Structure-Function Study of the N-terminal Domain of
Exocyst Subunit Sec3*□
Kyuwon Baek‡1, Andreas Kno ¨dler§1,2, Sung Haeng Lee‡3, Xiaoyu Zhang§4, Kelly Orlando§, Jian Zhang§,
Trevor J. Foskett‡, Wei Guo§5, and Roberto Dominguez‡6
subunit of the exocyst acts as a spatial landmark for exocytosis
through its ability to bind phospholipids and small GTPases.
The structure of the N-terminal domain of Sec3 (Sec3N) was
determined ab initio and defines a new subclass of pleckstrin
homology (PH) domains along with a new family of proteins
that mediate dimerization through domain swapping. The
structure identifies residues responsible for phospholipid bind-
ing, which when mutated in cells impair the localization of exo-
exocytosis. Through its ability to bind the small GTPase Cdc42
and phospholipids, the PH domain of Sec3 functions as a coin-
cidence detector at the plasma membrane.
complex composed of subunits Sec3, Sec5, Sec6, Sec8, Sec10,
Sec15, Exo70, and Exo84. This complex was first identified by
genetic and biochemical methods in the budding yeast Saccha-
romyces cerevisiae (1, 2). A homologous complex was subse-
quently discovered in mammalian cells (3). The exocyst medi-
ates initial tethering of post-Golgi secretory vesicles to the
plasma membrane, a step that precedes SNARE7-driven mem-
brane fusion (4, 5). The exocyst is regulated by numerous cel-
lular factors, and in particular small GTPases, which are pri-
marily responsible for the spatiotemporal control of exocytosis
Recent studies have provided insights into the molecular
architecture and function of tethering proteins. Crystal struc-
tures of nearly full-length Exo70 (7–9) and large fragments of
Sec6 (10), Sec15 (11), and Exo84 (7) have been determined.
Despite the lack of sequence similarity, these structures all
reveal a similar fold, consisting of elongated tandem repeats of
helical bundles, which are predicted to pack against one
another during assembly of the exocyst complex (4). The
recently determined structure of the yeast Dsl1p complex
implicated in Golgi-to-endoplasmic reticulum transport pro-
sisting of helical bundles similar to those of exocyst subunits
(12). The structure suggested a similar architecture, and possi-
bly a common origin, among multisubunit tethering com-
ing domains of the mammalian exocyst subunits Sec5 (13) and
Exo84 (14), which display immunoglobulin-like and pleckstrin
homology (PH) folds, respectively. However, these two do-
mains are missing in the yeast complex and are not considered
part of the conserved core of the exocyst (4).
Studies in yeast suggest that subunit Sec3 plays a pivotal role
in exocyst function and vesicle tethering. Sec3 localizes,
together with Exo70, to the growing end of the daughter cell
(known as the “bud tip”). Although the localization of other
exocyst components relies on the actin cables that serve as
tracks for motor-driven vesicle transport to the daughter cell,
bly (15–17). Genetic analyses and live cell imaging have shown
for localization of the exocyst to the plasma membrane and
exocytosis (18). This region has also been implicated in the
binding of small Rho-family GTPases and phosphatidylinositol
As an important step toward understanding exocyst-medi-
ated vesicle tethering, we identified the precise domain of Sec3
involved in plasma membrane and Cdc42 binding, determined
Grants R01-GM073791 (to R. D.) and R01-GM64690 (to W. G.). Use of the
Industrial Macromolecular Crystallography Association-Collaborative
Macromolecular Crystallography Association through a contract with
Hauptman-Woodward Medical Research Institute. The Advanced Photon
the Protein Data Bank, Research Collaboratory for Structural Bioinformatics,
supplemental Figs. S1–S4 and Movie S1.
1Both authors contributed equally to this work.
2Supported by a fellowship from the Deutsche Forschungsgemeinschaft.
3Present address: Chosun University School of Medicine, 375 Seosuk-dong,
Dong-gu, Gwangju, Korea 501-759.
