From the Department of Biochemistry, University of
Cambridge, 80, Tennis Court Road, Cambridge CB2 1GA, United Kingdom and
the ¶ Institute Curie, 26, Rue d'Ulm,
75248 Paris Cedex 05, France
Received for publication, January 7, 2003, and in revised form, March 6, 2003
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ABSTRACT |
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The exocyst complex is involved in the final
stages of exocytosis, when vesicles are targeted to the plasma membrane
and dock. The regulation of exocytosis is vital for a number of
processes, for example, cell polarity, embryogenesis, and neuronal
growth formation. Regulation of the exocyst complex in mammals was
recently shown to be dependent upon binding of the small G protein,
Ral, to Sec5, a central component of the exocyst. This interaction is
thought to be necessary for anchoring the exocyst to secretory vesicles. We have determined the structure of the Ral-binding domain of
Sec5 and shown that it adopts a fold that has not been observed in a G
protein effector before. This fold belongs to the immunoglobulin
superfamily in a subclass known as IPT domains. We have mapped the Ral
binding site on this domain and found that it overlaps with
protein-protein interaction sites on other IPT domains but that it is
completely different from the G protein-geranyl-geranyl interaction face of the Ig-like domain of the Rho guanine
nucleotide dissociation inhibitor. This mapping, along with
available site-directed mutagenesis data, allows us to predict how
Ral and Sec5 may interact.
Spatial regulation of exocytosis is crucial for a variety of
cellular processes, including embryogenesis, establishment and maintenance of cell polarity, and neuronal growth cone formation (reviewed in Ref. 1). The exocyst complex is involved in the final
stage of exocytosis, when post-Golgi vesicles are targeted and dock to
the plasma membrane. The exocyst consists of an assembly of eight
proteins: Sec3, -5, -6, -8, -10, and 15, and Exo70 and -84, which form
a complex localized to sites of vesicle docking to the plasma membrane
during exocytosis.
In Saccharomyces cerevisiae the exocyst directs vectorial
targeting of secretory vesicles to sites of membrane expansion, such as
bud sites. It appears that Sec3p, which is always localized to the
plasma membrane, forms a targeting patch and is the spatial landmark
for polarized exocytosis (reviewed in Ref. 1). A subcomplex of Sec15p
and Sec10p is localized to secretory vesicles (2) and a network of
protein-protein interactions among the exocyst components bridges
Sec15p-Sec10p to the targeting patch made by Sec3p (3).
In mammals the exocyst is involved in the targeting of Golgi-derived
vesicles to the basolateral membrane of polarized epithelia. The
regulation of the exocyst seems to be somewhat different in higher
eukaryotes, because Sec3 does not have the same role. Rather, in
polarized epithelial cells, Exo70 is localized to the plasma membrane
(4), whereas the other components of the exocyst remain cytosolic,
implying that exocyst assembly at the membrane is dependent upon Exo70,
rather than Sec3 as in yeast.
Small GTPases of the Ras superfamily are involved in the regulation of
a variety of cellular processes, including growth, differentiation,
actin cytoskeleton, nuclear transport, and vesicle transport. Because
many of these processes involve exocytosis, it was likely that small
GTPases could play a role in exocyst regulation (5). In yeast, Sec4p, a
homologue of Rab3A GTPase, anchors Sec15p to secretory vesicles (2).
The exocyst is anchored to the plasma membrane via the interaction of
Sec3p with another GTPase, Rho1p (6). Sec3p has also been shown to bind
to Cdc42, another member of the Rho family (7). Finally, it was shown that Exo70p interacts with Rho3p at the plasma membrane (8). The role
of the Rho family GTPases, which regulate the actin cytoskeleton, in
exocyst regulation implies a coordination of cytoskeletal changes with exocytosis.
In mammalian cells, the exocyst components do not seem to interact with
Rab3A or Rho family members. Rather, it was recently shown that the
exocyst is regulated by yet another GTPase, RalA (9-12). Ral is a Ras
family small G protein that is not present in S. cerevisiae.
Both activated Ral and Ral inhibition disrupt polarized exocytosis in
epithelial cells, suggesting it is necessary for the GTPase to cycle
between the GDP- and GTP-bound forms to direct vesicle movement. Ral,
like Rab3A, is localized to secretory vesicles and the plasma membrane,
but the exocyst component responsible for interacting with RalA was
found to be Sec5. In yeast, Sec5p is at the center of the
exocyst complex, linking the Sec10p-Sec15p subcomplex to the rest of
the exocyst via its interactions with Exo70p, Sec3p, and Sec6p (3).
