infotrieve]
  5. Machesky, L. M., and Hall, A. (1996) Trends Cell Biol. 6, 304-310[CrossRef]
  6. Narumiya, S. (1996) J. Biochem. 120, 215-228[Abstract]
  7. Takai, Y., Sasaki, T., Tanaka, K., and Nakanishi, H. (1995) Trends. Biochem. Sci. 20, 227-231[CrossRef][Medline] [Order article via Infotrieve]
  8. Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., Yamamori, B., Feng, J., Nakano, T., Okada, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 273, 245-248[Abstract]
  9. Matsui, T., Amano, M., Yamamoto, T., Chihara, K., Fukuta, Y., Nakafuku, M., Ito, M., Nakano, T., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) EMBO J. 15, 2208-2216[Abstract]
  10. Amano, M., Ito, M., Kimura, K., Fukata, Y., Chihara, K., Nakano, T., Matsuura, Y., and Kaibuchi, K. (1996) J. Biol. Chem. 271, 20246-20249[Abstract/Free Full Text]
  11. Amano, M., Chihara, K., Kimura, K., Fukata, Y., Nakamura, N., Matsuura, Y., and Kaibuchi, K. (1997) Science 275, 1308-1311[Abstract/Free Full Text]
  12. Tan, J. L., Ravid, S., and Spudich, J. A. (1992) Annu. Rev. Biochem. 61, 721-759[CrossRef][Medline] [Order article via Infotrieve]
  13. Leung, T., Chen, X. Q., Manser, E., and Lim, L. (1996) Mol. Cell. Biol. 16, 5313-5327[Abstract]
  14. Ishizaki, T., Maekawa, M., Fujisawa, K., Okawa, K., Iwamatsu, A., Fujita, A., Watanabe, N., Saito, Y., Kakizuka, A., Morii, N., and Narumiya, S. (1996) EMBO J. 15, 1885-1893[Abstract]
  15. Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 271, 648-650[Abstract]
  16. Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y., Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648[Abstract]
  17. Reid, T., Furuyashiki, T., Ishizaki, T., Watanabe, G., Watanabe, N., Fujisawa, K., Morii, N., Madaule, P., and Narumiya, S. (1996) J. Biol. Chem. 271, 13556-13560[Abstract/Free Full Text]
  18. Madaule, P., Furuyashiki, T., Reid, T., Ishizaki, T., Watanabe, G., Morii, N., and Narumiya, S. (1995) FEBS Lett. 377, 243-248[CrossRef][Medline] [Order article via Infotrieve]
  19. Ren, X.-D., Bokoch, G. M., Traynor-Kaplan, A., Jenkins, G. H., Anderson, R. A., and Schwartz, M. A. (1996) Mol. Biol. Cell 7, 435-442[Abstract]
  20. Watanabe, N., Madaule, P., Reid, T., Ishizaki, T., Watanabe, G., Kakizuka, A., Saito, Y., Nakao, K., Jockusch, B. M., and Narumiya, S. (1997) EMBO J. 16, 3044-3056[Abstract/Free Full Text]
  21. Fujisawa, K., Fujita, A., Ishizaki, T., Saito, Y., and Narumiya, S. (1996) J. Biol. Chem. 271, 23022-23028[Abstract/Free Full Text]
  22. Manser, E., Leung, T., Salihuddin, H., Tan, L., and Lim, L. (1993) Nature 363, 364-367[CrossRef][Medline] [Order article via Infotrieve]
  23. Manser, E., Leung, T., Salihuddin, H., Zhao, Z. S., and Lim, L. (1994) Nature 367, 40-46[CrossRef][Medline] [Order article via Infotrieve]
  24. Fabian, J. R., Vojtek, A. B., Cooper, J. A., and Morrison, D. K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5982-5986[Abstract]
  25. Aktories, K. (1997) Trends Microbiol. 5, 282-287[CrossRef][Medline] [Order article via Infotrieve]
  26. Navara, J. (1994) Acta Crystallogr. Sec. A 50, 157-163[CrossRef]
  27. Brunger, A. T., Kuriyan, J., and Karplus, M. (1990) Acta Crystallogr. Sec. A 46, 585-593[CrossRef][Medline] [Order article via Infotrieve]
