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
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ABSTRACT |
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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 (GTPS),
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
-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 GTP
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).
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INTRODUCTION |
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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, GTPS, and we
compare it with the structures of H-Ras and other related GTPases.
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EXPERIMENTAL PROCEDURES |
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Preparation and Crystallization of RhoAV14--
The
cloning, expression, and purification of the dominantly active form of
recombinant human RhoAV14 complexed with GTPS 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
GTP
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 GTP
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
CuK 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 -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
-helix. The GTP
S molecule and the
Mg2+ ion were identified unequivocally by their appearance
in 2Fo-Fc maps. The
-sulfur atom of GTP
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 GTP
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).
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RESULTS |
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Overall Structure--
The major features of the fold, consisting
of a six-stranded -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
-sheet is formed by the anti-parallel association of two extended
-strands (B2 and B3) and the parallel association of five extended
-strands (B3, B1, B4-B6). RhoAV14 contains five
-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 C
-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 C
-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
-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 C
-carbon atoms of
RhoAV14-GTP
S and RhoA-GDP superimpose with a r.m.s.
deviation of 0.48 Å. The nonhydrolyzable nucleotide GTP
S binds to
the protein with a Mg2+ ion that has a typical octahedral
coordination sphere (Fig. 4).
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Insertion Regions--
The N-terminal segment
(Glu125-Lys133) of the 13-residue insertion
forms an -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.
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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-GTPS and RhoA-GDP is small (0.31 Å) and that
between RhoAV14-GTP
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 C
-carbon atom of Val14 from the
corresponding atom of the wild-type RhoA (Fig. 5B).
Switch I--
The switch I region in RhoAV14-GTPS
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 G
subunits of the trimeric GTPases
such as transducin-
(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 -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
-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
-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
-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
GTPS 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 GTPS 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
- and
-phosphates from those of GDP, 0.75 Å and 0.67 Å, respectively.
Two oxygen atoms of the
-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
-phosphate to orient the
-sulfur atom toward Val14,
Tyr34, and Pro36. Similar configurations of the
-thiophosphate were also observed in the crystal structures of
transducin-
(44) and Gi
1 (45) complexed with GTP
S.
In all these crystals complexed with GTP
S, the
-sulfur atom is
the closest atom to the side-chain amide group of Gln63
(Gln200 of transducin-
and Gln204 of
Gi
1) among the
-thiophosphate atoms.
Putative Nucleophilic Water Molecule--
We identified one water
molecule (Wat-3) that is close enough to the -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
-sulfur
atom, although the distance to the
-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
-phosphate in the crystal structures of transducin-
and
Gi
1 complexed with GTP
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.
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DISCUSSION |
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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 GTPS 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
-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.
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 G
subunits of trimeric GTPases, as described for H-Ras (31),
transducin-
(41), and Gi
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
-strand, and Ran
has a completely different orientation of switch II that contains a
short
-strand. All these results indicate that small GTPases from
different families may have a similar fold but with significant
variations in the switch regions.
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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 -Sulfur Atom and the G14V Mutation on the GTPase
Activity--
The GTP
S molecule exhibits its resistance to
hydrolysis, which is conferred by the
-thiophosphorothioate. In the
present structure, the
-thiophosphate turns the sulfur-phosphorus
bond toward Gln63 and positions the
-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
-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 GTP
S molecule
to hydrolysis, which is conferred by the
-thiophosphorothioate, are
possible for transducin-
and Gi
1. Based on the
crystal structure of transducin-
complexed with GTP
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).
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
Supported by a research fellowship from the Japan Society for the
Promotion of Science.
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; GTPS, 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'-(
-imino)triphosphate; GMP-PCP,
guanosine-5'-(
-methylene)triphosphate; Arf, ADP-ribosylation factor; smgGDS, small GTP-binding protein guanine nucleotide
dissociation stimulator.
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REFERENCES |
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