Thus, Sec5 may have a different role in the mammalian exocyst.
Ral small GTPases are members of the Ras superfamily of small G
proteins implicated in oncogenesis, endocytosis, actin dynamics, and
membrane trafficking. Downstream effector proteins identified for Ral
include Ral BP11 (or
RLIP-76), which mediates the effects of Ral on endocytosis (13, 14) and
interactions with the Rho family GTPases (15), filamin, an actin
filament cross-linking protein, and phospholipase D1, which is involved
in vesicle trafficking (16). The discovery that Ral is involved in
regulation of exocytosis provides a link between secretory and
cytoskeletal pathways.
The exocyst was found to bind specifically to GTP-bound RalA (11). One
region of small G proteins that is sensitive to the state of the bound
nucleotide is the effector loop, which interacts with downstream
effectors. The effector loop mutant D49E does not bind to Sec5 and has
been shown to disrupt transport of proteins to the basolateral surface
in polarized epithelial cells (9).
The region of Sec5 responsible for Ral binding was identified and
comprises the first 80 amino acids (10). The first 95 residues of Sec5
contain a putative domain, the IPT (17), which is found in some
cell-surface receptors such as Met and Ron and in intracellular
transcription factors, e.g. NF- We have solved the structure of the Ral-binding domain of Sec5 using
solution NMR techniques and find that it forms an IPT fold. We have
mapped the binding of Ral·GMPPNP to this domain and found that the
surface of Sec5 that interacts with Ral is similar to that used in
other IPT domains for protein-protein interactions. Furthermore, this
surface is different from that used in the Rho GDI Ig domain for
contacting the Rho family proteins and their geranyl-geranyl
moiety. How Ral may bind to the IPT domain of Sec5 will be
discussed and compared with other G protein-effector interactions.
Protein Expression--
The IPT domain of murine Sec5 (residues
5-97) was expressed as a His-tagged fusion protein. Labeled protein
was produced by growing Escherichia coli BL21 in a medium
based on MOPS buffer, containing 5% Celtone (Spectra Stable Isotopes),
and 15NH4Cl and/or
13C6-glucose. The fusion protein was affinity
purified on a Ni2+ column and cleaved from its His-tag with
Factor Xa (Roche) followed by gel filtration. The NMR buffer was 20 mM sodium phosphate, pH 6.0, 50 mM NaCl, 10 mM d10-dithiothreitol, 0.05%
NaN3. NMR experiments were run with a protein
concentration of ~1 mM.
Ral was expressed as a His-tagged fusion protein and affinity purified
in the same manner as Sec5. The purified protein was concentrated and
the bound nucleotide exchanged for the non-hydrolyzable GTP analogue,
GMPPNP (Sigma) as described previously (22).
NMR Spectroscopy--
NMR spectra were recorded at 25 °C on a
Bruker DRX600 spectrometer, except for 13C- and
15N-separated NOESY experiments, which were recorded on a
Bruker DRX800. The following experiments were recorded: 15N
HSQC; 15N-separated NOESY (100 ms mixing time);
15N-separated TOCSY (36 ms DIPSI-2 mixing); intra-HNCA
(23); HN(CO)CA; HNCACB; CBCA(CO)NH; HNCO; (H)CC(CO)NH; H(CC)(CO)NH;
HCCH-TOCSY (18 ms FLOPSY-16 mixing); 13C HSQC; and
13C-separated NOESY (100 ms mixing time) (see Ref. 24 and
references therein). Backbone torsion angles were estimated
from CA, CO, CB, N, and HA chemical shifts using the program
TALOS (25). NMR data were processed using the
AZARA package and analyzed using ANSIG (26).
Structure Calculation--
Structures were calculated
iteratively, using CNS 1.0 and ARIA 1.0 (27). The
parameters used for the calculation were essentially those described in
Ref. 27 except that the length of the high temperature dynamics was
increased to 45 ps and the cooling to a total of 39 ps. The NMR Titration--
The buffer for the titration was 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.05% NaN3. A
0.4-mM sample was prepared and used to record the first
15N HSQC in the Ral titration. Ral·GMPPNP was then added
into the Sec5 sample to give titration points at the following ratios; 1:0.1, 1:0.25, 1:0.38, 1:0.5, 1:0.8, 1:1, and 1:1.5
(Sec5:Ral·GMPPNP). 15N HSQC spectra were recorded
for each titration point.
