From the Crystallography Research Program of Oklahoma
Medical Research Foundation, Oklahoma City, Oklahoma 73104 and the
§ Department of Biochemistry and Molecular Biology,
University of Oklahoma Health Sciences Center, Oklahoma City,
Oklahoma 73104
Received for publication, October 29, 2002, and in revised form, November 12, 2002
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ABSTRACT |
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GTPase domain crystal structures of Rab5a wild
type and five variants with mutations in the phosphate-binding loop are
reported here at resolutions up to 1.5 Å. Of particular interest, the
A30P mutant was crystallized in complexes with GDP,
GDP+AlF3, and authentic GTP, respectively. The
other variant crystals were obtained in complexes with a
non-hydrolyzable GTP analog, GppNHp. All structures were solved in the
same crystal form, providing an unusual opportunity to compare
structures of small GTPases with different catalytic rates. The A30P
mutant exhibits dramatically reduced GTPase activity and forms a
GTP-bound complex stable enough for crystallographic analysis.
Importantly, the A30P structure with bound GDP plus AlF3
has been solved in the absence of a GTPase-activating protein, and it
may resemble that of a transition state intermediate. Conformational changes are observed between the GTP-bound form and the transition state intermediate, mainly in the switch II region containing the
catalytic Gln79 residue and independent of A30P
mutation-induced local alterations in the P-loop. The structures
suggest an important catalytic role for a P-loop backbone amide group,
which is eliminated in the A30P mutant, and support the notion that the
transition state of GTPase-mediated GTP hydrolysis is of considerable
dissociative character.
As essential regulators of intracellular vesicle trafficking
between subcellular compartments of eukaryotic cells, Rab proteins comprise the largest branch in the monomeric Ras-related GTPase superfamily (1, 2) and mediate membrane fusion and possibly vesicle
budding as well (3-7). This group of 20-25 kDa proteins share ~30%
amino acid sequence identity (8). Like other Ras-related GTPases (small
GTPases), Rab proteins serve as molecular switches by cycling between
GTP-bound (on/active) and GDP-bound (off/inactive) conformations. Upon
GTP binding, an extensive hydrophobic interface forms between two
so-called switch regions (I and II) (9), resulting in presentation of
ordered structural features characteristic for the active state that
binds and responds to effectors/regulators (10, 11). The inactive form
usually has displaced and mobile switch regions (11, 12). The off-to-on
process requires dissociation of GDP, which is an intrinsically slow
and reversible process, and association of GTP. This process can be
accelerated by guanidine nucleotide exchange factors (GEF) (13, 14) and
regulated by other proteins such as GDP dissociation inhibitors (GDI)
(15). The on-to-off process is also an intrinsically slow but
irreversible process, which involves hydrolysis of GTP to GDP and is
stimulated by GTPase-activating proteins
(GAP)1 (16-20). Despite the
conserved catalytic machinery, the intrinsic GTP hydrolytic rates in
the Rab family vary by more than an order of magnitude. For example,
Rab5a exhibits a rate 20-fold higher than that of Rab6 or Rab7 (21).
The intrinsic GTP hydrolytic rate of a GTPase is important for the
association duration with its GTP-specific partners, and thus for its
functions in vivo. The structural determinants responsible
for the large variation in the intrinsic rates of GTP hydrolysis remain
elusive. In addition to the common GTPase fold, Rabs usually possess
hypervariable N- and C-terminal peptides; the C-terminal peptides are
often isoprenylated for targeting to specific membranes (8, 22). These
peptides may participate directly in protein-protein contacts with some
effectors or regulatory proteins but are not essential for nucleotide
binding or intrinsic GTP hydrolysis.
Rab5, a member of the Rab family, regulates early endosome fusion in
endocytosis (23-25) and possibly the budding process (26). It is
widely distributed in many tissues. Rab5 physically changes locations
during its GTP hydrolysis cycle. In its active stage, Rab5 is localized
on the cytoplasmic side of early endosomes, while in its inactive
stage, Rab5 stays in cytosol presumably associated with GDI. The
biological functions of Rab5 are evident from the following
observations. Anti-Rab5 antibodies and dominant negative Rab5 mutants
are inhibitory in several early endosome fusion assays reconstituted
in vitro (24, 27, 28). GTP-bound, but not GDP-bound Rab5,
can stimulate early endosome fusion in vitro (27, 29, 30).
Expression of dominant negative Rab5 mutants in intact cells causes
fragmentation of early endosomes and reduced endocytosis (23, 28).
Furthermore, overexpression of wild-type (WT) Rab5 or a
constitutively activated Rab5 mutant results in enlargement of
early endosomes and increased endocytosis (23, 28, 30). The intrinsic
GTPase hydrolytic rate human Rab5a (one of three Rab5 isoforms) has
been reported to be about 0.1 min Structures of a number of small GTPases and effects of mutations have
been investigated over the last two decades (31, 32). Besides their
similar overall folding, these proteins share a number of conserved
structural motifs. Among them, the phosphate-binding loop (P-loop),
i.e. the GXXXXGK(S/T) motif (where X
stands for any amino acid residue), is found to play crucial roles in
GTP hydrolysis. In Rab5a, the P-loop consists of residues
27GESAVGKS34, corresponding to residues
[10]GAGGVGKS[17] in the prototypic small
GTPase Ras (residue numbers in brackets denote positions in Ras).
