From the Program in Molecular Medicine and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
Received for publication, October 26, 2000, and in revised form, January 11, 2001
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ABSTRACT |
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Rab GTPases function as regulatory components of
an evolutionarily conserved machinery that mediates docking, priming,
and fusion of vesicles with intracellular membranes. We have previously shown that the active conformation of Rab3A is stabilized by a substantial hydrophobic interface between the putative conformational switch regions (Dumas, J. J., Zhu, Z., Connolly, J. L., and Lambright, D. G. (1999) Structure 7, 413-423). A triad of invariant hydrophobic residues at this switch
interface (Phe-59, Trp-76, and Tyr-91) represents a major interaction
determinant between the switch regions of Rab3A and the Rab3A-specific
effector Rabphilin3A (Ostermeier, C., and Brunger, A. T. (1999) Cell 96, 363-374). Here, we report the crystal
structure of the active form of Rab5C, a prototypical endocytic Rab
GTPase. As is true for Rab3A, the active conformation of Rab5C is
stabilized by a hydrophobic interface between the switch regions.
However, the conformation of the invariant hydrophobic triad (residues
Phe-58, Trp-75, and Tyr-90 in Rab5C) is dramatically altered such that
the resulting surface is noncomplementary to the switch interaction
epitope of Rabphilin3A. This structural rearrangement reflects a set of
nonconservative substitutions in the hydrophobic core between the
central As general regulators of intracellular vesicle transport between
donor and acceptor membranes, Rab proteins comprise the largest GTPase
family with 11 distinct homologues in yeast and more than 50 known Rab
GTPases in mammals (3-7). As is true of other GTPases of the Ras
superfamily, Rabs cycle between active (GTP-bound) and inactive
(GDP-bound) conformations (3, 8, 9). A key question concerns the
molecular and structural mechanisms by which Rab GTPases generate
specificity for a diverse spectrum of effectors and regulatory factors.
Biochemical and genetic studies of chimeric and mutant Rab proteins
have identified several hypervariable regions, including the N and C
termini and the Crystallographic studies of Rab GTPases have identified
structural motifs and modes of effector interaction that are distinct from those of other GTPase families. The active conformation is stabilized by additional hydrogen bonding interactions with the Rab5 is an essential regulator of early endosome fusion (18, 19).
Several putative Rab5 effectors have been identified including EEA1
(early endosomal auto antigen), a
large protein consisting of an N-terminal Zn2+ finger, four
long heptad repeats having weak homology with myosins, and a compact
C-terminal region containing an IQ motif (putative calmodulin binding
site), a Rab5 interaction site, and a FYVE domain that specifically
binds phosphatidylinositol 3-phosphate (20-26). Interactions with both
phosphatidylinositol 3-phosphate and Rab5 are essential for the fusion
of endocytic vesicles with early endosomes in an in vitro
reconstitution assay (27, 28). Although lacking the corresponding
C-terminal regions, the FYVE domain of EEA1 is structurally similar to
the ring finger domain of Rabphilin3A (29). Moreover, a predicted
helical region N-terminal to the FYVE domain of EEA1 exhibits weak
homology with the N-terminal helix of Rabphilin3A and has been
implicated in the interaction with Rab5 (25, 30). However, critical
hydrophobic residues in Rabphilin3A are not conserved in EEA1. Thus,
the extent to which the Rab3A-Rabphilin3A complex can be regarded as a
paradigm for other Rab-effector complexes remains uncertain
(31).
The crystallographic and biochemical studies cited above are consistent
with the hypothesis that the relatively conserved switch regions convey
information regarding the state of the bound nucleotide, whereas
independent Rab CDRs determine the specificity of Rab-effector
interactions. Here, we report the crystal structure of Rab5 bound to
GppNHp. Despite strong homology, the characteristic RabF motifs in the
switch regions of Rab5 and Rab3A do not encode the same active
conformation. In particular, the invariant triad of partially exposed
hydrophobic residues located at the switch I/switch II interface adopts
a dramatically different conformation in Rab5 such that the resulting
hydrophobic surface is noncomplementary to Rabphilin3A. These changes
reflect an alternative packing arrangement in response to a concerted
set of nonconservative substitutions in the hydrophobic core. Moreover,
the packing constraints on the conformation of the invariant
hydrophobic triad appear to be engaged only in the active form. Thus,
structural plasticity within the relatively conserved switch regions is
evolutionarily coupled to the conformational switching mechanism and
represents a potentially general determinant of Rab-effector recognition.