Description of the Structure--
Backbone resonances of the Sec5
IPT domain were assigned using intra-HNCA (23), HN(CO)CA, HNCACB, and
CBCA(CO)NH experiments (reviewed in Ref. 24). Side chain resonances
were assigned using (H)CC(CO)NH, H(CC)(CO)NH, and HCCH-TOCSY
experiments. NOEs were measured from 15N-separated NOESY
(961 NOEs) and13C-separated NOESY (2,662 NOEs) experiments.
Initial structures were calculated using a total of 2,941 unique
(non-degenerate) NOE restraints, (1,350 unambiguous and 1,591 ambiguous) and 42 pairs of dihedral restraints from TALOS.
After 8 iterations, there were 2,052 unambiguous NOEs and 828 ambiguous NOEs. In the final iteration, 100 structures were calculated; the 25 with the lowest energy were selected for analysis.
The structure of the Sec5 IPT is well defined by the NMR data and has
good covalent geometry (Table I). The
family of structures and the closest structure to the mean are shown in
Fig. 1. The domain forms an Ig-like
Comparison to Other IPT Domains--
The first structures of IPT
domains determined were of the DNA-binding domains of transcription
factors such as NF-
The transcription factor IPT domains have two major roles; they are
involved in DNA binding and homo- and heterodimerization. The IPT
domains whose structures have been solved by x-ray crystallography in
complex with either DNA, another IPT domain, or other protein domains
were analyzed using the program CONTACT (32) with a cutoff
distance of 4.0 Å. The residues involved in DNA binding are generally
basic or polar (Fig. 4, shaded blue) and are not conserved
in IPT domains that do not interact with nucleic acids, such as
Delineation of the Ral Binding Site--
The Sec5 IPT domain binds
to Ral, and the binding contacts on Sec5 were mapped by titrating
unlabeled Ral into 15N-labeled Sec5. The resulting changes
in the 15N HSQC spectrum are shown in Fig.
5A. When two proteins
interact, the chemical environment of the backbone amides changes, and
this usually causes a change in chemical shift. Such changes can be grouped into three regimes: fast, intermediate, and slow. The regime
observed for any amide depends on the relationship between the chemical
shift difference and the rate of exchange between the free and bound
states. If the exchange rate were higher than the chemical shift
difference, a single peak would appear at a position between the
chemical shift of the free and bound forms. If the exchange rate is
lower than the chemical shift difference, two peaks would be observed,
one for the free form and one for the bound. In the intermediate case
(the exchange rate is comparable with the chemical shift difference),
the peaks become broadened and may be unobservable. In the Sec5-Ral
complex spectra, all the resonances that change significantly are in
slow or intermediate exchange. None of the resonances appear to be in
fast exchange, because they do not shift gradually as more G protein is
added (Fig. 5A). In addition, new resonances appear as the
ratio of Sec5:Ral approaches 1:1, for example that of Leu-92 (Fig.
5A). There is a trend for the signal intensities to decrease
because of the increase in correlation time between 10 kDa Sec5
and the 30 kDa Sec5-Ral complex. Some resonances in Sec5 disappear
completely in the 1:1 complex, e.g. Arg-37 (Figs. 4 and
5A). It should be noted that NMR mapping implicates a larger
surface than the actual contact site, because secondary effects will be
observed. Thus, changes in backbone amides that are not exposed to the
solvent have been excluded from Figs. 3 and 5B. In Sec5, the
changes are concentrated on one face of the domain, comprising strands
a, b, e, and g (Figs. 2, 3, and 5B). There are no changes in
strands c, c', and f.
The structural and sequence alignments reveal that, although the
overall folds are similar, the Sec5 domain is more similar to the
bacterial The residues involved in dimerization in the transcription factors map
to one surface of the domain, comprising residues from The Ral binding surface on the Sec5 IPT domain can be compared with the
regions of the IPT domains involved in interactions with other
molecules (Figs. 4 and 5B, shown in green). The
A number of G protein-effector complex structures have been solved and
have shown that the way that proteins can contact G proteins varies
significantly. In most G protein-effector complexes, the effector
contacts switch I (the effector loop). The Sec5 domain also makes
contacts with the effector loop, because mutation of Asp-49 of Ral
abrogates its interaction with Sec5 (9). In other G protein-effector
complexes, the structural motifs that interact with the effector loop
vary widely, so it is not possible to predict which region of Sec5 may
be contacting this region of Ral.