4Supported by a Scientist Development Grant from the American Heart
Pennsylvania, Philadelphia, PA 19104. E-mail: firstname.lastname@example.org.
Medicine, University of Pennsylvania, A507 Richard Building, 3700 Hamil-
ton Walk, Philadelphia, PA 19104. Tel.: 215-573-4559; Fax: 215-573-5851;
7The abbreviations used are: SNARE, soluble NSF attachment protein recep-
green fluorescent protein; Bgl2, endo-?1,3-glucanase; 5-FOA, 5-fluoroo-
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 14, pp. 10424–10433, April 2, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
10424 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 14•APRIL 2, 2010
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its crystal structure, and studied its function in cells. We show
that the core of this domain, consisting of residues 71–241 and
referred to as Sec3N, defines a new subclass of PH domains.
the PH domain through domain swapping. A phosphate ion
and the C terminus of a neighboring molecule in the crystal
bind in the predicted phospholipid-binding pockets at the dis-
tions. Mutations of positively charged residues identified by
these interactions impair the polarized localization of exocyst
and secretion defects. Based on this structure, a new family of
proteins was identified containing Sec3-like PH domains,
including amisyn, a protein implicated in the regulation of
SNARE complex assembly.
Protein Preparation—The cDNA encoding for yeast Sec3N
(residues 71–241) was amplified by PCR and cloned into the
vector comprises a glutathione S-transferase affinity purifica-
tion tag and a thrombin-cleavage site. BL21(DE3) competent
cells (Invitrogen) were transformed with this construct and
grown in LB medium at 37 °C to A600? 0.6. Expression was
side and carried out overnight at 20 °C. Cells were harvested by
centrifugation, resuspended in phosphate-buffered saline (140
mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4(pH
Corp.). Affinity purification on a glutathione-Sepharose col-
umn (Amersham Biosciences) was done according to the man-
ufacturer’s protocol. Sec3N-glutathione S-transferase was
eluted with 50 mM Tris-HCl (pH 7.5), 10 mM glutathione and
dialyzed against 20 mM Tris-HCl (pH 7.5), 20 mM NaCl, 1 mM
of 1:1000 (Hematologic Technologies Inc.). Further purifica-
sham Biosciences) in 20 mM Tris-HCl, (pH 7.5), 1 mM dithio-
threitol, and 1 M NaCl gradient. Selenomethionine-substituted
Sec3N was obtained by a similar procedure, growing cells in
SelenoMet medium (Athena Enzyme Systems), supplemented
with 70 mg/ml selenomethionine (Acros Organics).
(pH 7.5), 50 mM NaCl, 5 mM dithiothreitol and concentrated to
Biotech). Crystals were obtained using the hanging drop,
vapor-diffusion method. A typical setup consisted of a 1:1
(v/v) mixture of protein solution and a well solution contain-
ing 100 mM Bicine (pH 9.5), 14% polyethylene glycol
monomethyl ether 5000, 1 mM dithiothreitol at 20 °C. Crys-
tal quality was improved with addition of 10 mM of CdCl2or
BaCl2. Crystals were flash-frozen in liquid nitrogen using
Paratone-N (Hampton Research) as cryoprotectant.
Data Collection, Structure Determination, and Analysis—X-
crystals (Table 1) using beamline 17-BM at the Industrial Mac-
Team facility of the Advance Photon Source (Argonne, IL).