Expression and Purification--
The GTPase domain of Rab5C
(residues 16-186) was expressed in Escherichia
coli using a modified pET15b vector containing an N-terminal
His10 peptide (MGHHHHHHHHHHGS). BL21(DE3) cells
harboring the modified pET15b plasmid containing the Rab5 insert were
grown at 37 °C in 2× YT medium (16 g of Bacto tryptone, 10 g of
Bacto yeast extract, and 5 g of sodium chloride liter of water)
induced at an A600 of ~0.6 by the addition of
1 mM
isopropyl-1-thio- Crystallization and Data Collection--
The GppNHp-bound form
of Rab5C was prepared as described for Rab3A (1). Crystals of the
Rab5-GppNHp complex were grown at 4 °C by vapor diffusion in hanging
drops containing 10% polyethylene glycol-6000, 50 mM MES, pH 6.0, 0.2 M NaCl, 0.5 mM
MgCl2, and 0.1% 2-mercaptoethanol. Single crystals
appeared overnight and grew to maximum dimensions of 0.3 × 0.3 × 0.2 after several days. The crystals are in the primitive
orthorhombic space group P212121 with cell constants a = 35.9 Å, b = 64.0 Å, and c = 65.9 Å. The volume of the unit cell
is consistent with one molecule in the asymmetric unit and a solvent
content of 35%. Crystals were soaked for 5 min at 4 °C in a
cryoprotectant-stabilizer solution (30% polyethylene glycol-6000 and
20% glycerol) prior to flash freezing in a nitrogen cryostream. A
native data set complete to 1.8 Å was collected on a Rigaku RUH3R/Mar
30-cm image plate detector equipped with focusing mirrors (Charles
Supper). The crystal was maintained at 100 K using a nitrogen
cryostream (Oxford Cryosystems). All data were processed with Denzo and
scaled with Scalepack (32).
Structure Determination and Refinement--
The structure of
GppNHp-bound Rab5C was solved by molecular replacement using a
polyalanine search model derived from the coordinates of the Rab3A
structure (1). A translation search that included the top 50 rotation function solutions yielded a unique solution with an
R value of 50.5% after rigid body refinement against
data from 8 to 3 Å. Initial difference maps calculated with Sigma A
weights to reduce model bias revealed poor density for the
putative switch I and II regions and several loop regions. Multiple
rounds of simulated annealing and manual fitting were interleaved with
gradual extension of the resolution to 1.8 Å. Manual fitting of the
omitted regions and placement of the side chains were followed by
additional rounds of simulated annealing, positional, and B factor
refinement. The final refined model includes residues 19-182, one
molecule of GppNHp, a Mg2+ ion, and 153 ordered water
molecules and has a crystallographic R value of 0.198 and a
free R value of 0.246 based on a 5% subset of reflections
randomly omitted prior to refinement. Molecular replacement was
conducted with AMORE as implemented in CCP4, refinement with X-PLOR,
and interactive model building with O (33-36). Structural images
were generated with MOLSCRIPT or GRASP, combined with GL RENDER (provided by Dr. L. Esser), and rendered with RASTER 3D
(37-40).
Other Crystallographic Models--
For comparison with Rab5C,
models for Rab3A, Ypt51, Rab6, and the Rab3A-Rabphilin3A complex were
rendered from the coordinates of the corresponding crystal structures
(PDB ID codes 3RAB, 1EK0, 1D5C, and 1ZBD, respectively).