Mutations at Ras Gly[12] often reduce its GTPase activity
and increase its biological activity in cellular transformation (33);
however, some of these variations can be found at the equivalent
position in other functional WT small GTPases. To investigate
structural roles of P-loop in GTP hydrolysis and biological function,
Rab5a has been used as a model system to replace both
Ser29[12] and Ala30[13] with all the other
19 amino acids (34, 35). The choice of mutation sites was partly made
based on their proximity to the Despite considerable kinetic, structural, and theoretical studies on
the reaction mechanism of small GTPases, there still lacks definitive
structural evidence to resolve different mechanistic hypotheses. It has
been shown that GTP hydrolysis in solution occurs via a dissociative,
metaphosphate-like transition state (39). Such a reaction pathway
implies that there is little bond formation between the nucleophilic
water and GTP but substantial cleavage of the bond between the
Expression and Purification of Recombinant Rab5a Proteins from
Escherichia coli--
Human Rab5a WT and P-loop mutants with Arg, Lys,
Glu, Leu, and Pro at position 30 were subcloned into the bacterial
expression vector pET11 from the corresponding pGEX-3X constructs
containing the Rab5a cDNAs described previously (35). The native
Rab5 protein consists of 215 residues. After comparison with the
canonical folding of small GTPases, we deleted the N- and C-terminal
hypervariable regions to promote crystallization. As a result, the
recombinant proteins contain a starting codon-derived methionine
residue, followed by residues 15-184 of Rab5a or its mutants. The
recombinant proteins were expressed in the BL21 strain of E. coli. and purified as soluble proteins from the cytoplasm.
Briefly, cell cultures were grown at 30 °C to an OD600
of 0.6-0.9, and then induced with isopropyl- Crystallization of Rab5--
Rab5a recombinant proteins were
crystallized using hanging drop vapor diffusion methods under
conditions similar to those reported previously (10). The protein
sample at ~15 mg/ml concentration in a buffer containing 20 mM Tris-HCl (pH 8.0) and 5 mM GppNHp was mixed
with an equal volume of the reservoir solution containing 10% (w/v)
polyethylene glycol 6000 (PEG 6000), 50-100 mM MES (pH 6.0), 0.2 M NaCl, 1 mM MgCl2, and
0.1% (v/v) Data Collection and Structure Refinement--
A complete data
set of WT Rab5a complexed with GppNHp was collected at 1.5 Å resolution from our in-house MAR345 image-plate data collection
system (Mar Research Inc., Norderstedt, Germany). Data sets of other
variants were also collected at comparable resolutions and processed
with the program suite HKL (47). The structures were solved by the
difference-Fourier method. The previously reported isomorphous crystal
structure of rat WT Rab5c at 1.8 Å resolution (PDB file 1huq) (10)
served as the initial model for phasing and refinement. The structures
were refined with CNS (48), and model building was performed with
Turbo-Frodo (49). Difference-Fourier maps calculated with coefficients
of (Fobs(crystal 1) The Overall Structure--
A previous biochemical study comparing
the GTP hydrolytic rates of Rab5a WT and P-loop mutants replacing the
Ala30[13] residue with 19 different amino acid residues
showed a range of over 50-fold variation in their intrinsic GTPase
activities (35). We selected five mutants from this group, namely A30P, Lys, Leu, Glu, and Arg, as well as the WT, for further structural studies. To facilitate crystallization, we deleted the hypervariable N-
and C-terminal peptides (14 and 31 residues, respectively). The
resulting recombinant proteins had comparable GTPase activities as
their full-length counterparts (data not shown). Since both termini are
located at one end of the
All crystals belonged to the
P212121 space group with similar
unit cell parameters. There was one Rab5a molecule per asymmetric unit,
with a low VM of 2.0 Å3/Da (50), reflecting a
tight crystal packing. The crystal structures of Rab5a were refined at
resolutions ranging from 1.8 to 1.5 Å. Statistics of data collection
and structural refinement of the eight crystal structures are
summarized in Table I. All coordinates have been deposited to the Protein Data Bank (see Table I for the PDB
IDs). The overall structure of Rab5a (Fig.
1) possessed a typical Ras-like small
GTPase folding (51) and was essentially the same as that of Rab5c, a
Rab5a isoform (10). In all structures, residues 18-181 were visible in
the electron density map. The corresponding root mean square deviations
(rmsd) for C Summary of WT and Non-proline Substitution
Variants--
The mutation site, residue 30[13] in the
P-loop, was not involved in any crystal contact in the present crystal
form; the closest distance between C P-loop and Nucleotide in A30P Complexes--
We were able to
obtain crystal structures for three A30P complexes, including
GDP·A30P, (GDP+AlF3)·A30P, and GTP·A30P. In contrast
to all other mutants, the Ala30[13] to Pro substitution
results in significant changes in the local three-dimensional structure
(Fig. 3A). The changes were,
however, confined at Ser29[12] and
Pro30[13], while the P-loop retained an overall low
thermal B-factor; for example, the GTP·A30P structure had a 10 Å2 average backbone B-factor for the P-loop, compared with
the 13 Å2 one of the overall backbone.