Data indexation and scaling were carried out with the program
HKL2000 (HKL Research Inc.). The structure was determined
using the multiwavelength anomalous dispersion method at
Sec3N monomer in the asymmetric unit) were found using the
program SnB (23). The positions of the selenium atoms were
refined, and phases were calculated with the program Phenix
2.0-Å resolution, and subsequent model building and refine-
ment iterations were performed with the programs Coot and
Phenix. Illustrations were prepared with the program PyMOL
(DeLano Scientific LLC.). The solvent-accessible area buried at
the dimer interface, defined as the locus of the center of a sol-
vent probe of radius 1.4 Å as it rolls over the van der Waals
surface of the protein, was calculated with the CCP4 program
Plasmids and Yeast Strains—Sec3 mutations were generated
with the QuikChange site-directed mutagenesis kit (Agilent
Technologies) using a CEN-LEU2 plasmid (pG1273) contain-
ing the full-length Sec3 gene as a template. To generate exo70
sec3 double mutants, yeast cells bearing the sec3?N mutation,
exo70::HIS3 deletion, and carrying an EXO70 balancer plasmid
(CEN-URA3) and plasmids with exo70–45 or exo70-47 were
transformed with various sec3 mutant variants. The transfor-
mants were tested for growth upon losing the EXO70 balancer
on 5-fluoroorotic acid (5-FOA) plates as described previously
Bgl2 Secretion Assay—Wild-type, exo70-47, and exo70-47
sec3-303 cells were grown to early log phase at 25 °C. sec10-2
mutant cells were grown at 25 °C and moved for 1 h to 37 °C.
NaF and NaN3were added to a concentration of 10 mM. An
amount of cells equivalent to 10 A600/ml was collected and
washed three times with washing buffer (20 mM Tris-HCl, pH
7.5, 10 mM NaN3, and 10 mM NaF). Cells were resuspended in
300 ?l of spheroplast solution (50 mM Tris-HCl, pH 7.5, 1.4 M
sorbitol, 10 mM NaF, 10 mM NaN3, 30 mM 2-mercaptoethanol,
and 0.2 mg/ml zymolyase), and incubated for 30 min at 37 °C.
The resulting spheroplasts were pelleted at 2000 rpm, and
supernatants (external pools) were collected. After washing,
7.5, 100 mM NaCl, 2 mM MgCl2, 0.5% Triton X-100, and prote-
ase inhibitor) on ice for 10 min. The samples were centrifuged
blot using a rabbit anti-Bgl2 polyclonal antibody.
Microscopy—Chromosomal GFP tagging of exocyst compo-
nents was performed as described (15). Cells were grown to
using a Leica DM IRB microscope equipped with a 100? oil
immersion objective and a high resolution charge-coupled
device camera (ORCA-ER, Hamamatsu Photonics). The fluo-
rescence distribution in cell images was analyzed with the pro-
APRIL 2, 2010•VOLUME 285•NUMBER 14JOURNAL OF BIOLOGICAL CHEMISTRY 10425
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gram ImageJ (rsb.info.nih.gov/ij). A straight line was drawn
used to determine the gray value of each point along the line, a
measure of the fluorescence intensity.
Sec3N Folds into a PH Domain—We had previously mapped
the GTPase and membrane-binding activities of Sec3 to an
N-terminal fragment comprising residues 1–320 (18, 19).
Sequence analysis suggested that Sec3-(1–320) contained N-
and C-terminal disordered, non-conserved regions. The frag-
ment 71–250 appeared to form an independent globular
ordered regions. Our genetic analysis also indicated that dele-
in cells (supplemental Fig. S1). Attempts to crystallize Sec3-
(71–250) yielded needle-like crystals of poor quality that could
not be improved. Analysis of protein samples from old crystal-
lization setups showed partial protein degradation, a condition
known to affect crystallization. A stable fragment of slightly
smaller mass (residues 71–241, referred to here as Sec3N) was
identified using a combination of mass spectrometry and
N-terminal sequencing. This fragment was subcloned and
yielded excellent quality crystals, diffracting the x-rays to
?2.0-Å resolution (Table 1 and “Experimental Procedures”).
shown below, accounts for the plasma membrane-binding
activity of Sec3.
the Protein Data Bank. Therefore, the structure was deter-
of selenomethionine-substituted protein. Sec3N has a single
methionine residue in 171 amino acids, resulting in a weak
anomalous signal. However, the crystal contained four Sec3N
molecules in the asymmetric unit, and the implementation of
4-fold non-crystallographic symmetry averaging improved sig-
was built and refined to 2.0-Å resolution (Table 1).
During model building, the core domain of the Sec3N mon-
omer was recognized as a PH domain. The PH domain belongs
to a structural superfamily that includes the phosphotyrosine-
binding, Ena/VASP homology, and Ran-binding domains (27).