Overall Structure and Comparison with Other Rab GTPases--
The
GTPase domain of Rab5C bound to GppNHp was crystallized, and the
structure was determined by molecular replacement (Table I). The final refined model, which
includes residues 19-182, one molecule of GppNHp, a Mg2+
ion, and 153 ordered water molecules, has a working R value
of 0.198 and a free R value of 0.246. The stereochemistry is
excellent, and there are no backbone torsion angles outside the allowed
regions of the Ramachandran plot. Residues 16-18 at the N terminus,
residues 65-69 in the
Like other members of the GTPase superfamily, Rab5C possesses a
characteristic nucleotide binding fold consisting of a six-stranded
Although the overall fold of Rab5C resembles that of Rab3A, significant
structural differences are evident throughout the GTPase domain (Fig.
2). The structures are most similar in
the regions in contact with the nucleotide, whereas the largest
differences occur in or adjacent to regions implicated in the
interaction with effectors and/or regulatory factors. The GTPase
domains of Rab5C and Rab3A share 37% sequence identity overall;
however, the homology within the switch regions is considerably higher. For example, the switch II regions of Rab5C and Rab3A are 63% identical, and substitutions either are conservative or occur at
exposed positions. Moreover, the majority of intramolecular interactions with the switch II region involves residues from the
highly conserved RabF motifs. Interestingly, the magnitude of the
displacement of C Structural Variability in the Rab CDRs--
Although the large
structural differences in the variable Rab CDRs are due in part to the
inherent disorder in the absence of interactions with effectors,
critical changes can be attributed to specific sequence determinants.
For example, proline residues from a PXXXP motif in Rab5
result in an Structural Rearrangement of an Invariant Hydrophobic Triad at the
Switch Region Interface--
As shown in Fig.
3, A and B, a triad
of invariant hydrophobic residues encoded by three of the
characteristic RabF motifs undergoes a dramatic structural
rearrangement. In Rab5, the aromatic ring of Tyr-90 in the switch II
region is buried in the hydrophobic core formed between the switch
regions and the central
Because of crystal contacts involving the hydrophobic surfaces at the
switch interface, it is reasonable to question whether the observed
active conformations might be influenced by crystal packing or by their
engagement with effectors. Several lines of evidence indicate that the
structural plasticity of the invariant hydrophobic triad is not a
crystallographic artifact but rather an inherent property of Rab
GTPases. First, the hydrophobic triad in the independently determined
structure of the yeast Rab5 homologue Ypt51 (16) adopts a nearly
identical conformation despite crystallizing in a different space group
(Fig. 3C). This observation eliminates crystal packing as a
likely explanation. Second, the conformation of the invariant
hydrophobic triad in Rab3A is not altered by the interaction with
Rabphilin3A (2), indicating that these residues are preoriented (Fig.
3C). Consistent with this observation, the side chains of
the invariant triad are well ordered as a result of intramolecular
packing constraints (Fig. 3D). Finally, as described below,
a concerted set of nonconservative substitutions in the hydrophobic
core between the switch regions and the central Sequence Determinants of the Active Conformation--
As
illustrated in Fig. 4, exocytic Rab
GTPases conserve either valine or isoleucine at the equivalent of
position 57 in Rab3A, whereas a subset of endocytic Rab GTPases (Rab5,
Rab7, Rab20, Rab21, Rab22, and Rab24) substitutes residues with small
side chains (alanine or glycine). In yeast, the distinction is
absolute; all exocytic Rab homologues conserve an isoleucine or valine, and all endocytic Rab homologues conserve an alanine. Although the
alanine to isoleucine substitution in the switch I region represents an
obvious proximal determinant of the observed differences, other
nonconservative substitutions could contribute to the stabilization of
the alternative conformation.
To determine whether the conformation of the invariant hydrophobic
triad is likely to vary from one Rab to another or simply reflects two
general arrangements, we have systematically analyzed each position in
or adjacent to the switch regions. Residues were segregated into three
groups based on the sequences of all known Rab GTPases and the
available structural data for the active conformations of Rab3A and
Rab5. The first group includes invariant residues as well as residues
that are conservatively substituted. Although these residues are
not determinants of the active conformation, it is nevertheless clear
that structural rearrangements involving conserved residues
(e.g. the invariant hydrophobic triad) can mediate and
amplify the effects of adjacent nonconservative substitutions. The
second group consists of nonconservative substitutions of residues that
occupy solvent-exposed positions. Such substitutions could well
contribute to the conventional mechanism of specificity determination;
however, the effects are not expected to propagate beyond the immediate
vicinity. The third group corresponds to nonconservative substitutions
of residues that are substantially buried in either the Rab3A or Rab5
structures. The effects of such substitutions will necessarily
propagate to surrounding regions and thus have the potential to be key
conformational determinants of specificity. A surprising number of
nonconservative substitutions occur at interior positions. As shown in
Fig. 5, the majority of such
substitutions involve a cluster of hydrophobic residues situated in the
hydrophobic core formed between the switch regions, the Structural Plasticity in the Switch Regions Is a Determinant of
Effector Recognition--
As shown in Fig.