In several complexes, for example the Ras-effector complexes (18, 48,
49) and the Cdc42-CRIB effector complexes (41, 50, 51), an
intermolecular The other Ral effector that has been studied in some detail is Ral BP1
or RLIP. The Ral-binding domain of this protein has been delineated and
comprises residues 403-499 (15). Although there is no structure
available for this protein, secondary structure predictions show that
RLIP is predominantly Although the IPT fold exists in most phyla, the IPT domain of Sec5
seems to appear with Ral in evolution. In S. cerevisiae and
Arabidopsis thaliana, where Ral does not exist, Sec5 lacks the IPT. In S. cerevisiae the exocyst components that bind
to G proteins do not contain any regions with homology to the IPT domains, so it is possible that Ral is the only small GTPase
controlling the exocyst that binds an IPT. The other Ral effector
proteins, Ral BP1, filamin, and phospholipase D, do not contain an IPT
domain. It remains to be seen whether other Ral effectors are isolated that use an IPT to mediate their interaction with Ral.
Directed exocytosis is crucial to the regulation of several cellular
processes. Central to the understanding of regulation of exocytosis is
the role of small G proteins in recruitment of the components of the
exocyst complex. We have solved the structure of the Ral-binding domain
of the mammalian exocyst protein, Sec5. This is the first structure of
a Ral effector domain and the first G protein effector that utilizes an
IPT fold to contact the GTPase. We have shown that the Sec5 Ral-binding
domain forms an IPT domain that is topologically closer to
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B, where it is responsible
for DNA binding. A domain of this type has not been found in a G
protein effector so far, and it is not possible to predict how it will
interact with Ral. In contrast, the topology of the Ras-binding domain
of the Ras effectors is structurally conserved and forms a
ubiquitin-like fold (18, 48, 49). It is thus of great interest to study
how the Ral GTPases (the closest homologues of Ras) interact with their
own effectors. There is some precedent for Ig domain-G protein
interactions. A guanine nucleotide dissociation inhibitor (GDI) for Rho
family proteins contains an Ig domain, whose structure in complex with Cdc42 and Rac has been solved (19-21).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
restraints from TALOS were included with errors
of ±30° or twice the S.D., whichever was greater.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sandwich, consisting of eight
-strands that pack together into
two
-sheets. The topology of the Sec5 domain is shown in Fig.
2, along with the topology of an Ig
V-type domain and that of the IPT domain of the transcription factor NF-
B. The first
-strand is split into two, a and a', connected by
three residues that form a bulge centered around Pro-15. This is
followed by strand b, which packs against strand a in an anti-parallel fashion and then strands c and c', which form the edge of the second
-sheet. The b-c loop is interrupted by a single turn of 310 helix. The c' strand is followed by strand d, which
forms the edge of the first
-sheet, packing against strand e. This is followed by strands f and g, which complete the second
-sheet. The last strand is also split into two: g, which forms an anti-parallel connection with strand f and g', which forms a parallel connection with
strand a'.
Experimental restraints and structural statistics
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Fig. 1.
The structure of the Sec5 IPT domain. On
the left is a backbone trace of the 25 lowest energy
structures. On the right is a ribbon representation of the
structure that is closest to the mean. The -strands are labeled.
This figure was generated with Molscript (45) and Raster3D (46).
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Fig. 2.
Topologies of Ig-like domains. The
topology of the Sec5 IPT domain (a), the NF- B
transcription factor IPT domain (b), and the Ig V-type
domain (c). Although the cores of the two
-sheets are the
same in all the domains, the strands at the edges of the sheets are
different and in the IPT domains there are variable short stretches of
310 helix within the inter-strand loops.
B (28, 29) and revealed a 7-stranded
-sandwich, which differs from that of the Ig V-type domains in that
it is missing strand c' from the first
-sheet and strand d is much
shorter (Figs. 2, b and c, and
3). A sequence alignment of Sec5
IPT with other IPT domains (Fig. 4) was
constructed after the structure of Sec5 IPT was solved, on the basis of
tertiary structure alignment of Sec5 with NF-
B (28) and
Bacillus stearothermophilus
-amylase (30) (PDB codes 1bfs
and 1qho), using TOP (31). Alignment on the basis of
sequence alone was not accurate, even when the secondary structures of
IPT domains such as NF-
B were used to guide the alignment. The
sequence alignment available from data bases such as Pfam is also
incompatible with this structure-based alignment, probably because the
sequence identity between Sec5 and the other IPT domains is extremely
low.
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Fig. 3.
Comparison of the structures of Sec5 with
other IPT domains and Rho GDI. The -strands are labeled in the
Sec5 domain. The e-f loop, the site of an insertion in NF-
B, is
labeled.