These domains lack sequence similarity, but all share a core
structure of 100–120 amino acids consisting of a seven-
stranded, semi-open, antiparallel ?-barrel (strands ?1 to ?7),
capped at one end by a C-terminal ?-helix (?1). At the other
end of the barrel three inter-strand variable loops (VL1, VL2,
Sec3N shares this core structure, although it lacks statistically
significant sequence identity with classic PH domains. For
instance, the PH domain of Sec3 and that of the prototypical
PH-domain-containing protein phospholipase C? (28) (PDB
code: 1mai) superimpose with r.m.s.d. of 2.15 Å for 87 equiva-
lent C? positions, whereas the two proteins share only 7%
sequence identity (Fig. 1A). The PH fold appears to be con-
(supplemental Fig. S3), including human Sec3, which shares
16.4% sequence identity (37.4% sequence similarity) with yeast
to be involved in phospholipid binding are well conserved (dis-
In addition to Sec3-related sequences from different species,
a BLAST search identified proteins previously unsuspected to
contain PH domains (examples are given in Fig. 1 and
supplemental Fig. S3). Most of these proteins are still unchar-
acterized. However, an interesting example is the protein ami-
syn (also known as syntaxin-binding protein 6 or STXBP6), a
210-amino acid polypeptide implicated in the regulation of
SNARE complex assembly and exocytosis through its C-termi-
nal coiled-coil, a vesicle-SNARE-homology domain (29, 30).
Although its characterization is still limited, amisyn has been
proposed to form part of a vesicle-docking complex and is
its relationship with Sec3N (Fig. 1B), we now predict that the
presence of an N-terminal PH domain (residues 1–153).
Dimerization of Sec3N—The Sec3N fragment crystallized
here is 50–70 amino acids longer than the canonical PH
domain, and the structure includes an additional ?-helix at the
Crystallographic data, phasing, and refinement statistics
Values in parentheses correspond to the highest resolution shell.
Unit cell a/b/c, Å
Unit cell ?/?/?, °
Root mean square bonds, Å
Root mean square angles, °
B-factor protein, Å2
B-factor solvent, Å2
aRmerge? ?hkl(I ? ?I?)/?I, where I and ?I? are the observed and mean intensities of all observations of reflection hkl, including its symmetry-related equivalents.
bRfactor? ?hkl?Fobs? ? ?Fcalc?/??Fobs?, where Fobsand Fcalcare the observed and calculated structure factors of reflection hkl.
cRfreeand Rfactorwere calculated for a randomly selected subset of the reflections (5%) that were omitted during refinement.
10426 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 14•APRIL 2, 2010
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N terminus (?0) and two ?-strands at the C terminus (?8 and
?9). To our surprise, the four Sec3N molecules in the asym-
antiparallel dimers (Fig. 2). Dimerization involves domain
swapping of strands ?8 and ?9 of each monomer, which
overlap in “crossed arms” arrangement (supplemental
movie S1). The dimer interface is extensive, burying 4074 Å2
(2037 Å2per monomer) of solvent-accessible surface area
(“Experimental Procedures”). This value is much larger than
the average crystal packing contact (570 Å2) or protein-pro-
tein complex interface (1910 Å2), and even greater than the
average homodimer interface (3880 Å2) (31–33). The dimer-
ization interface is hydrophobic in character and involves
residues from strands ?8 and ?9 and helix ?1 (Tyr-217, Ile-
218, Phe-221, Val-229, Trp-231, Phe-236, and Leu-238).
Additional stabilization of the dimer interface results from
incorporation of strands ?8 and ?9 from two different
monomers into extended antiparallel ?-sheets with strands
?5, ?6, and ?7 of each of the PH domains (Fig. 2).