Pro30[13] assumes a cis conformation, thus
distorting the Shift of the Switch I Region in A30P Structures--
In the
structures of all three A30P complexes, part of the switch I region
(i.e. residues 49-51) shifted away from the nucleotide by
0.5-0.8 Å relative to WT (Fig. 3A). This displacement was
plausibly caused by a solvent network rearrangement between the P-loop
and switch I region as a consequence of the A30P mutation. In the WT
structure, the water molecule that hydrogen bonded with the
Located in the switch I region, the Ser51[34] side chain
assumed a trans rotamer in the WT structure and formed a
hydrogen bond with a Comparison of GDP-, (GDP+AlF3)-, and GTP-bound A30P
Structures--
The overall structures of GDP·A30P complexes both
with and without AlF3 were similar to those of other Rab5a
variants, and their switch I regions were essentially identical to that
in the GTP·A30P complex. However, noticeable conformational changes
were found in the (i) Other Structural Features--
In addition to the structural
changes in the vicinity of the nucleotide-binding site, we observed
some alternative packing in a hydrophobic cluster among the variant
Rab5a structures. In A30E, A30K, and A30L, the side chain of
Phe21[4] was partially exposed to solvent. However, in
other crystal structures, this side chain assumed mainly a buried
conformation inside a hydrophobic core. The Phe71[53]
side chain had to make a 20° The biological and medical importance of GTPases continue to
stimulate structural studies on their functional specificity and
interactions with effectors/regulators. Despite considerable mutagenesis, kinetic and structural efforts, a consensus remains to be
reached regarding key mechanistic aspects of catalysis by small
GTPases. Here, we report high resolution crystal structures of a number
of Rab5a mutants with substitutions in the catalytically important
P-loop, which provide new insight into the GTPase catalysis. Whereas
some of the variants (e.g. the A30R mutant) demonstrate noticeable difference in their intrinsic GTPase activity, the structural differences between non-proline substitution variants are
marginal and mainly restricted to the mutation site, with a uniform
gauche+ Structural flexibility in solution, especially for a side chain at
position 30[13] in the P-loop, may modify the static picture we have
obtained here from the crystal structures, thus contributing to the
variation in hydrolytic rates. For example, A30R hydrolyzes GTP with a
5-fold higher rate than WT (35). It was hypothesized that
Arg30[13] may accelerate the hydrolysis by contributing
an arginine finger-like motif to the catalytic site. Such a motif might
stabilize the transition state intermediate through electrostatic
interaction with negatively charged groups in either the Our GTP·A30P complex crystal structure provides a high resolution
picture of the binding of an authentic GTP molecule to a GTPase,
confirming that GTP assumes a similar conformation to GppNHp inside the
nucleotide-binding site, thus justifying most conclusions obtained from
structural studies on the GTP analog-GTPase complexes. Given the fact
that A30P can crystallize with both GTP and GDP in the same crystal
form, it raises the question of why WT and other Rab5a variants do not
crystallize in the GDP-bound form. One possible explanation is that
A30P possesses a quasi-stable active conformation that does not require
The A30P structures support the dissociative transition state
hypothesis for GTPase-catalyzed GTP hydrolysis (43). One important prediction from this hypothesis is that the backbone amide group of
Gly[13] in Ras (equivalent of Rab5a Ala30)
plays the critical role in stabilizing a negative charge accumulation during the catalysis. In all GDP-small GTPase complex structures that
we have examined (PDB files 1an0, 1d16, 1d5c, 1ky3, 1q21, 1rrf, 1rrg,
and 4q21), this amide group forms a hydrogen bond with the
Another crucial residue in the GTPase catalysis is the frequently
conserved Gln79[61], which is located in the switch II
region. A mutation of this residue to leucine in a variety of GTPases
has been repeatedly shown to abrogate the GTPase activity, and thus
maintains the protein in its GTP-bound form (59). It is widely believed
that the function of this glutamine residue is to align the
nucleophilic water molecule in position for attacking the
We have successfully solved the structure of a small GTPase complexed
with GDP and AlF3. While similar transition state
intermediate analog conformations have been observed in a number of
crystal structures of (GDP+AlFx)-bound forms of small
GTPases complexed with GAPs (PDB files 1grn, 1k5d, 1tx4, 1wq1, and
2ngr) and of trimeric GTPases (PDB files 1agr and 1tad), no crystal structure of a small GTPase complexed with both GDP and
AlFx in the absence of a GAP partner has been reported. In
our (GDP+AlF3)·A30P complex structure, the
Ser29[12] hydroxyl group participates in the
hexacoordination to the aluminum ion. The interaction between
Ser29[12] and AlF3 may contribute to the
stability of the complex which would otherwise be provided by a more
extensive interaction with GAPs. The geometry of this aluminum complex
is similar to those of AlF The two Rab5a regions corresponding to switch I and II in Ras are
Gln49[32]-Ala56[38] and
Ala77[59]-Leu85[67], respectively.
In conclusion, our study has emphasized the importance of P-loop in
catalysis. Particularly, the amide group of residue 30 is likely to
play a critical role in the dissociative-like transition state, because
elimination of this group seems to be a major, if not the only, reason
for the A30P mutant to lose GTP hydrolytic activity. Structurally, the
(GDP+AlF3)·A30P complex resembles the reaction
intermediate, although not necessarily in the sense of an associative
transition state. It provides an unusual opportunity to visualize the
transition state intermediate in a small GTPase in the absence of
stabilization by a GAP partner.
INTRODUCTION
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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES
1 (21), which is in the
high range among Rab family members.