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Fig. 4.
Sequence alignment of IPT domains whose
structures have been determined with the Sec5 IPT domains from various
organisms. The murine Sec5 IPT domain is identical to that of
human Sec5. The residue numbers for murine Sec5 are shown
above the sequences. The basis of this alignment was taken
from Pfam, but Sec5 was added by using an alignment of the
three-dimensional structure with that of -amylase and mouse NF-
B
p50. The secondary structures of these three proteins are shown in
blue above the sequences, with cylinders
representing
-helices and arrows representing
-strands. The solvent-exposed residues in murine Sec5 that
shift when Ral is added are colored green and are
boxed if they are conserved between species. The other IPT
domain residues involved in interacting with other molecules are
colored as follows: DNA interactions, blue; dimer interface,
red; interactions with other proteins or other domains,
green; interactions with DNA and other proteins,
cyan; dimer interface and interactions with DNA,
magenta; dimer interface and interaction with other
proteins, yellow. This figure was produced with Alscript
(47).
-amylase and Sec5. The IPT domains are also involved in contacting
other protein domains within the same molecule. Although the residues
are not likely to be conserved between different proteins, they
highlight regions of the IPT domain that may generally be involved in
protein-protein interactions (Fig. 4, shaded green). They
include the a'/g' surface (Figs. 3 and 4) and the b/e surface in both
-amylase and the transcription factors.
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Fig. 5.
A, the Sec5-Ral NMR titration. A section
of the 15N HSQC is shown at three points in the titration
as follows: black contours 1:0; green
contours 1:0.5; red contours 1:1.0; blue contours
1:1.5. In each case the ratios denote Sec5:Ral·GMPPNP. Resonances
that are in slow exchange are marked with a dashed box. An
expansion is shown for the backbone amide of Leu-92; as the titration
proceeds, the intensity of the peak on the left decreases
until the final point when it has disappeared. At the same time, the
intensity of the peak on the right increases. B,
interaction surfaces in the IPT and GDI domains: Sec5 with Ral
(a); -amylase with domains in the rest of the protein
(b); NF-
B with DNA and other domains (c);
Rho-GDI with Cdc42 (d). Location of residues involved in
interactions with other molecules are denoted by balls, the
color coding of which is the same as the shading of residues in Fig.
4.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amylases than to NF-
B (Figs. 3 and 4). One of the
major differences between these domains is the region between strands c
and e. In NF-
B there is a loop containing a single turn of a
310 helix, then the c' strand, then another turn of 310 helix and strand d, which is only 2 residues; in Sec5
and
-amylase the loop between c and c' is much shorter and does not contain any helix and strand d is longer (Fig. 4). The other main difference between Sec5/
-amylase and the transcription factors is
that there is a 6-residue insertion in the e-f loop in the DNA-binding
proteins. This insertion is visible in the structures, because the loop
protrudes from the surface in NF-
B (Fig. 3).
-strands a,
b, d, and e (Figs. 4 and 5B, shown in red). This is in contrast to Ig-Ig interactions, such as the Cd2-Cd58 complex, where the interaction surface is on the opposite surface of the
-sandwich and is composed of residues in strands g, f, c, and c'
(33). The residues involved in dimerization are generally hydrophobic,
although some salt bridges are also found in the dimer interfaces.
These hydrophobic residues are not conserved in Sec5 except where they
are involved in packing (e.g. Ile-13). There is one notable
exception, Val-61 in Sec5, which is not conserved in
-amylase and is
equivalent to Val-313 in NF-
B. This side chain is exposed on the
surface of the Sec5 even though it is hydrophobic. Despite this, it is
not likely that Sec5 dimerizes via the IPT domain in a manner similar
to the transcription factors; the correlation time of this domain,
determined from the T1/T2 ratios of residues in secondary
structure, is 5.9 ns (data not shown), which is consistent with a
monomeric protein of this size.
-amylase IPT domain uses the same face to contact other domains
within the same protein (30). NF-
B and the other DNA-binding
proteins use the same face both for contacting other IPT domains and
for contacting other domains within the same protein (28, 29, 34-40).
The DNA-binding residues in these IPT domains are close to the
protein-protein interaction surface but only partially overlap.
Interestingly, the other Ig-like domain that has been found to contact
G proteins, that from Rho GDI, uses the opposite face to contact the
Rho family proteins (Fig. 5B) (19-21). It is perhaps not
surprising that this divergence in binding interfaces exists between
the GDI and Sec5, because the GDI domain is quite different; it has
several extra
-strands, one of which is involved in G protein
binding (Figs. 3 and 5B). In addition, the GDI domain, although it interacts with the G protein, makes the majority of its
contacts with the geranyl-geranyl moiety that is covalently attached to
the C terminus of the Rho family proteins.
