Conservation of Helical Bundle Structure between the
Nicole J. Croteau1, Melonnie L. M. Furgason1, Damien Devos2, Mary Munson1*
1Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America, 2EMBL,
Background: The exocyst is a large hetero-octomeric protein complex required for regulating the targeting and fusion of
secretory vesicles to the plasma membrane in eukaryotic cells. Although the sequence identity between the eight different
exocyst subunits is less than 10%, structures of domains of four of the subunits revealed a similar helical bundle topology.
Characterization of several of these subunits has been hindered by lack of soluble protein for biochemical and structural studies.
Methodology/Principal Findings: Using advanced hidden Markov models combined with secondary structure predictions,
we detect significant sequence similarity between each of the exocyst subunits, indicating that they all contain helical
bundle structures. We corroborate these remote homology predictions by identifying and purifying a predicted domain of
yeast Sec10p, a previously insoluble exocyst subunit. This domain is soluble and folded with approximately 60% a-helicity,
in agreement with our predictions, and capable of interacting with several known Sec10p binding partners.
Conclusions/Significance: Although all eight of the exocyst subunits had been suggested to be composed of similar helical
bundles, this has now been validated by our hidden Markov model structure predictions. In addition, these predictions
identified protein domains within the exocyst subunits, resulting in creation and characterization of a soluble, folded
domain of Sec10p.
Citation: Croteau NJ, Furgason MLM, Devos D, Munson M (2009) Conservation of Helical Bundle Structure between the Exocyst Subunits. PLoS ONE 4(2): e4443.
Editor: Dafydd Jones, Cardiff University, United Kingdom
Received November 24, 2008; Accepted January 2, 2009; Published February 13, 2009
Copyright: ? 2009 Croteau et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the EU grant 3D-Repertoire (LSHG-CT-2005-5120828) to D.D., and by the US National Institutes of Health grant GM068803
to M.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
The exocyst is a large, eight protein complex localized to sites of
polarized secretion that is required for exocytosis and cytokinesis
in eukaryotes ([1–3]; and references therein). Its specific function(s)
is unclear, but it interacts with small Ras superfamily GTPases on
secretory vesicles and the plasma membrane, where it is
hypothesized to tether vesicles to the plasma membrane prior to
membrane fusion . The complex also interacts with the
regulatory protein, Sec1p , and the plasma membrane SNARE
(soluble N-ethylmaleimide sensitive protein receptor) protein
Sec9p . These interactions indicate a role for the exocyst and
its subunits in the quality control of exocytic trafficking, as well as
in facilitating SNARE complex assembly and vesicle fusion at the
plasma membrane. Elucidation of the exocyst’s function, and that
of other related tethering complexes [7–10], requires biochemical
and structural analyses of the individual subunits, as well as various
protein-protein interactions within the complex.
Each of the exocyst subunits is predicted to be a-helical. They
possess less than 10% sequence identity with each other, although
limited sequence similarity has been detected using PSI-BLAST
analyses [7,11]. They also show little similarity to other proteins or
domains, except for short regions of predicted coiled coils [1,7].
Several recent crystal structures of domains from individual
subunits have been determined: nearly full-length yeast and
human Exo70 [12–14], and the C-terminal domains of yeast
Exo84p , yeast Sec6p  and Drosophila Sec15 . They
show similar structures containing multiple helical bundles,
yielding an overall similar shape (Figure 1A). Specific details of
the bundles differ, especially the surface residues, but the helical
bundle topologies are identical, suggesting divergent evolution
from an ancient exocyst ancestor protein for these four exocyst
Progress for the other subunits has been hindered by lack of
soluble protein. Insolubility can occur for many different reasons:
recombinant proteins may not fold correctly when overexpressed
in Escherichia coli cells, they may not have the correct post-
translational modifications, or they may be insoluble in the
absence of co-factors or binding partners. Much effort has been
spent to develop methods to address these issues ([17–21]; and
references therein), including the use of different strains, selective
growth conditions, fusion tags, co-expression with binding
partners, and expression of independently folded structural
domains. Often, the latter strategy is approached by using
secondary structure predictions to deduce a domain, making
numerous constructs with slight variations at the N- and/or C-
termini, or by using limited proteolytic digestions to cleave
unstructured or floppy regions, thereby defining a core domain.