-phosphate group of the substrate
GTP. The resulting variants have been analyzed for GTP hydrolysis, GTP
binding, GTP dissociation, and biological activity. At position 30 [13], only the substitution with proline reduces the GTPase activity
significantly (at least 12-fold) (35). Whereas most of the other
substitutions at this position show either a small negative effect or
no effect on the GTPase activity, the arginine substitution stimulates
the intrinsic GTP hydrolysis by 5-fold. It was proposed that this
introduced arginine residue may mimic the function of an arginine
finger motif (35), which enhances GTPase activity in trimeric GTPases (36, 37) and small GTPase-GAP complexes (38) by positioning the
positively charged guanidinium group close to the GTP
-phosphate group.
-phosphoryl moiety and the GDP-leaving group. Recent results from
Fourier transform infrared spectroscopy experiments suggest that the
transition state seems to have a considerable amount of dissociative
character (40). Binding of GTP to Ras has been shown to shift negative
charge from the
- to
-phosphate (41), which is a characteristic
feature of dissociative-like transition states, and such a shift can be
enhanced by GAP binding (42). The charge shift is interpreted as a key factor contributing to catalysis by Ras in addition to correct positioning of the nucleophilic water. These observations seriously challenge the long held dominant mechanistic hypothesis that GTP hydrolysis occurs via an associative-like pathway. A fully associative mechanism involves a trigonal bipyramidal intermediate, followed by the
departure of the leaving group. It is characterized by an accumulation
of negative charge on the
-phosphate in the transition state.
Structural observations, that catalytically important residues in
GTPases interact extensively with the trigonal bipyramidal or
hexacoordinating transition site analog GDP+AlFx (where x
is 3 or 4), form a cornerstone of associative transition state
hypothesis. A compromising view has been put forward to describe the
phosphoryl transfer by a structure somewhere between dissociative and
associative extremes (40). However, there is still a controversial
debate as to what extent the reaction proceeds via a dissociative or an
associative mechanism. The dissociation transition state hypothesis of
GTPase catalysis predicts that the enzyme stabilizes accumulation of
negative charge at the
-
bridge oxygen in the transition intermediate through an important hydrogen bond between this oxygen and
the backbone amide group at the residue equivalent to Ras Gly[13] (43). In short, the associative transition
hypothesis emphasizes the catalytic roles of the nucleophilic water and
structural features that stabilize negatively charged GTP
-phosphate
(44, 45), whereas the dissociative transition hypothesis proposes that
the P-loop is crucial for the reaction (41, 43). A few crystal structures of Ras variants with mutations at Gly[12]
inside the P-loop have been reported (44, 46), which in general support
the notion that correct alignment of the nucleophilic water is critical
for the GTP catalytic hydrolysis. To identify further the structural
determinants in the P-loop that may regulate the intrinsic GTPase
activity and potentially, the GAP-accelerated GTP hydrolytic rates, we
have carried out a comprehensive crystallographic study to analyze a
number of crystal structures of human Rab5a and five variants.
Particularly, we have determined crystal structures of a variant that
contains an Ala30[13] to Pro substitution (A30P) in its
GTP-bound, (GDP+AlF3)-bound, and GDP-bound forms, taking
advantage of its very low hydrolytic activity. High resolution crystal
structures of WT and other P-loop mutants have also been solved in
complexes with a non-hydrolyzable GTP analog; they provide a clean
background for discussion of the novel structural features observed in
A30P complexes. Our results support the dissociation transition state
hypothesis (43).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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DISCUSSION
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-D-thiogalactoside at final concentration of
0.5 mM, with an additional 3 h of growth at
35-37 °C. Cells were harvested by centrifugation and resuspended in
4 °C TN buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl) containing EDTA-free protease inhibitor mixture (Roche Molecular Biochemicals, Mannheim, Germany). Cells were then
lysed with lysozyme and a freeze-thaw cycle and treated with DNase in
the presence of 1 mM MgCl2. The cell lysate was
centrifuged, and the supernatant was loaded to a Sephacryl-200-HR
sizing column (Amersham Biosciences) equilibrated in TN buffer
containing 0.1% (v/v)
-mercaptoethanol and 0.02% (w/v)
NaN3. High A280 fractions were
pooled and loaded onto a Resource-Q ion-exchange column (Amersham Biosciences). Rab5a was eluted at ~100 mM salt
concentration with a NaCl gradient.
-mercaptoethanol. The A30P mutant crystals were grown
with GDP or GTP instead of GppNHp. In order to grow
(GDP+AlFx)·Rab5a complex crystals, NaCl in the above
reservoir solution was replaced with 2 mM AlCl3
and 80 mM NaF. Crystals appeared 1 day after microseeding.
Reservoir solution with additional 30% (v/v) glycerol (for WT, A30E,
and A30L), 30% PEG 6000 (for A30K), or 20% 2-methyl-2,4-pentanediol (for A30R and A30P) served as the cryoprotection solution to soak the
crystal before being cooled in a 100 K nitrogen gas stream for data collection.