  28. Cowtan, K., and Main, P. (1996) Acta Crystallogr. Sec. D 52, 43-48[CrossRef]
  29. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sec. A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve]
  30. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef]
  31. Milburn, M. V., Tong, L., de Vos, A. M., Brunger, A., Yamaizumi, Z., Nishimura, S., and Kim, S. H. (1990) Science 247, 939-945[Medline] [Order article via Infotrieve]
  32. Pai, E. F., Krengel, U., Petsko, G. A., Goody, R. S., Kabsch, W., and Wittinghofer, A. (1990) EMBO J. 9, 2351-2359[Abstract]
  33. Amor, J. C., Harrison, D. H., Kahn, R. A., and Ringe, D. (1994) Nature 372, 704-708[CrossRef][Medline] [Order article via Infotrieve]
  34. Scheffzek, K., Klebe, C., Fritz-Wolf, K., Kabsch, W., and Wittinghofer, A. (1995) Nature 374, 378-381[CrossRef][Medline] [Order article via Infotrieve]
  35. Nassar, N., Horn, G., Herrmann, C., Scherer, A., McCormick, F., and Wittinghofer, A. (1995) Nature 375, 554-560[CrossRef][Medline] [Order article via Infotrieve]
  36. Hirshberg, M., Stockley, R. W., Dodson, G., and Webb, M. R. (1997) Nat. Struct. Biol. 4, 147-152[Medline] [Order article via Infotrieve]
  37. Krengel, U., Schlichting, L., Scherer, A., Schumann, R., Frech, M., John, J., Kabsch, W., Pai, E. F., and Wittinghofer, A. (1990) Cell 62, 539-548[Medline] [Order article via Infotrieve]
  38. Wei, Y., Zhang, Y., Derewenda, U., Liu, X., Minor, W., Nakamoto, R. K., Somlyo, A. V., Somlyo, A. P., and Derewenda, Z. S. (1997) Nat. Struct. Biol. 4, 699-703[Medline] [Order article via Infotrieve]
  39. Tong, L., de Vos, A. M., Milburn, M. V., and Kim, S. H. (1991) J. Mol. Biol. 217, 503-516[Medline] [Order article via Infotrieve]
  40. Schlichting, I., Almo, S., Rapp, G., Wilson, K., Petratos, K., Lentfer, A., Wittinghofer, A., Kabsch, W., Pai, E. F., Petsko, G. A., and Goody, R. S. (1990) Nature 345, 309-315[CrossRef][Medline] [Order article via Infotrieve]
  41. Lambright, D. G., Noel, J. P., Hamm, H. E., and Sigler, P. B. (1994) Nature 369, 621-628[CrossRef][Medline] [Order article via Infotrieve]
  42. Der, C. J., Finkel, T., and Cooper, G. M. (1986) Cell 44, 167-176[Medline] [Order article via Infotrieve]
  43. Marshall, M. S. (1993) Trends Biochem. Sci. 18, 250-254[CrossRef][Medline] [Order article via Infotrieve]
  44. Noel, J. P., Hamm, H. E., and Sigler, P. B. (1993) Nature 366, 654-663[CrossRef][Medline] [Order article via Infotrieve]
  45. Coleman, D. E., Berghuis, A. M., Lee, E., Linder, M. E., Gilman, A. G., and Sprang, S. R. (1994) Science 265, 1405-1412[Medline] [Order article via Infotrieve]
  46. Berchtold, H., Reshetnikova, L., Reiser, C. O. A., Schirmer, N. K., Sprinzl, M., and Hillgenfeld, R. (1993) Nature 365, 126-132[CrossRef][Medline] [Order article via Infotrieve]
  47. Sekine, A., Fujiwara, M., and Narumiya, S. (1989) J. Biol. Chem. 264, 8602-8605[Abstract/Free Full Text]
  48. Morii, N., Kawano, K., Sekine, A., Yamada, T., and Narumiya, S. (1991) J. Biol. Chem. 266, 7646-7650[Abstract/Free Full Text]
  49. Chong, L. D., Traynor-Kaplan, A., Bokoch, G. M., and Schwartz, M. A. (1994) Cell 79, 507-513[Medline] [Order article via Infotrieve]
