(Received for publication, April 17, 1995; and in revised form, May 24, 1995)
From the
To gain further insight into structural elements involved in
Rab5 function, differences in the intrinsic tryptophan fluorescence of
the GDP- and guanosine 5`-O-(3-thiotriphosphate)
(GTPS)-bound forms of the protein were examined. When excited at
290 nm, Rab5 displays emission maxima at 339.7 nm for the GDP-bound and
336.7 nm for the GTP
S-bound forms. The tryptophan fluorescence
intensity is quenched by
25% in the GTP
S-bound form relative
to the GDP-bound conformation. Variant Rab5 molecules were created by
site-directed mutagenesis to convert the protein's two
tryptophans to phenylalanine residues. Fluorescence studies reveal that
the observed changes upon GDP/GTP
S exchange are due to a blue
shift in the emission spectra for both Trp
(342.0 to 339.5
nm) and Trp
(335.3 to 333.7 nm) and fluorescence
quenching of Trp
. Consistent with the blue shift in the
emission spectra, both tryptophans are more resistant to oxidation by N-bromosuccinimide in the GTP
S-bound state. These data
indicate that both of Rab5's tryptophans are brought into a more
sequestered, hydrophobic environment upon conformational changes
promoted by guanine nucleotide exchange. Since Trp
lies
adjacent to Rab5's cognate switch II domain, local conformational
changes would be predicted based on the known structure of Ras.
However, Trp
lies within a region of Rab5 potentially
related to the switch III domain unique to heterotrimeric
G
. Thus, changes in the fluorescence properties of
Trp
upon guanine nucleotide exchange suggest that Rab
proteins may have structure-function relationships similar to those
described for heterotrimeric GTP-binding proteins.
Rab proteins are a family of Ras-like small molecular weight
GTP-binding proteins localized to distinct subcellular compartments (1, 2, 3, 4, 5, 6) .
Rabs have been shown to regulate specific steps of intracellular
membrane
trafficking(7, 8, 9, 10, 11, 12) .
One member of the Rab family, Rab5, is localized on plasma membrane,
clathrin-coated vesicles, and early
endosomes(2, 9, 10) . Even though it has been
shown to play an important role in early events of
endocytosis(9, 10, 11, 12) , the
exact mechanism of Rab5 function remains to be determined. The current
model for the GTPase cycle of Rab5 is that a GDP dissociation inhibitor
(GDI) ()delivers the GDP-bound protein to target
membranes(13, 14) , where a guanine nucleotide
exchange factor catalyzes GDP/GTP exchange(14) . After GTP
hydrolysis, GDP-bound Rab5 is retrieved from the membrane by
GDI(13, 15) . Thus, membrane association and
dissociation is correlated with the GTP- and GDP-bound states,
respectively.
The transition between GDP- and GTP-binding states is
a universal molecular switch adopted by many GTP-binding proteins to
regulate a diverse set of biological functions. An understanding of the
structural differences between these guanine nucleotide binding
conformations can help define the basis for interactions with
regulatory molecules like GDI and guanine nucleotide exchange factor.
Comparison of the two guanine nucleotide-bound conformations of Ras and
EF-Tu indicates significant structural changes in two regions: switch I (residues 30-38 in Ras and residues 41-62
in EF-Tu) and switch II (residues 60-76 in Ras and
residues 84-96 in EF-Tu)(16, 17) . Both domains
are on the surface of Ras and EF-Tu and are proposed to interact with
the molecules' accessory proteins. In contrast, studies on
G revealed three switch regions. Besides counterparts
of switch I (residues 173-183) and switch II (residues
195-215), a third region or switch III (residues
227-238) was identified to undergo marked structural
rearrangements and is thought to be unique to heterotrimeric
GTP-binding proteins(18) .
Based on this crystallographic
information, intrinsic tryptophan fluorescence measurements have proven
to be a sensitive means to detect local conformational changes in the
switch regions of G(19, 20) ,
G
(21) , and Ras(22, 23) .
G
and G
both contain two tryptophans,
one of which resides in the molecules' switch II domain. The fact
that fluorescence properties of G
and G
are sensitive to GDP/GTP exchange(19, 20, 21) indicates that these residues can detect conformational
changes within this domain. In contrast, native Ras does not contain
any tryptophan residues, but three altered forms have been studied with
tryptophans introduced at position 28 (immediately upstream of the
switch I domain), position 56 (four amino acids upstream of the switch
II domain), or position 64 (within the switch II domain); each of these
Ras variants displays fluorescence properties sensitive to GDP/GTP
exchange(22, 23) .