Fobs(crystal 2))
exp(i
calc) were used in structural
comparison to minimize model bias.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet that is opposite from the
nucleotide-binding site, the truncations are unlikely to contribute to
the conformational changes we observed in the different mutant crystal
structures. In the following, we will refer to these truncation
constructs as WT Rab5a and its mutants. All constructs were
overexpressed in E. coli. as soluble proteins and purified
for structural studies using x-ray crystallography. The intrinsic GTP
hydrolytic rates of these mutants ranged from the highest (A30R) to the
lowest (A30P) among the original 20 variants. The purification yields
of these variants were about the same as the WT (5-10 mg per liter of
cell culture). The recombinant proteins of all variants were able to be
crystallized under the same or very similar conditions. Of particular
interest, three A30P complex crystals were obtained with GDP,
(GDP+AlF3), and authentic GTP, respectively. For the other
variants, crystals were obtained only in complexes with GppNHp. Like
the GppNHp·Rab5a variant crystals, the GTP·A30P complex crystals
were stable at 20 °C for at least 50 days; GDP·A30P crystals,
however, appeared less stable (i.e. <10 days).
atoms between WT structure and those of non-proline
variants range from 0.10 (A30R) to 0.34 Å (A30E). The structures of
GTP (or its analog) and Mg2+ ligands were essentially
identical in all variant crystals. Even in the GDP- and
(GDP+AlF3)-bound complex structures, the GDP, Mg2+ ion, and its ligand atoms were perfectly
superimposable with the corresponding parts from other structures.
Crystallography data collection and refinement statistics
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Fig. 1.
Ribbon diagram of Rab5a catalytic
domain. The GppNHp molecule is shown in a stick model.
Residue 30 C position and the active site Mg2+ are
marked with spheres. The P-loop, Mg2+, switch I
(SW1) and II (SW2) as well as the N and C termini are labeled. All
figures were drawn with the programs Molscript and Raster3D (64,
65).
of residue 30[13] and
symmetry-related protein molecules was larger than 8 Å. In each
structure, residues in the P-loop had lower-than-average backbone
B-factors. They ranged between 9 and 16 Å2 for the P-loop,
compared with those between 13 and 21 Å2 of the overall
backbone B-factors. Structural differences between the non-proline
mutants and WT were restricted to the mutated side chain only; the
P-loop backbone rmsd was below 0.1 Å for all structures in this group.
As an example, Fig. 2 shows the differences in electron density map between A30R and WT. The
differences included the side chain mutation (the large blue density
block) as well as the shift of a water molecule nearby. The P-loop
contained a type-II tight turn (52) formed by residues
28[11]-31[14]. Atoms of the 29[12]-30[13] peptide plane and
-
bridge nitrogen atom in GppNHp were located perfectly in the
same plane. The
-
bridge nitrogen atom hydrogen-bonded as an
acceptor with the Ala30[13] amide group (N-N distance of
3.02 ± 0.03 Å averaged over the five structures ± S.D.)
and as a donor with a water molecule (N-O distance of 2.90 ± 0.02 Å), respectively. Together with the
- and
-phosphate atoms,
they formed a nearly perfect tetrahedral geometry centered at the
-
bridge atom. A similar network should form around the bridge
oxygen atom if an authentic GTP molecule had been bound, except that
the oxygen atom would function as an acceptor in each hydrogen bond. At
the mutation site in each structure, the side chains were mobile beyond
the C
atom because of lacking supportive interaction, while the
1 torsion angles of A30E, A30K, A30L, and A30R variants
all assumed a gauche+ rotamer. The side chain
terminal group in both A30R and A30E pointed to solvent with a
trans
2 rotamer; and in A30K, the N
group
of lysine side chain hydrogen bonded with the hydroxyl group of
Ser51[34] in the switch I region. In no case did we see
that a side chain at position 30 pointed to the nucleotide. Thus any
influence of the non-proline mutations at this position on the GTPase
activity would most likely be indirect in nature. In the following, we use the WT structure as a representative of this group for comparison with the A30P structures, unless otherwise specifically mentioned. Furthermore, in all GTP(analog)-bound complex structures, including GTP·A30P, the nucleophilic water was located in the vicinity of the
-phosphoryl group and hydrogen bonded with the backbone amide group
of residue 79 (O-N distance of 3.2 ± 0.1 Å). The side chain of
catalytic residue Gln79[61] protruded into solvent and
became mobile at its carbonyl tip, although it might form a weak
hydrogen bond with the Arg81[63] side chain.
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Fig. 2.
A30R compared with WT. Crystal
structures of A30R (blue) and WT (red) are
superimposed, and their catalytic sites are shown. Also superimposed
are the Fobs(A30R) Fobs(WT) difference electron densities
contoured at 3.5
, with positive density colored in cyan
and negative in pink. These densities represent the most
significant features in the entire difference map and are
typical for all mutants of non-proline substitutions reported here.
Mg2+ and ordered water molecules are shown as
large and small crosses, respectively.
-turn in which the mutated residue was at the third
position (52). Because of the cyclic proline side chain, the backbone
amide group was no longer available for hydrogen bonding with the
-
bridge oxygen in GTP/GDP. The corresponding proline imide group
moved 1.5 Å in the direction away from the nucleotide. The C
atom
of Ser29[12] in the GTP·A30P complex moved 0.4 Å relative to the GTP·WT complex, tilted toward to the
-phosphoryl
group. In the GDP-complex, this C
atom moved even further (1.2 Å relative to WT) presumably due to loss of contact with the
-phosphoryl group, and the hydroxyl group assumed double
conformations (gauche+/trans). In all
three structures, the nucleotides had well defined electron densities,
and the P-loop conformations were similar but not identical to each
other (Fig. 3B), suggesting a structural adjustment
associated with nucleotide binding. In the GTP-bound structure,
B-factors of all atoms in GTP, including the
-phosphate group,
ranged between 6 and 12 Å2 and were lower than that of the
average protein backbone (13 Å2), indicating that no
significant hydrolysis occurred inside the crystal.