6, the invariant hydrophobic triad
represents a major site of interaction between Rabphilin3A and the
switch regions of Rab3A (2). The protruding aromatic side chains of Phe-59 and Tyr-91 engage complementary hydrophobic surfaces in Rabphilin3A. Because of the structural rearrangement described above,
the corresponding surface in Rab5 is noncomplementary to the switch
interaction epitope of Rabphilin3A. These observations clearly
demonstrate that structural plasticity involving a partially exposed
invariant hydrophobic triad contributes to the specificity of
Rab-effector interactions. Thus, in addition to signaling the state of
the bound nucleotide, the active conformation of the switch regions
conveys information about the subfamily of a particular Rab GTPase.
A General Model for Activation of Rab GTPases--
The
recent crystal structure of a GDP-bound Rab6 homologue provides the
opportunity to assess whether the structural changes accompanying
activation are likely to influence the conformation of the invariant
hydrophobic triad. Interestingly, the invariant hydrophobic triad in
GDP-bound Rab6 adopts a conformation distinct from that of the active
forms of either Rab3A or Rab5 (Fig. 7). In particular, the conformation determining isoleucine in the switch I
region and invariant tyrosine in the switch II region are displaced
relative to their counterparts in Rab3A and Rab5. Although the main
chain atoms of the invariant tryptophan and phenylalanine are not
substantially displaced, the aromatic side chains of these residues
adopt different rotomer conformations. Moreover, crystallographic and
NMR studies of a large number of GTP-binding proteins support the
general conclusion that the switch regions are highly flexible in the
GDP-bound form but adopt specific ordered conformations in the
GTP-bound form because of the additional hydrogen bonding interactions
with the Given the large number of Rab GTPases, it is plausible that the
structural plasticity of the invariant hydrophobic triad evolved primarily as a mechanism to augment other specificity-determining interactions with the Rab CDRs. In addition to the obvious implications with respect to effector interactions, differences in the active conformation of the invariant hydrophobic triad could potentially contribute to the specificity of interactions with Rab GAPs (42-47). On the other hand, a comparison of the GppNHp-bound Rab5C and Rab3A
structures with the GDP-bound Rab6 structure suggests that the
constraints on the conformation of the invariant hydrophobic triad are
engaged only in the active form (1, 16). Consequently, the conformation
of the invariant hydrophobic triad is unlikely to affect the
specificity of interactions with GDI, guanine nucleotide exchange
factors, GDI displacement factors, or other factors that interact with
the GDP-bound form (12, 48-57). Indeed, one or more residues from the
invariant hydrophobic triad could well contribute to the interaction
with Rab GDI, which recognizes the GDP-bound conformation of all Rab
GTPases (12).
The use of invariant hydrophobic residues in the switch regions as
nucleotide-dependent recognition determinants appears to be
unique to the Rab family. For example, invariant hydrophobic residues
in the switch II region of heterotrimeric G protein When compared with other Rab family GTPases, the Rab5 structure
provides compelling evidence for an evolutionarily conserved mechanism
of effector recognition in which the structural plasticity of an
invariant hydrophobic triad at the switch region interface is coupled
to nonconservative substitutions in the hydrophobic core. The
conformation of the invariant hydrophobic triad reflects the state of
the bound nucleotide as well as the Rab subfamily identity and thus
appears to be an integral component of the switching mechanism.