  50. Just, I., Mohr, C., Schallehn, G., Menard, L., Didsbury, J. R., Vandekerckhove, J., van Damme, J., and Aktories, K. (1992) J. Biol. Chem. 267, 10274-10280[Abstract/Free Full Text]
  51. Just, I., Richter, H. P., Prepens, U., von Eichel-Streiber, C., and Aktories, K. (1994) J. Cell Sci. 107, 1653-1659[Abstract/Free Full Text]
  52. Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503[CrossRef][Medline] [Order article via Infotrieve]
  53. Schmidt, G., Sehr, P., Wilm, M., Selzer, J., Mann, M., and Aktories, K. (1997) Nature 387, 725-729[CrossRef][Medline] [Order article via Infotrieve]
  54. Flatau, G., Lemichez, E., Gauthier, M., Chardin, P., Paris, S., Florentini, C., and Boquet, P. (1997) Nature 387, 729-733[CrossRef][Medline] [Order article via Infotrieve]
  55. Mixon, M. B., Lee, E., Coleman, D. E., Berghuis, A. M., Gilman, A. G., and Sprang, S. R. (1995) Science 270, 954-960[Abstract]
  56. Cherfils, J., Menetrey, J., Le Bras, G., Le Bras, G., Janoueix-Lerosey, I., de Gunzburg, J., Garel, J.-R., and Auzat, I. (1997) EMBO J. 16, 5582-5591[Abstract/Free Full Text]
  57. Self, A. J., Paterson, H. F., and Hall, A. (1993) Oncogene 8, 655-661[Medline] [Order article via Infotrieve]
  58. Nassar, N., Horn, G., Herrmann, C., Scherer, A., McCormick, F., and Wittinghofer, A. (1996) Nat. Struct. Biol. 3, 723-729[Medline] [Order article via Infotrieve]
  59. Freeman, J. L., Abo, A., and Lambeth, J. D. (1996) J. Biol. Chem. 271, 19794-19801[Abstract/Free Full Text]
  60. Cerione, R. A., and Zheng, Y. (1996) Curr. Biol. 8, 216-222
  61. Zheng, Y., Olson, M. F., Hall, A., Cerione, R. A., and Toksoz, D. (1995) J. Biol. Chem. 270, 9031-9034[Abstract/Free Full Text]
  62. Habets, G. G., Scholtes, E. H., Zuydgeest, D., van der Kammen, R. A., Stam, J. C., Berns, A., and Collard, J. G. (1994) Cell 77, 537-549[Medline] [Order article via Infotrieve]
  63. Hart, M. J., Eva, A., Evans, T., Aaronson, S. A., and Cerione, R. A. (1991) Nature 354, 311-314[CrossRef][Medline] [Order article via Infotrieve]
  64. Li, R., and Zheng, Y. (1997) J. Biol. Chem. 272, 4671-4679[Abstract/Free Full Text]
  65. Kaibuchi, K., Mizuno, T., Fujioka, H., Yamamoto, T., Kishi, K., Fukumoto, Y., Hori, Y., and Takai, Y. (1991) Mol. Cell. Biol. 11, 2873-2880[Medline] [Order article via Infotrieve]
  66. Yaku, H., Sasaki, T., and Takai, Y. (1994) Biochem. Biophys. Res. Commun. 198, 811-817[CrossRef][Medline] [Order article via Infotrieve]
  67. Kikuchi, A., Kuroda, S., Sasaki, T., Kotani, K., Hirata, K., Katayama, M., and Takai, Y. (1992) J. Biol. Chem. 267, 14611-14615[Abstract/Free Full Text]
  68. Araki, S., Kikuchi, A., Hata, Y., Isomura, M., and Takai, Y. (1990) J. Biol. Chem. 265, 13007-13015[Abstract/Free Full Text]
  69. Gosser, Y. Q., Nomanbhoy, T. K., Aghazadeh, B., Manor, D., Combs, C., Cerione, R. A., and Rosen, M. K. (1997) Nature 387, 814-819[CrossRef][Medline] [Order article via Infotrieve]
  70. Keep, N. H., Barnes, M., Barsukov, I., Badii, R., Lian, L. Y., Segal, A. W., Moody, P. C., and Roberts, G. C. (1997) Structure (Lond.) 5, 623-633[Medline] [Order article via Infotrieve]
  71. Rittinger, K., Walker, P. A., Eccleston, J. F., Nurmahomed, K., Owen, D., Laue, E., Gamblin, S. J., and Smerdon, S. J. (1997) Nature 388, 693-697[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.