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Fig. 3.
Structures of A30P mutant.
A, reduce structural comparison between GppNHp·WT and
GTP·A30P. WT is shown in red and A30P in blue.
The water hydrogen-bond network around the active site and
Mg2+ coordination are shown in dashed lines.
Mg2+ and ordered water molecules are shown as
large and small crosses, respectively.
B, structural comparison among the three A30P
complexes. The active sites of GTP- (blue),
(GDP+AlF3)- (red), and GDP- (yellow)
forms of A30P are superimposed on each other. C, active site
structure of the (GDP+AlF3)·A30P complex.
2Fobs Fcalc map
was contoured at 1.0
and superimposed with a
ball-and-stick model of the final refined structure. Carbon
(yellow), nitrogen (blue), oxygen
(red), phosphate (white), aluminum
(cyan), fluoride (magenta), and magnesium
(green) ions are colored, respectively. The coordination of
both Mg2+ and Al3+ (except the Al-F bonds) are
shown in thin lines. Orientations are the same as that of
Fig. 2.
-
bridge nitrogen atom in GppNHp simultaneously formed another hydrogen
bond with an
-phosphoryl oxygen atom but did not interact with the
switch I region directly. In A30P structures, this water molecule was
not seen because of a close contact from the Pro30[13]
C
atom. It was replaced by two new water molecules at slightly shifted positions. One of them formed hydrogen bonds in a tetrahedral geometry with the
-phosphoryl oxygen, the backbone carbonyl oxygen of Gln49[32] in the switch I region, and two water
molecules including the other new one. Similarly, the second water
molecule formed four hydrogen bonds with a
-phosphoryl oxygen atom,
the hydroxyl group of Ser51[34] side chain again in the
switch I region, and two water molecules. Through interactions with
residues 49[32] and 51[34], this new solvent network resulted in a
shift in the switch I region, making the nucleotide-binding pocket
slightly more open, which was consistent with the higher GTP
dissociation rate (3-fold) of this mutant than that of WT (35).
-phosphoryl oxygen atom. In A30K,
Ser51[34] switched to the gauche+
rotamer, which might weaken the hydrogen bond with the
-phosphoryl oxygen; the potential hydrogen bond with Lys30[13] in
A30K might provide some compensation for the energy loss. Furthermore,
in A30R, A30E, and A30L, the Ser51[34] side chain
appeared to have a double conformation, switching between
trans and gauche+. Collectively, the
greatest difference was observed in A30P, where the hydrogen bond
between a
-phosphoryl oxygen and the hydroxyl group of
Ser51[34] was abrogated, accompanied by the backbone
shift of the switch I region and a Ser51[34] side chain
rotamer change to gauche+.
-phosphate-binding site, (ii)
Gln79[61] side chain, (iii) backbone of the switch II
region, and (iv) Lys33[16] side chain, accompanying
deformation/removal of the
-phosphoryl group (see Fig.
3B). First, in the GDP·A30P complex, the
-phosphoryl group was replaced by two water molecules, including one that coordinated with Mg2+ at the position of a GTP
-phosphoryl oxygen. In the (GDP+AlF3)-bound complex, the
aluminum ion coordinated with a hexavalent octahedral symmetry, namely
three fluoride ions, the hydroxyl group of Ser29[12], one
-phosphoryl oxygen, and a nucleophilic water (Fig. 3C). The rmsd of the aluminum and three fluoride ions and
Ser29[12] O
atom from an ideal plane was 0.07 Å, and
the rmsd values of the other two principal planes of the octahedron
were 0.09 and 0.18 Å. Two of the fluoride ions occupied similar
positions to those of
-phosphoryl oxygen atoms that bind with the
Mg2+ and Lys33[16] side chain, respectively.
These two fluoride ions formed covalent bonds with the aluminum ion
(1.83 ± 0.04 Å), while the third one was 2.1 Å from the
aluminum ion and had a 23 Å2 B-factor, which was almost
twice as high as that of the other two fluoride ions. Thus, this third
ion could be interpreted as a water molecule, although it is reported
here as a fluoride ion for simplicity. Distances from the aluminum ion
to the
-phosphoryl oxygen, Ser29[12] hydroxyl group,
and the nucleophilic water molecule were 2.0, 2.1, and 2.4 Å,
respectively. Secondly, while Gln79[61] retained the same
gauche+ rotamer in its
1 torsion
angle in both GDP and (GDP+AlF3) complexes as in the WT
structure, its
2 rotamer changed so that the side chain
tip plunged into the phosphate-binding pocket. The side chain carbonyl
oxygen of Gln79[61] simultaneously formed hydrogen bonds
with its own backbone amide group and the nucleophilic water, replacing
the hydrogen bond between the latter two. The water molecule was pushed
~1.5 Å toward the
-phosphate atom as well as its symmetry axis
along which the nucleophilic attack would occur. In the GDP-bound form,
the water molecule further hydrogen bonded with a
Mg2+-coordinating water molecule; in the
(GDP+AlF3)-bound form, the bound water molecule mimicked
the nucleophile and coordinated to the aluminum ion. The nucleophilic
water molecules in both GDP and (GDP+AlF3) complexes
occupied almost identical positions, indicating that the
Gln79[61] side chain can align the nucleophilic water
molecule almost perfectly even in the absence of
-phosphate.