Since crystallographic
information is not yet available for Rab5, we have examined the
intrinsic tryptophan fluorescence of the protein to detect structural
changes promoted by guanine nucleotide exchange. Sequence alignment of
Rab5, Ras, and heterotrimeric G shown in Fig. 1indicates that Trp
is located immediately
adjacent to the region that is cognate to the switch II of Ras (16) and G
(18) , whereas
Trp
is within a domain potentially related to the switch
III region of G
(18) . Thus, conformational
rearrangement in both regions of Rab5 should be reported by
Trp
and Trp
, respectively. Our
characterization of the intrinsic tryptophan fluorescence properties of
Rab5 and two altered forms with Trp
Phe substitutions suggests
that the nature of conformational changes promoted by GDP/GTP exchange
may be similar to features observed for heterotrimeric GTP-binding
proteins.
Figure 1:
Sequence
alignment of Ras, G, and Rab5 in the switch II and III
regions. Protein sequences of Rab5, H-Ras, and G
were
aligned using the GCG package, version 7.3 (Genetics Computer Group,
Madison, WI); shown is the region adjacent to the highly conserved
phosphoryl-binding domain present in all GTP-binding proteins as
indicated. Double and single dots represent identical
and semi-conserved amino acids, respectively, determined based on
identities tabulated by Dayhoff et al.(24) . The
switch II domains of Ras and G
and the switch III
region of G
are highlighted. The highlighted regions in Rab5 represent the L4/
2/L5
(predicted fourth loop, second
-helix, fifth loop as switch II)
and
3/L7 (predicted third
-helix, seventh loop as switch III)
domains, adopting the nomenclature of Stenmark et al. (29) .
To examine the fluorescence properties of Rab5, samples of
the protein were loaded in parallel with GDP and GTPS exactly as
described under ``Experimental Procedures,'' and tryptophan
fluorescence emission spectra were recorded. As shown in Fig. 2,
the GTP
S-bound form exhibits a blue shift relative to the
GDP-bound form of the protein (maxima are at 336 and 339 nm,
respectively). Moreover, the fluorescence intensity is markedly
quenched in the GTP
S-bound state. Results obtained in several
identical experiments are summarized in Table 1, with the
fluorescence intensity normalized to that measured for GDP-bound
samples.
Figure 2:
Emission spectra of wild type Rab5 in
GTPS- and GDP-bound states. An aliquot of Rab5
was
rapidly thawed and exchanged into buffer A, and protein concentration
was determined as described under ``Experimental
Procedures.'' Equivalent amounts of protein were removed from this
stock to load with GDP or GTP
S in parallel. Protein (500
nM) was incubated for 2 h at 37 °C in buffer A in the
presence of 20 µM guanine nucleotide (GTP
S or GDP)
and 0.5 mM MgCl
. Fluorescence emission spectra
were recorded at an excitation wavelength of 290 nm. Fluorescence
intensities are shown in arbitrary units and the
values for the GTP
S- and the GDP-bound forms of the protein
are 336 and 339 nm, respectively. The ratio of the peak intensity of
the GTP
S- and the GDP-bound forms is
0.74.
To identify whether the fluorescence properties of one or
both of Rab5's tryptophan residues are affected by guanine
nucleotide exchange, Rab5 and Rab5
were
constructed by site-directed mutagenesis. As shown in Fig. 3and
summarized in Table 1, the emission maxima for the two variant
Rab5 molecules are also blue-shifted when they are bound with
GTP
S, indicating that environmental changes for both Trp
and Trp
must contribute to the blue shift observed
for wild type. The fluorescence intensity is markedly quenched for
Rab5
, indicating that the local environment of
Trp
is altered upon guanine nucleotide exchange. The right panels of Fig. 3compare the differences in
between wild type and the two Trp
Phe
variants by normalizing their intensities to the same level in the GDP-
and GTP
S-bound states. The
of Rab5
is red-shifted while Rab5
is blue-shifted relative
to Rab5
in both guanine nucleotide binding states (see
also Table 1). This difference in the emission maxima between the
two variants indicates that Trp
may be located in a more
non-polar environment than Trp
.