Limited proteolysis proved critical for most of the current exocyst
structures; however, this strategy relies on availability of at least
PLoS ONE | www.plosone.org1February 2009 | Volume 4 | Issue 2 | e4443
slightly soluble protein. An alternative approach is to computa-
tionally predict domain structures based on the similarity to
proteins with known structural domains [20,22–24]. However, this
approach is challenging if the protein has little or no similarity with
proteins of known structures. In that case, more sensitive
computational methods, such as hidden Markov model (HMM)
predictions , may be successful.
Based on the structural conservation observed in the exocyst
subunits, we hypothesized that the other subunits would have
similar helical bundle structures . Therefore, we examined the
conservation of structural similarity between the subunits by
profile HMM analyses using the HHSearch program . Profile
HMMs are similar to simpler sequence profiles, but in addition to
the amino acid frequencies in the columns of a multiple sequence
alignment, they contain information about the frequency of inserts
and deletions at each column, plus transition probabilities. In
addition, secondary structure can be included in the HMM-HMM
comparison, leading to another increase in sensitivity. We applied
state-of-the-art HMM-HMM comparisons to the exocyst complex
and detected structural similarity between all of the exocyst
We verified these structure predictions by identifying a
structural domain in one of the exocyst subunits, Sec10p
(YLR166C), from Saccharomyces cerevisiae. Sec10p is one of the core
subunits in the complex and has previously been shown to interact
in vitro with its partner exocyst subunits Sec6p, Exo70p and Sec15p
Figure 1. The exocyst subunits have similar helical bundle structures. (A) The known structures of the exocyst subunits are shown: Exo70p
(PDB ID 2B1E), Exo84CT (PDB ID 2D2S), Sec15CT (PDB ID 2A2F), Sec6CT (PDB ID 2FJI). Molecular graphics were generated with PyMOL (http://pymol.
sourceforge.net/). Exo84CT is aligned with the N-terminal helical bundles of Exo70p, while Sec15CT and Sec6CT are aligned with the C-terminal
bundles of Exo70p. (B) Secondary structure predictions for all of the exocyst subunits. The black horizontal lines represent the sequence of each yeast
exocyst subunit. The predicted a-helices (magenta) and b-strands (cyan) are indicated by vertical bars above each line. The height of the bars is
proportional to the confidence of the secondary structure prediction . Red blocks underline regions of the known structures. Green blocks
underline the best hits to exocyst structures (see Table 1).
Exocyst Helical Bundles
PLoS ONE | www.plosone.org2 February 2009 | Volume 4 | Issue 2 | e4443
[2,12,15]. The specific function(s) of Sec10p is unknown; however,
overexpression of N- and C-terminal truncated constructs show
dominant negative secretory and morphogenic defects in vivo .
Further characterization of Sec10p by biophysical and structural
methods has been hindered by the lack of soluble recombinant
Sec10p protein. We expressed and purified the predicted Sec10p
structural domain, and show that it is folded and helical in
solution. This domain is functional—it retains the ability to
interact with both Sec6p and Exo70p. In addition, we show that it
interacts directly with the C-terminal domain of Exo84p, an
interaction previously shown only by yeast two-hybrid studies .
Thus, our bioinformatic analyses have revealed structural
similarity between all exocyst components and have additionally
defined a soluble domain of the exocyst complex subunit Sec10p
for further biochemical and structural characterization.