-sheet is observed, formed by an interaction between
the
2-strand of the G protein and a strand from the effector. In the
case of Sec5, it is tempting to speculate that a similar intermolecular
-sheet will be formed in this effector complex, because many of the
residues implicated in binding to the Ral are within strands a, b, e,
and g. If an intermolecular
-sheet is formed in this complex, it
must involve a
-strand of the Sec5 IPT that is available to make
hydrogen bonds. The
-strand that fulfils this criterion and
experiences chemical shift changes on Ral binding is strand d. If,
however, this were involved in an intermolecular
-sheet interaction,
there would have to be some structural rearrangement of the Sec5
because there is not enough space between strand d and the single turn
of helix in the b-c loop to insert part of the G protein there.
-helix and that the Ral-binding domain
partially overlaps a potential coiled-coil region. Thus, the
interaction between Ral and RLIP is likely to be significantly
different from that between Ral and Sec5. There are other G
protein-effector complexes where the effector domain is
predominantly
-helix, e.g. Rho-PKN (42), Rac-Arfaptin
(43), and Rab-Rabphilin (44), but the structures and interactions are
highly variable.
-amylase
than to the transcription factors such as NF-
B. Mapping of the Ral
binding site in the Sec5 domain reveals that, within the family of IPT
domains, a similar region is used for protein-protein interactions and
that this region is not the same as that used for interacting with DNA.
Comparison of the Ral-binding surface of Sec5 with the region of GDI
that interacts with Rho family G proteins shows that the contacts
between Sec5-Ral and GDI-Cdc42 are significantly different. A detailed
analysis of the contacts that Sec5 makes with Ral awaits determination
of the three-dimensional structure of the complex. The information
presented here can be used to help design mutants that disrupt Ral-Sec5
interactions. Such mutants could be used to elucidate the role of the
Sec5-Ral interaction in exocyst assembly at the plasma membrane.
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ACKNOWLEDGEMENT |
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We thank Prof. Ernest Laue for constant support and encouragement.
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FOOTNOTES |
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* This work was supported by Cancer Research UK and the European Commission. This work is a contribution from the Cambridge Centre for Molecular Recognition, which is supported by the Biotechnology and Biological Sciences Research Council and the Wellcome Trust.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 1hk6) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ A Medical Research Council Career Development Fellow. To whom correspondence may be addressed. Tel.: 44-1223-766018; Fax: 44-1223-766002; E-mail: mott@bioc.cam.ac.uk.
Present address: Harvard Medical School, Massachusetts General
Hospital Cancer Center, Bldg. 149, 13th St., Charlestown, MA 02129.
** To whom correspondence may be addressed. Tel.: 44-1223-766018; Fax: 44-1223-766002; E-mail: do@bioc.cam.ac.uk.
Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M300155200
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ABBREVIATIONS |
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The abbreviations used are: Ral BP1, Ral-binding protein 1; Ig, immunoglobulin; IPT, Ig-like, plexins, transcription factors; GDI, guanine nucleotide dissociation inhibitor; NMR, nuclear magnetic resonance; HSQC, heteronuclear single quantum correlation; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; MOPS, 3-(N-morpholino)propanesulfonic acid.
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1. | Hsu, S. C., Hazuka, C. D., Foletti, D. L., and Scheller, R. H. (1999) Trends Cell Biol. 9, 150-153[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Guo, W.,
Roth, D.,
Walch-Solimena, C.,
and Novick, P.
(1999)
EMBO J.
18,
1071-1080 |
3. |
Guo, W.,
Grant, A.,
and Novick, P.
(1999)
J. Biol. Chem.
274,
23558-23564 |
4. |
Matern, H. T.,
Yeaman, C.,
Nelson, W. J.,
and Scheller, R. H.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
9648-9653 |
5. | Novick, P., and Guo, W. (2002) Trends Cell Biol. 12, 247-249[CrossRef][Medline] [Order article via Infotrieve] |
6. | Guo, W., Tamanoi, F., and Novick, P. (2001) Nat. Cell Biol. 3, 353-360[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Zhang, X. Y.,
Bi, E. F.,
Novick, P.,
Du, L. L.,
Kozminski, K. G.,
Lipschutz, J. H.,
and Guo, W.