Figure 3:
Emission spectra of Rab5,
Rab5
, and Rab5
. Rab5 proteins were
rapidly thawed and exchanged into buffer A, and protein concentrations
were determined as described under ``Experimental
Procedures.'' For each protein, equivalent aliquots were were
removed from this starting stock to load with GDP or GTP
S in
parallel. Proteins (500 nM) were incubated for 2 h at 37
°C in buffer A in the presence of 20 µM GDP or
GTP
S and 0.5 mM MgCl
. Fluorescence emission
spectra were then recorded as described for Fig. 1. Left
panels show spectra for Rab5
(top) and
Rab5
(bottom) in the GDP- and GTP
S-bound
states as indicated. Right panels present fluorescence spectra
of Rab5
and Rab5
normalized to that of
Rab5
in the GDP-bound (top) and GTP
S-bound (bottom) forms. The
values for the
GDP-bound forms are: Rab5
, 339 nm; Rab5
,
336 nm; Rab5
, 342 nm; and for the GTP
S-bound forms
the
values are: Rab5
, 336 nm;
Rab5
, 334 nm; Rab5
, 340
nm.
It is important to note
that our interpretation of the fluorescence data is based on the
assumption that the Trp Phe conversions do not alter the Rab
protein's structure. The two independent Trp
Phe
substitutions do not functionally impair guanine nucleotide binding
capacity as demonstrated by overlay blots of
[
P]GTP binding (data not shown); however, we
cannot exclude the possibility for minor conformational alterations
that might contribute to the fluorescence properties of each variant.
For example, data to suggest that Trp
's environment
is more hydrophobic relative to that of Trp
could simply
reflect subtle conformational differences in the altered protein(s).
Nonetheless, our conclusion that both regions of Rab5 undergo
conformational changes upon guanine nucleotide exchange is strongly
supported by the spectral differences between the GDP- and
GTP
S-bound forms observed for both Rab5
and
Rab5
, as well as Rab5
. A second caveat to
the interpretation of our data involves the possibility of
ligand-induced aggregation contributing to changes in the
proteins' fluorescence properties. To examine potential artifacts
caused by ligand binding, light scattering peak intensities were
determined for each protein in the GDP- and GTP
S-bound states.
Because significant differences were not detected and no obvious trend
was apparent with respect to GTP
S binding (data not shown), we
conclude that the formation of large protein aggregates does not occur.
Although lower order ligand-induced oligomerization cannot be excluded,
biochemical characterization of Rab5 (11) and other Rab
proteins (28) has yet to reveal functional evidence to suggest
this possibility.
To provide further support for our conclusions,
the sensitivity of Rab5's tryptophans to NBS oxidation was
measured. Since highly accessible residues typically are located on the
protein surface or in a hydrophilic pocket, resistance to NBS oxidation
indicates sequestration of a tryptophan within a more buried,
hydrophobic environment. Fig. 4shows the dose-dependent loss of
tryptophan fluorescence as a function of the molar ratio of NBS to
protein. A measurable increase in resistance to oxidation was observed
for the GTPS-bound form relative to the GDP-bound structure of
each protein, consistent with the blue shift of the fluorescence
emission maxima upon GTP
S binding (Table 1). Although it is
possible that modification of other amino acids by NBS at high
concentrations might cause conformational effects to influence the
intrinsic tryptophan fluorescence, we presume these alterations would
have an equivalent contribution to measurements made for the GDP- and
GTP
S-bound forms of each protein. If this assumption is valid,
then the NBS oxidation results indicate that upon GDP/GTP
S
exchange, both tryptophans are sequestered into more nonpolar
surroundings.
Figure 4:
NBS
oxidation of Rab5, Rab5
, and
Rab5
. Rab5 proteins were exchanged into buffer A;
equivalent samples were removed and then loaded in parallel with 20
µM GDP (open symbols) or GTP
S (filled
symbols) as described for Fig. 3. NBS, freshly prepared in
50 mM Tris acetate, pH 5.0, was then added to samples at the
indicated molar ratios, and oxidation reactions were carried out as
described under ``Experimental Procedures.'' The fluorescence
intensity of each sample was converted to a percentage of the initial
fluorescence intensity (no NBS added) and plotted against the ratio of
NBS/protein. circles, Rab5
; squares,
Rab5
; triangles,
Rab5
.
Based on the results of our fluorescence experiments, we
conclude that Rab5 undergoes conformational changes upon guanine
nucleotide exchange in two regions of the molecule as reported by
spectral differences noted for Trp and Trp
.