Results and Discussion
All of the exocyst subunits have similar helical bundle
The exocyst complex is composed of eight large proteins
(between 71 and 155 kD) that are predicted to be predominantly
helical by secondary structure predictions (Figure 1B). They form a
complex at sites of exocytosis, have been proposed to tether
secretory vesicles to the plasma membrane, and may serve as a
quality control mechanism to ensure proper membrane fusion
[3,4]. Little is known about the structure of the intact complex,
except a series of images taken by quick freeze-deep etch EM .
With less than 10% sequence identity between them, the exocyst
subunits were originally thought to be unrelated. However, several
short stretches in each of the subunits are predicted to form coiled
coil (or amphipathic helical) structures [1,7,30], and regions of
similarity with subunits from other tethering complexes have been
detected [7,11,30]. When the high resolution crystal structures of
several exocyst subunits (Sec15p, Exo70p, Exo84CT, and
Sec6CT) were determined, it became clear that they are
structurally and topologically similar (Figure 1A; ). Searches
of the Protein Data Bank (PDB;  using Dali (http://www.ebi.
ac.uk/dali) indicated that these helical bundle structures are
considerably more similar to each other than to other proteins.
The structures of the cargo binding domain of Myo2p, the
unconventional type V myosin that transports yeast secretory
vesicles , as well as the Conserved Oligomeric Golgi tethering
complex subunit, COG2 (residues 61–262; ), also show
structural similarity to the exocyst helical bundles. It is unclear
whether the similarity of COG2 represents divergence or
functional convergence of the COG complex subunits from the
exocyst subunits [7,9,11,30]. The structural relatedness between
the different exocyst subunits, combined with similar patterns of
predicted helical secondary structures (Figure 1; also [12,15,16])
led us to predict that all the exocyst structures would be similar,
and to create a new working model for the exocyst complex
structure . This structural similarity was recently supported
using multiple iterations of PSI-BLAST , for four of the
exocyst subunits .
Here, we use hidden Markov models to examine the relatedness
of the exocyst subunits at the sequence level. These analyses use
comparisons of different HMM profiles generated from the
individual exocyst families combined with secondary structure
predictions and the known structures; this method has previously
been shown to be more sensitive than other current analyses .
Indeed, statistically significant P-values were detected between all
the exocyst components of known structure (Table 1). Two
families of exocyst structures can be distinguished in the helical
bundle structures of the exocyst components [not including the
Ral binding domains of mammalian Sec5 [34,35] and Exo84
]: the Exo70/84 and Sec6/15 families. The Exo70 (2B1E_A &
2PFT_A) HMM detects the Exo84 (2D2S_A) HMM with a P-
value of 1024and reciprocally, while the Sec6 (2FJI_1) and Sec15
(2A2F_X) HMMs detect each other with 1023and 1027P-values.
All exocyst sequences of unknown structures can be linked to at
least one exocyst sequence of known structure (Table 1).
The exocyst subunits show a striking pattern with many of their
N-terminal regions detecting the Exo70/84 families, and many of
their C-terminal regions detecting the Sec6/15 families (Table 1).
The exceptions are Exo84 and Sec3, which differ in their N-
terminal regions; both contain coiled coil and b-sheet domains.
The similarities suggest that the Sec3/5/8/10 proteins are formed
by tinkering with modules based on the exocyst families of known
structures: N-terminal modules are derived from the Exo70/84
families and C-terminal modules from the Sec6/15 families. These
analyses suggest an ancient gene duplication event, followed by
fusion of these genes and divergence, then followed by multiple
gene duplication events and divergence to create the different
subunits. Additional modules, such as the coiled coil and b-sheet
regions of Sec3p and Exo84p and the Ral-binding domains of the
Table 1. Similarity to exocyst subunits of known structures.