Thirdly, the Gln79[61] conformational change between GDP-
(as well as GDP+AlF3) and GTP-bound forms was associated
with a ~0.5 Å backbone shift of residues 79[61]-81[63] in the
switch II region, which seemed required in order to dip the
Gln79[61] side chain into the phosphate-binding pocket.
Finally, in the GTP-bound form, Lys33[16] side chain
formed hydrogen bonds with oxygen atoms from both
- and
-phosphoryl groups; it retained the same interactions in the
(GDP+AlF3)-bound complex. However, it changed side chain conformation to hydrogen-bond with the carbonyl oxygen atoms of residues 27[10] and 76[58] in the GDP form; and its previous
position was occupied by a solvent molecule. A similar
Lys[13] side chain conformational change had been
observed in a crystal structure of the GDP·Cdc42Hs complex (PDB file
1an0).
1 adjustment to
accommodate the insertion of Phe21[4]. Considering the
fact that the C
-C
distance between residues 21 and 30 was over
25 Å, these structural changes more likely reflect intrinsic
flexibility of Rab5a GTPase domain rather than response to the
mutations introduced. Whether this hydrophobic repacking was of any
functional significance, for example in the presence of N- and
C-terminal peptides, remains an open question.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 rotamer. The equivalent
position in Ras is a glycine. Mutations that add a side chain at
position Gly[13] in Ras have little effect on the
intrinsic GTPase activity (53). On the other hand, Rab3A has a serine
at this position, which assumes a gauche
rotamer to avoid clashing with the side chain of
Phe51[32] from the switch I region. In Rab5a, the
corresponding Gln49[32] side chain in the switch I region
points in a different direction, thus having no interaction with
whatever residue is at position 30[13] in the P-loop.
-
bridge
oxygen or
-phosphoryl group. Our crystal structure of the A30R
mutant argues against a static model of such a structural motif,
because no hydrogen bond or salt bridge was observed between the
Arg30[13] side chain and the nucleotide or catalytic
apparatus. However, favorable interactions of a similar nature,
directly or indirectly through a solvent network, may occur transiently
in solution where the Arg30[13] side chain may exhibit
dynamic movement. One could argue that the Arg30[13]
guanidine group might not attack the
-
bridge atom in the crystal structure because of the GTP analog used; an authentic GTP has an
oxygen atom at this position, which may function as an acceptor to two
hydrogen bonds and allow an interaction with the guanidine group in
addition to the hydrogen bond with P-loop. However, it is noteworthy
that the rate increase of A30R is significantly smaller than the
200-105-fold increase stimulated by an arginine finger
from GAPs (54, 55). It indicates that the arginine side chain
introduced by the single point mutation is far from optimal for such a
catalytic function. Another possibility is that a mutation at position
30[13] may change the association/dissociation rate of the
substrate/product, thus modifying the apparent hydrolytic rate. This
might happen to A30R as observed for A30I and A30V (34).
-phosphate. This is consistent with the observation that the A30P
mutant exhibits dominant positive phenotype (i.e. it is
locked in the active conformation) in stimulating endocytosis (34).
Furthermore, the altered water network observed in the A30P crystal
structures may favor such a conformation, which is associated with a
small (~0.5 Å) and localized (residues 48-53) shift in the switch I region.
-phosphoryl oxygen corresponding to the bridge oxygen in GTP,
consistent with the charge-shifting theory. NMR spectroscopic studies
of Ras in solution also suggest that the Gly[13] amide
proton forms a hydrogen bond in a GTP[S]-Ras complex (56). Our
GTP·A30P complex crystal structure provides direct evidence supporting the dissociative transition state hypothesis from a different angle. In this case, the proposed important hydrogen bond
between the backbone amide group and the
-
bridge oxygen does not
exist because of the nature of the mutation. Elimination of the
backbone amide group of residue 30[13] in A30P is accompanied by the
loss of GTPase activity, strongly supporting the mechanism requiring a
hydrogen bond to the
-
bridge oxygen of GTP (43). The fact that
we have been able to obtain the complex crystal of GTP·A30P confirms
that the GTP hydrolytic rate must be very low and suggests that other
hydrolytic mechanisms are unlikely to function in the absence of this
hydrogen bond. Inside the crystals, the most noticeable structural
change in GTP·A30P complex relative to GppNHp·WT complex is located
in residues 29[12] and 30[13]. Therefore, the decrease in the
intrinsic GTPase activity can be explained solely by loss of the key
catalytic component, particularly the peptide plane between residues
29[12] and 30[13]. This conclusion is consistent with the
observation that a proline substitution at Rab5 Ser29[12]
does not reduce the intrinsic GTPase activity (34). This residue is the
closest neighbor to Ala30[13] but whose backbone atoms
assume no direct role in catalysis. Furthermore, our GTP·A30P
structure shows no conformational change in the rest of the P-loop,
including residues 31[14]-33[16], which were proposed to be
important for a charge shift from the
- to
-phosphate on GTP
hydrolysis (41); thus, it is unlikely that the A30P mutation might
disrupt the catalysis via this region. In addition, unlike the backbone
amide group of Ras Gly[13] that retains its hydrogen bond
with the
-phosphoryl oxygen in GDP (9), the Lys33[16]
side chain in GDP·A30P moves away from GDP. It suggests that this
lysine residue is less important than the backbone amide group of
residue 30[13] in the charge shift during GTP hydrolysis.