Although the extent to which Rab effectors other than Rabphilin3A will
interact with the invariant hydrophobic triad remains to be established
experimentally, the presence of a substantial nucleotide-dependent hydrophobic surface adjacent to the
Rab CDRs suggests the possibility of a general role with respect to
activation and effector recognition.
sheet and the
2 helix. These observations demonstrate
that structural plasticity involving an invariant hydrophobic triad at
the switch interface contributes to the mechanism by which effectors
recognize distinct Rab subfamilies. Thus, the active conformation of
the switch regions conveys information about the identity of a
particular Rab GTPase as well as the state of the bound nucleotide.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3/
5 loop, that play an important role in
determining functional specificity (10, 11). However, interactions
involving hypervariable regions cannot explain the ability of Rab
GDI1 (GDP
dissociation inhibitor) to recognize most or
all Rab GTPases yet still discriminate against other GTPase families or
the ability of certain regulatory factors to recognize particular Rab
subfamilies (12-15). These observations imply the existence of
specificity determinants that are common to all Rab GTPases but not
other GTPase families, as well as determinants that are conserved only within particular Rab subfamilies.
phosphate of GTP, mediated by serine residues in the P-loop and switch
I region, as well as an extensive hydrophobic interface between the
switch I and II regions (1, 16). The structure of the complex between a
constitutively active mutant of Rab3A and a putative effector,
Rabphilin3A, revealed an interaction epitope that extends from the
relatively conserved switch interface to an adjacent pocket formed by
three hypervariable "complementary determining regions" (CDRs)
corresponding to the N and C termini of the GTPase domain and the
3/
5 loop (2). A triad of invariant hydrophobic residues at the
switch I/switch II interface of Rab3A mediates a central interaction
with Rabphilin3A. In a recent analysis of the primary structure of all
known Rab GTPases, five distinctive motifs (known as the Rab family
motifs or RabFs) were identified that distinguish Rab proteins from
other GTPase families (17). The RabF motifs are located either in the
putative switch I and II regions or in the adjacent
3 and
4
strands. It was also noted that the Rab family could be further
subdivided based on the presence of four subfamily motifs, which
include all three Rab CDRs as well as a region corresponding to the
1 helix and most of the
1/
2 loop.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside, and
harvested after 3 h at 37 °C. The cell pellet was resuspended in 50 mM Tris, pH 8.5, and 0.1% 2-mercaptoethanol, lysed
by sonication, and was centrifuged at 35,000 × g for
1 h. The supernatant was loaded onto a nickel-nitrilotriacetic
acid-agarose column (Qiagen). After washing with 11 column
volumes of 50 mM Tris, pH 8.5, 500 mM NaCl, 10 mM imidazole, and 0.1% 2-mercaptoethanol, the fusion protein was eluted with a gradient of 10-500 mM imidazole.
The protein was further purified by ion exchange chromatography on Resource Q and Resource S (Amersham Pharmacia Biotech) followed by gel filtration on Superdex-75 (Amersham Pharmacia Biotech). Roughly
30 mg of >99% pure protein were obtained from a 6-liter culture.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2/
3 loop, and residues 183-186 at the C
terminus are poorly ordered.
Structure determination and refinement
sheet surrounded by five
helices (Fig.
1). In the absence of a structure for the
GDP-bound form of Rab5C, a precise definition of the conformational
switch regions is not possible. However, a comparison of the
GppNHp-bound structure of Rab3A with that of the GDP-bound form of Rab6
from the malaria parasite Plasmodium falciparum suggests
that the conformational changes accompanying nucleotide exchange will
be localized to regions analogous to those in p21ras (41). As a
working hypothesis, we will assume that the conformational changes in
Rab5 are localized to the corresponding regions, although the precise
nature and extent of the conformational changes will not alter the
observations described below. To facilitate discussion, we will also
refer to the Rab CDRs as defined for Rab3A based on the complex with
Rabphilin3A (2). Whether these and/or other variable regions mediate
effector interactions with Rab5C remains to be determined.
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Fig. 1.
Overall structure of Rab5C.