It is anticipated that the local environment of Trp
should
be altered upon GTP
S binding due to its proximity to Rab5's
cognate switch II domain. This prediction is based on the
conformational changes noted in crystal structures of Ras(16) ,
EF-Tu(17) , and G
(18) . However, the
guanine nucleotide-dependent fluorescence properties of Trp
are more informative. The sequence alignment of Fig. 1reveals that Trp
is in a domain of Rab5
potentially related to the flexible switch III region of
G
. In fact, molecular modeling by Stenmark et al.(29) predicts nucleotide-dependent conformational changes
in three structural elements of Rab5 upon GDP/GTP exchange, including
L2 (the predicted switch I or effector loop), L4/
2/L5 (cognate to
switch II), and
3/L7 (Fig. 1). The fluorescence
characteristics we observe for Trp
provide further
experimental evidence demonstrating conformational changes in the
3/L7 region of Rab5 upon GTP
S binding.
Switch III of
G is a loop domain that detects molecular changes in
switch II primarily via ionic interactions (18) . In the
GTP
S-bound state, switch II and III become juxtaposed to close a
cavity in G
's tertiary structure. While the
3/L7 domain of Rab5 lacks the charged residues highly conserved
among G protein
-subunits, it may nonetheless functionally mimic
the switch III domain to swing closer to L4/
2/L5 upon GTP
S
binding, thereby occluding a cleft in Rab5's tertiary structure.
It is important to note that
3/L7 corresponds to domains in Ras
and EF-Tu, which are also rearranged in response to molecular changes
in the switch II domain of these GTP-binding proteins (16, 17) (for review, see (30) ). Sequence and
structure alignment by Lambright et al.(18) place the
latter domains immediately adjacent to the switch III loop of
G
.
Our prediction that 3/L7 fulfills a
``switch III-like'' function is based on the fluorescence
properties of Rab5, which reveal environmental changes for both
Trp
and Trp
upon GTP
S binding. We
envision that in the GTP
S-bound state, L4/
2/L5 and
3/L7
may closely align with one another, mimicking structural changes
observed for switch II and switch III domains of
G
(18) . This conformational change would
create a hydrophobic pocket within which Rab5's tryptophans
become buried. The latter idea is supported by the blue shift in
observed for both Trp
and Trp
in the GTP
S-bound state. The fact that these residues become
resistant to NBS oxidation upon GDP/GTP
S exchange is a further
indication that both tryptophans become sequestered.
Compelling
functional similarities between heterotrimeric G protein -subunits
and Rab proteins support the structural rearrangement of ``switch
II'' and ``switch III-like'' domains in Rab5. In the
GDP-bound conformation, both switch II and III domains of G
are solvent-exposed, lining a cavity that is thought to serve as
a binding site for G
(18) . This
heterotrimeric complex associates with rhodopsin and, upon activation,
the receptor catalyzes GDP/GTP exchange and the dissociation of
G
from G
. Hence, the closure of
the switch II and III domains is thought to result in dissociation of
the G
subunit from G
. By analogy,
we theorize the functional counterpart of G
,
guanine nucleotide dissociation inhibitor (GDI), may interact with Rab
proteins in a similar fashion. It is well established that GDI binds to
the prenylated GDP-bound forms of Rab proteins(31) . Bound GDI
strongly inhibits GDP release(32, 33) and delivers
the Rab proteins to the correct membrane
compartment(14, 34, 35, 36) . GDI
dissociates upon catalyzed GDP/GTP exchange of the Rabs at the membrane
surface (14, 35) but will retrieve the GDP-bound
proteins from the membrane after GTP
hydrolysis(37, 38) .
Given the nature of the strong
functional relationships between Rab/GDI and
G/G
, the ``switch II''
and ``switch III'' domains of GDP-bound Rab5, as identified
by Trp
and Trp
, are likely to provide a
binding pocket for GDI. This hypothesis is consistent with functional
evidence indicating that both L4/
2/L5 and
3/L7 domains of
Rab5 are required for membrane association(28) . Our model also
is in agreement with the proposition that equivalent structural domains
in GDP-bound Ras may interact with signaling molecules that become
activated, much like G
, only after GDP/GTP
exchange(29) . Since among the Ras superfamily, only Rab, Rho,
and Rac family members are known to interact with
GDIs(31, 32, 39, 40, 41) ,
it is interesting to further speculate that all of these GTP-binding
proteins may display structure-function relationships similar in nature
to those of heterotrimeric G proteins.