(2001)
J. Biol. Chem.
276,
46745-46750 |
8. |
Robinson, N. C. G.,
Guo, L.,
Imai, J.,
Toh-E, A.,
Matsui, Y.,
and Tamanoi, F.
(1999)
Mol. Cell. Biol.
19,
3580-3587 |
9. | Moskalenko, S., Henry, D. O., Rosse, C., Mirey, G., Camonis, J. H., and White, M. A. (2002) Nat. Cell Biol. 4, 66-72[CrossRef][Medline] [Order article via Infotrieve] |
10. | Sugihara, K., Asano, S., Tanaka, K., Iwamatsu, A., Okawa, K., and Ohta, Y. (2002) Nat. Cell Biol. 4, 73-78[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Brymora, A.,
Valova, V. A.,
Larsen, M. R.,
Roufogalis, B. D.,
and Robinson, P. J.
(2001)
J. Biol. Chem.
276,
29792-29797 |
12. |
Polzin, A.,
Shipitsin, M.,
Goi, T.,
Feig, L. A.,
and Turner, T. J.
(2002)
Mol. Cell. Biol.
22,
1714-1722 |
13. |
Jullien-Flores, V.,
Mahe, Y.,
Mirey, G.,
Leprince, C.,
Meunier-Bisceuil, B.,
Sorkin, A.,
and Camonis, J. H.
(2000)
J. Cell Sci.
113,
2837-2844 |
14. |
Nakashima, S.,
Morinaka, K.,
Koyama, S.,
Ikeda, M.,
Kishida, M.,
Okawa, K.,
Iwamatsu, A.,
Kishida, S.,
and Kikuchi, A.
(1999)
EMBO J.
18,
3629-3642 |
15. |
Jullienflores, V.,
Dorseuil, O.,
Romero, F.,
Letourneur, F.,
Saragosti, S.,
Berger, R.,
Tavitian, A.,
Gacon, G.,
and Camonis, J. H.
(1995)
J. Biol. Chem.
270,
22473-22477 |
16. |
Luo, J. Q.,
Liu, X.,
Frankel, P.,
Rotunda, T.,
Ramos, M.,
Flom, J.,
Jiang, H.,
Feig, L. A.,
Morris, A. J.,
Kahn, R. A.,
and Foster, D. A.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
3632-3637 |
17. | Bork, P., Doerks, T., Springer, T. A., and Snel, B. (1999) Trends Biochem. Sci. 24, 261-263[CrossRef][Medline] [Order article via Infotrieve] |
18. | Nassar, M., Horn, G., Herrmann, C., Scherer, A., McCormick, F., and Wittinghofer, A. (1995) Nature 375, 554-560[CrossRef][Medline] [Order article via Infotrieve] |
19. | Hoffman, G. R., Nassar, N., and Cerione, R. A. (2000) Cell 100, 345-356[Medline] [Order article via Infotrieve] |
20. | Scheffzek, K., Stephan, I., Jensen, O. N., Illenberger, D., and Gierschik, P. (2000) Nat. Struct. Biol. 7, 122-126[CrossRef][Medline] [Order article via Infotrieve] |
21. | Gosser, Y. Q., Nomanbhoy, T. K., Aghazadeh, B., Manor, D., Combs, C., Cerione, R. A., and Rosen, M. K. (1997) Nature 387, 814-819[CrossRef][Medline] [Order article via Infotrieve] |
22. | Owen, D., Mott, H. R., Laue, E. D., and Lowe, P. N. (2000) Biochemistry 39, 1243-1250[CrossRef][Medline] [Order article via Infotrieve] |
23. | Nietlispach, D., Ito, Y., and Laue, E. D. (2002) J. Am. Chem. Soc. 124, 11199-11207[CrossRef][Medline] [Order article via Infotrieve] |
24. | Ferentz, A. E., and Wagner, G. (2000) Q. Rev. Biophys. 33, 29-65[CrossRef][Medline] [Order article via Infotrieve] |
25. | Cornilescu, G., Delaglio, F., and Bax, A. (1999) J. Biomol. NMR 13, 289-302[CrossRef][Medline] [Order article via Infotrieve] |
26. | Kraulis, P. J., Domaille, P. J., Campbell-Burk, S. L., Vanaken, T., and Laue, E. D. (1994) Biochemistry 33, 3515-3531[Medline] [Order article via Infotrieve] |
27. | Linge, J. P., O'Donoghue, S. I., and Nilges, M. (2001) 339, 71-90 |
28. | Ghosh, G., Vanduyne, G., Ghosh, S., and Sigler, P. B. (1995) Nature 373, 303-310[CrossRef][Medline] [Order article via Infotrieve] |