Ribbon representation of the Rab5C structure with the putative switch
regions highlighted in purple, the regions
corresponding to the CDRs of Rab3A highlighted in
orange, and the nucleotide highlighted in
green.
atoms does not strictly correlate with sequence
variability. Indeed, the displacements in the relatively conserved
switch II region are considerably larger than the overall root-mean-square deviation and comparable with the displacements in the poorly conserved
4 helix. Moreover, the largest main chain rearrangement within the switch II region is centered on an invariant Tyr residue in one of the highly conserved RabF motifs. The origin and
functional consequences of these changes with respect to effector recognition are considered below.
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Fig. 2.
Comparison of the active conformations of
endocytic and exocytic Rab GTPases. A, superposition of
the Rab5C structure (blue) with the structures of Rab3A
alone (purple), the yeast Rab5 homologue Ypt51
(semitransparent blue), and Rab3A from the complex with
Rabphilin3A (semitransparent purple). The superposition is
based on C atoms. Models for Rab3A alone (1), Ypt51 (16), and Rab3A
from the Rab3A-Rabphilin3A complex (2) were derived from the
coordinates of the corresponding crystal structures. B,
distance between pairs of C
atoms in the Rab5C and Rab3A structures
following the superposition. Also shown is the secondary structure with
the putative switch regions highlighted in
magenta and the CDRs of Rab3A highlighted in
orange.
5 helix that is truncated by two turns relative to
Rab3A. The additional turns of the
5 helix in Rab3A comprise an
essential element of the binding pocket for a C-terminal hydrophobic
motif in Rabphilin3A (2). The differences in backbone conformation are
further augmented by nonconservative substitutions in which hydrophobic
residues in Rab3A are substituted for charged or polar residues in
Rab5. Thus, the specificity of effector interactions with the Rab CDRs
is determined by backbone conformational constraints as well as the stereochemical and electrostatic complementarity of the relevant molecular surfaces. These specificity determinants are strongly correlated with a high degree of variability in the sequences comprising the Rab CDRs.
sheet, whereas the aromatic ring of Phe-58
in the
2 strand is partially buried, leaving the indole ring of
Trp-75 in the
3 strand significantly exposed. In this packing
arrangement, the aromatic rings of Trp-75 and Tyr-90 lie in a van der
Waals contact with the methyl group of Ala-56 in the switch I region.
Consequently, the conformation adopted by the invariant hydrophobic
triad in Rab5 requires a residue with a small side chain at position
56. In Rab3A, a nonconservative alanine to isoleucine substitution at
the corresponding position displaces the aromatic side chains of Tyr-91
and Trp-76. The displacement of Trp-76 in turn forces the side chain of
Phe-59 to adopt an alternative rotomer conformation. Thus, the aromatic
side chains of Tyr-91 and Phe-59 in Rab3A protrude from either side of
the indole ring of Trp-76, which is considerably more buried than its
counterpart in Rab5. Despite the dramatic conformational rearrangement of the invariant hydrophobic triad, the alternative packing arrangement in Rab5 preserves the hydrophobic interface between the switch regions.
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Fig. 3.
Structural rearrangement of an invariant
hydrophobic triad at the switch region interface. A,
semitransparent molecular surface of Rab5C in the vicinity of the
invariant hydrophobic triad (Phe-58, Trp-75, and Tyr-90). B,
semitransparent molecular surface of Rab3A (1) in the vicinity of the
invariant hydrophobic triad (Phe-59, Trp-76, and Tyr-91). C,
overlay following the superposition of C atoms in the independently
determined active structures of Rab5C (blue and
orange), Ypt51 (semitransparent blue and
orange), Rab3A alone (gray and
purple), and Rab3A from the complex with Rabphilin3A
(semitransparent gray and purple). Models for
Rab3A alone (1), Ypt51 (16), and Rab3A from the Rab3A-Rabphilin3A
complex (2) were derived from the coordinates of the corresponding
crystal structures. D,
A-weighted 2Fo
Fc map countered at 1.2
showing residues in
the vicinity of the invariant hydrophobic triad.
sheet provides a
simple and plausible explanation for the observed structural rearrangement.
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Fig. 4.