The dimerization of several PH domains has been observed
in crystal structures, but never confirmed in solution (34–38).
ical size-exclusion chromatography, analytical ultracentrifuga-
tion, and multiangle light scattering (supplemental Fig. S4),
tration. However, we cannot totally exclude the possibility that
of phospholipase C? (28) (PDB code: 1MAI). Phospholipase C? (PLCD1) is colored blue and Sec3N is colored green (helices), yellow (?-strands in front), red
equivalent region of human Sec3, and the Sec3N-related proteins amisyn and maize roothairless (or rth1). PLCD1 was also aligned based on a structure
and colored according to A. Residues predicted to be important for phosphoinositide binding are highlighted (red contour). Uniprot accession codes:
SEC3_YEAST (P33332), SEC3_HUMAN (Q9NV70), AMISYN_HUMAN (Q8NFX7), rth1_MAIZE (Q5YLM3), and PLCD1_RAT (P10688).
APRIL 2, 2010•VOLUME 285•NUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 10427
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the region C-terminal to the PH domain contributes to dimer-
tallographically independent) dimers in the asymmetric unit
(r.m.s.d. of 0.85 Å between equivalent C? atoms) suggests that
dimerization is not a serendipitous crystallization artifact.
human sequence (39). Finally, recruitment at the plasma mem-
brane may increase the local concentration of Sec3 and facili-
PH domains (34–36).
Lipid-binding Pocket—By analogy with other PH domains,
the predicted phospholipid-binding site in Sec3 consists of a
large, positively charged pocket, surrounded by VL1–3 (Fig.
3A). Two such pockets are symmetrically disposed at the distal
ends of the Sec3N dimer (Fig. 2 and supplemental movie S1).
Despite multiple attempts, Sec3N
could not be co-crystallized with
bound phosphoinositides. Further-
more, crystals of the unliganded
protein soaked with various phos-
phoinositides dissolved immedi-
tides bound Sec3N in the crystals,
but their binding was incompatible
with crystal packing contacts.
It has been previously shown that
the locations of inorganic sulfate
and phosphate ions in phosphoino-
sitide-free structures of PH do-
mains, such as those of Grp1 and
Dapp1, coincide with the locations
of the phosphate groups of phos-
phoinositides in the structures of
is very informative that one of the
Sec3N dimers contains a phosphate
ion bound in each of the phospho-
lipid-binding pockets at the distal
phates, which are distinguishable
from solvent atoms at 2.0-Å resolu-
tion, present B-factors of ?40 Å2,
comparable to that of the neighbor-
ing residues, and were probably
incorporated during purification, as
cation buffer. What is more, the C
terminus and the carboxylic group
of Glu-240 from a neighboring
molecule in the crystal also bind in
the phospholipid-binding pocket
near the phosphate ion (Fig. 3B).
Although only one of the Sec3N
dimers shows this arrangement,
nearly identical interactions occur
Because the molecules of the
dimer are crystallographically independent, this corre-
sponds to two independent observations of the same set of
interactions. The presence of these crystal contacts probably
explains why the crystals could not withstand phosphoino-
sitide soaking. More importantly, these contacts probably
mimic protein-phosphoinositide interactions. We thus
attempted to model inositol 1,4,5-trisphosphate in the
phospholipid-binding pocket, so as to position the three
phosphate groups of the phosphoinositide on top of the
phosphate ion, the C terminus and the carboxylic group of
Glu-240 from the neighboring molecule (Fig. 3C). Although
the resulting model is unlikely to reflect the actual binding
orientation or phosphoinositide specificity of the Sec3N
pocket, it points to the residues most likely to be involved in
protein-phosphoinositide interactions (Arg-137, Lys-155,
Arg-157, Arg-168, Lys-194, and possibly Lys-135).
while the other is colored blue. The phosphate ions that bind in the predicted phosphoinositide-binding
FIGURE 3. Phosphoinositide-binding pocket. A, electrostatic surface representation of Sec3N near the pre-
the area corresponding to the yellow square in A. The C terminus of a symmetry-related molecule binds in the
phosphoinositide-binding pocket (green backbone and red electron density map). A phosphate ion, the C
model identifies some of the amino acids most likely to participate in protein-phosphoinositide interactions.
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