Based on the WT Rab5a structure, both the Ala30[13] amide
group and the water molecule that hydrogen bonds to the
-
bridge
group are likely to contribute to catalysis, supporting the
dissociative transition state hypothesis (43). This water molecule has
been observed in a number of small GTPase crystal structures,
e.g. those of Ypt51 (PDB file 1ek0) (57) and ARF1 (58), but
is disrupted in others often because of interference from the switch I
region. The water molecule occupies a position similar to the arginine
finger from the GAP protein in the Ras-GAP complex crystal structure
(45); its catalytic effect is likely, however, to be weaker.
-phosphoryl group from the axial direction (9, 43, 44, 46, 60, 61), although it has been argued that activation of the nucleophilic water
is not the rate-limiting step in the hydrolytic reaction (44). In our
GTP(analog)·Rab5a crystal structures, the Gln79[61]
side chain interacts with the nucleophilic water molecule only through
a Van der Waals contact. Therefore, the residue specificity is unlikely
to function at this stage. In contrast, in both GDP- and
(GDP+AlF3)-bound forms of A30P, the Gln79[61]
side chain carbonyl group points to the nucleotide-binding pocket. Particularly, in the (GDP+AlF3)-bound form, the
Gln79[61] side chain carbonyl group forms a hydrogen bond
ideal for orientating the nucleophilic water molecule to attack the
-phosphoryl group analog, in this case the AlF3 moiety.
The formation of such an intermediate is clearly vulnerable to a
mutation at Gln79[61]. Given the structural integrity of
this part of the catalytic apparatus in (GDP+AlF3)·A30P,
the dramatically reduced intrinsic GTPase activity of A30P strongly
argues that a perfect alignment of the nucleophilic water is not
sufficient for the catalysis. Furthermore, because the complex
structure preserves all required structural features predicted by an
associative transition-state mechanism, yet no hydrolysis is
detectable, such a mechanism is unlikely to function in A30P, and
possibly neither in WT Rab5 and other small GTPases.
-phosphoryl group would
go through during the transition state, the vicinity of
Ser29[12] side chain to the transition state intermediate
argues that similar hydrogen bond interaction could play roles in Rab5
functions. This serine residue is not present in any small GTPases
other than Rabs. Mutations including a Ser substitution at the cognate Gly[12] in Ras often reduce its GTPase activity and
increase its biological activity in cellular transformation (33, 46,
63). In a transition state mimicking Ras structure (38), the backbone
of Gln[61] moves toward the P-loop relative to the GTP
analog-bound form, instead of away from it as shown in our Rab5a
crystal structures. Thus, a side chain at the position [12] in
Ras is likely to interfere with Gln[61] in alignment of
the nucleophilic water. In the Rab5a structures, however, the
Ser29[12] side chain does not interfere with the
Gln79[61] in either the ground state or the transition
state intermediate analog, demonstrating that a subtle structural
adjustment can make a detrimental mutation in one protein become
acceptable or even favorable in others.
-Phosphoryl oxygen atoms form two hydrogen bonds with main chain
amide groups of residues Thr54[35] and
Gly78[60] in the switch I and II regions. These two
switch regions undergo significant conformational changes upon GTP
hydrolysis and the ability to distinguish upstream from downstream
partners by means of their dissimilar GDP and GTP structures is the
hallmark of GTPases. The comparison of (GDP+AlF3)- and
GTP-bound A30P structures has identified a backbone shift in the switch
II region (see Fig. 3B), demonstrating the conformational
change between the GTP-bound form and the intermediate state. This is
consistent with the notion that a glycine residue in the conserved
sequence ([57]DXXGQ[61]) (32)
may provide backbone flexibility in the switch II region in addition to
making proper contacts with the P-loop. Influence of crystal packing is
unlikely to play a role in the observed conformational change of the
switch II region because all structures are in one isomorphous crystal
form, thus sharing similar packing environments. Comparison of
(GDP+AlF3)- and GDP-bound A30P structures reveals no more
changes in either switch region, suggesting that the A30P mutant is
trapped at the transition state intermediate conformation, possibly due
to the loss of the catalytically important backbone amide group of
Ala30[13].
![]() |
ACKNOWLEDGEMENTS |
---|
We thank N. Wakeham and A. Howerton for technical assistance, Drs. G. Air, T. Mather, L. Hong, and G. Koelsch for critical reading of this article.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a Career Award from the National Science Foundation (to G. L.).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 the structure factors (code 1N6H, 1N6I, 1N6K, 1N6L, 1N6N, 1N6O, 1N6P, and 1N6R) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ To whom correspondence should be addressed. Tel.: 405-271-7402; Fax: 405-271-7953; E-mail: zhangc@omrf.ouhsc.edu.
Published, JBC Papers in Press, November 13, 2002, DOI 10.1074/jbc.M211042200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GAP, GTPase-activating protein;
GppNHp, guanosine-5'-(,
)-imidotriphosphate;
WT, wild type;
P-loop, phosphate-binding loop;
rmsd, root mean square deviation;
PDB, protein
data bank;
MES, 4-morpholineethanesulfonic acid.
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