Structure-based sequence alignment of
representative endocytic and exocytic Rab GTPases. Invariant
hydrophobic residues with significantly altered conformations are shown
in purple, and the corresponding conformation determining
residues are shown in green. Rab3A residues in the interface
with Rabphilin3A are shown in blue (2). Residues that are
highly conserved in the Rab GTPase family are indicated in
boldface type. Residue numbering and secondary structure
correspond to Rab5C. The complementary determining regions of Rab3A are
highlighted in orange. The conformational switch
regions defined by the comparison of GppNHp-bound Rab3A and GDP-bound
Rab6 are highlighted in purple. The
horizontal lines below the sequences denote the
RabF and Rab subfamily (RabSF) regions (17).
1-
4
strands and the
3 helix. Substitutions at these positions are
concerted and, to a first approximation, they can be correlated with
the overall similarity in the GTPase domains of the previously identified Rab subfamilies (17).
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Fig. 5.
Location of nonconservative substitutions
predicted to influence the active conformation of invariant residues in
the switch regions. A, Rab5. B, Rab3A from
the GppNHp-bound structure (1). Variable residues are
highlighted in green, and invariant residues are
highlighted in purple. The view is from the
interior of the protein.
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Fig. 6.
Complementarity of the invariant hydrophobic
triad in Rab3A and Rab5C with the switch region interaction epitope of
Rabphilin3A. A, molecular surface of Rabphilin3A in
contact with the switch regions of Rab3A (semitransparent
gray and purple). B, hypothetical
Rab5C-Rabphilin3A interface following the superposition of Rab5C
(semitransparent blue and orange) with Rab3A.
Note that the alternative conformation of the invariant hydrophobic
triad in Rab5C lacks complementarity with Rabphilin3A. The Rabphilin3A
and Rab3A models in this figure were generated from the coordinates of
the Rab3A-Raphilin3A structure (2).
phosphate (9). These observations suggest a simple
structural model for the activation of Rab GTPases, leading to distinct
active conformations for each Rab subfamily. Upon GTP binding, an
extensive hydrophobic interface forms between the switch I and II
regions. The formation of this interface engages the aromatic side
chains of the invariant hydrophobic triad in a Rab subfamily-specific
conformation determined by van der Waals interactions with the critical
conformation determining residues in the hydrophobic core. Finally, the
presence of a substantial exposed hydrophobic patch adjacent to the Rab
CDRs suggests that the nucleotide-dependent engagement of
the invariant hydrophobic triad could play a general role in Rab
activation and effector recognition.
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Fig. 7.
Distinct conformations for the invariant
hydrophobic triad in the active and inactive forms of Rab GTPases.
Overlay following the superposition of C atoms in GppNHp-bound Rab3A
(blue and purple) and GDP-bound Rab6
(gray and orange). The Rab3A and Rab6 models in
this figure were generated from the coordinates of the GppNHp-bound
Rab3A (1) and GDP-bound Rab6 structures (16).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits engage
hydrophobic residues in the
3 helix with little or no difference in
conformation in the crystal structures of the active forms of
Gt
, Gi
, and Gs
(58-60).
Interestingly, however, the specificity of the Gs
interaction with adenylyl cyclase is primarily attributable to a
conformational rearrangement in the
3/
5 loop (compared with
Gi
, which does not interact with the same site on
adenylyl cyclase) rather than differences in the composition of
residues in the effector interaction epitope (60). Thus, in both Rab
GTPases and G
subunits, the conformational plasticity of
invariant residues provides a structural mechanism that propagates and
amplifies the effects of key nonconservative amino acid substitutions.
It is likely that similar structural mechanisms of specificity
determination have evolved in other large protein or domain families.
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ACKNOWLEDGEMENTS |
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We thank Silvia Corvera, Michael Czech, and Jennifer O'Neil for comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM56324 (to D. 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 1HUQ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
National Institutes of Health postdoctoral fellow.
§ A Leukemia and Lymphoma Society Scholar. To whom correspondence should be addressed: Program in Molecular Medicine, Two Biotech, 373 Plantation St., Worcester, MA 01605. Tel.: 508-856-6876; Fax: 508-856-4289; E-mail: David.Lambright@umassmed.edu.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M009771200
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ABBREVIATIONS |
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The abbreviations used are: GDI, GDP dissociation inhibitor; CDR, complementary determining region; RabF, Rab family; MES, 4-morpholineethanesulfonic acid.
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