29. | Muller, C. W., Rey, F. A., Sodeoka, M., Verdine, G. L., and Harrison, S. C. (1995) Nature 373, 311-317[CrossRef][Medline] [Order article via Infotrieve] |
30. | Dauter, Z., Dauter, M., Brzozowski, A. M., Christensen, S., Borchert, T. V., Beier, L., Wilson, K. S., and Davies, G. J. (1999) Biochemistry 38, 8385-8392[CrossRef][Medline] [Order article via Infotrieve] |
31. | Lu, G. G. (2000) J. Appl. Crystallogr. 33, 176-183[CrossRef] |
32. | Bailey, S. (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760-763[CrossRef][Medline] [Order article via Infotrieve] |
33. | Wang, J., Smolyar, A., Tan, K. M., Liu, J., Kim, M. Y., Sun, Z. J., Wagner, G., and Reinherz, E. L. (1999) Cell 97, 791-803[Medline] [Order article via Infotrieve] |
34. | Chen, F. E., Huang, D. B., Chen, Y. Q., and Ghosh, G. (1998) Nature 391, 410-413[CrossRef][Medline] [Order article via Infotrieve] |
35. | Chen, Y. Q., Ghosh, S., and Ghosh, G. (1998) Nat. Struct. Biol. 5, 67-73[Medline] [Order article via Infotrieve] |
36. | Stroud, J. C., Lopez-Rodriguez, C., Rao, A., and Chen, L. (2002) Nat. Struct. Biol. 9, 90-94[CrossRef][Medline] [Order article via Infotrieve] |
37. | Chen, L., Glover, J. N. M., Hogan, P. G., Rao, A., and Harrison, S. C. (1998) Nature 392, 42-48[CrossRef][Medline] [Order article via Infotrieve] |
38. | Jacobs, M. D., and Harrison, S. C. (1998) Cell 95, 749-758[Medline] [Order article via Infotrieve] |
39. | Huxford, T., Huang, D. B., Malek, S., and Ghosh, G. (1998) Cell 95, 759-770[Medline] [Order article via Infotrieve] |
40. |
Cramer, P.,
Larson, C. J.,
Verdine, G. L.,
and Muller, C. W.
(1997)
EMBO J.
16,
7078-7090 |
41. | Mott, H. R., Owen, D., Nietlispach, D., Lowe, P. N., Manser, E., Lim, L., and Laue, E. D. (1999) Nature 399, 384-388[CrossRef][Medline] [Order article via Infotrieve] |
42. | Maesaki, R., Ihara, K., Shimizu, T., Kuroda, S., Kaibuchi, K., and Hakoshima, T. (1999) Mol. Cell. 4, 793-803[CrossRef][Medline] [Order article via Infotrieve] |
43. | Tarricone, C., Xiao, B., Justin, N., Walker, P. A., Rittinger, K., Gamblin, S. J., and Smerdon, S. J. (2001) Nature 411, 215-219[CrossRef][Medline] [Order article via Infotrieve] |
44. | Ostermeier, C., and Brunger, A. T. (1999) Cell 96, 363-374[Medline] [Order article via Infotrieve] |
45. | Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef] |
46. | Merritt, E. A., and Bacon, D. J. (1997) Methods Enzymol. 277, 505-524 |
47. | Barton, G. J. (1993) Protein Eng. 6, 37-40[Medline] [Order article via Infotrieve] |
48. | Vetter, I. R., Linnemann, T., Wohlgemuth, S., Geyer, M., Kalbitzer, H. R., Herrmann, C., and Wittinghofer, A. (1999) FEBS. Lett 451, 175-180[CrossRef][Medline] [Order article via Infotrieve] |
49. | Huang, L., Weng, X. W., Hofer, F., Martin, G. S., and Kim, S. H. (1997) Nat. Struct. Biol 4, 609-615[Medline] [Order article via Infotrieve] |
50. | Abdul-Manan, N., Aghazadeh, B., Liu, G. A., Majumdar, A., Ouerfelli, O., Siminovitch, K. A., and Rosen, M. K. (1999) Nature 399, 379-383[CrossRef][Medline] [Order article via Infotrieve] |
51. | Morreale, A., Venkatesan, M., Mott, H. R., Owen, D., Nietlispach, D., Lowe, P. N., and Laue, E. D. (2000) Nat. Struct. Biol 7, 384-388[CrossRef][Medline] [Order article via Infotrieve] |
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