scExo70pmusExo70 Exo84CTSec6CT dmSec15CT
2b1e_A 2pft_A 2d2s_A2fji_1 2a2f_X
YJL085WExo70p6220 (60–623)0 (70–622)2.1E-04 (94–288)1.7E-01 (12–342)6.2E-03 (241–383)
YBR102C Exo84p 752 3.8E-03 (549–747)8.5E-04 (549–707)0 (523–753)
YIL068CSec6p804 3.0E-01 (98–666)5.1E-01 (361–802) 2.5E-01 (174–385)0 (407–805)4.8E-07 (413–728)
YGL233 Sec15p909 2.6E-02 (159–684)2.7E-01 (432–746)4.6E-02 (81–252)4.2E-03 (491–861)0 (475–836)
YER008CSec3p9701.0E-07 (720–1333) 2.6E-08 (733–1329)4.7E-03 (1046–1305)
YDR166C Sec5p13359.4E-01 (221–660)4.4E-01 (153–551) 2.2E-06 (228–378) 2.1E-04 (687–937)4.8E-08 (625–920)
YPR055WSec8p 8701.3E-01 (157–686)7.7E-02 (404–677)1.4E-05 (161–228) 7.7E-03 (590–997) 3.6E-07 (864–967)
YLR166C Sec10p 10641.3E-02 (187–695) 3.1E-03 (186–465) 1.7E-03 (178–393)2.0E-04 (563–860)1.4E-06 (578–826)
HMM P-values of the comparisons are shown in bold, with the range of the aligned residues in parentheses below. Yeast protein lengths are indicated by the number of
amino acids (aa). SGD identifiers are indicated in the first column of the table, and PDB identifiers are indicated in the second row of the table. Blank cells have P-
Exocyst Helical Bundles
PLoS ONE | www.plosone.org3 February 2009 | Volume 4 | Issue 2 | e4443
mammalian Sec5 and Exo84 proteins [34–36] were perhaps later
The similarity of the N-terminal modules is intriguing. With the
exception of Exo70p, the N-terminal domains have not been soluble
enough for biophysical and structural studies. They may be unstable,
sticky, or perhaps natively unfolded in the absence of other exocyst
subunits. It is tempting to speculate that the N-terminal domains
might participate in protein-protein interactions at the core of the
assembled exocyst, as seen in the electron micrographs of the bovine
exocyst complex . The C-terminal domains, therefore, might
then play roles in interactions with small GTPases, the membrane,
and other potential binding partners [3,4].
Because the exocyst structures comprise only domains of the
proteins, we repeated the HMM predictions with the full-length
sequences. We determined that all the exocyst subunits can be
linked to each other (Table 2). The difference in P-values between
Table 1 and Table 2 is due to the usage of the complete sequence
versus only parts of it. These results corroborate the preliminary
analyses by , implying that all members of the exocyst complex
are sequence related, and indicating that all exocyst subunits have
a related structure.
Creation and purification of a soluble yeast Sec10p
In order to understand more about the structure and function of
the exocyst complex, it is imperative to have soluble purified subunits
for structural studies and for reconstitution of the functional complex
in vitro. Identification of a soluble structural domain for a previous
insoluble exocyst subunit would validate our structure predictions, as
well as provide soluble protein for further characterization. We chose
to examine a predicted domain of the yeast Sec10p. Sec10 is one of
the central subunits in the exocyst complex, and qualitative binding
studies and yeast two-hybrid analyses indicate that it interacts with
other exocyst subunits: Sec5, Sec6, Sec8, Sec15, Exo70 and Exo84
[3,12,15]. In addition, overexpression of either an N-terminal region
(1–589) or a C-terminal region (590–872) results in dominant
negative phenotypes in yeast .
Sec10p is one of the subunits that had previously been difficult
to produce in a soluble recombinant form. We attempted a
number of different strategies to produce soluble Sec10p protein.
Initial efforts included the design of twenty different truncations
using only secondary structure predictions. The N- and C-termini
of these constructs were chosen to reside in non-structured regions
so that predicted helices were not disrupted. Truncations were
expressed with one of several affinity tags (e.g. His6, MBP and
GST). The use of MBP as an N-terminal fusion tag appeared
promising, as milligrams of soluble Sec10p were produced.
However, removal of the MBP tag by proteolytic cleavage resulted
in immediate precipitation of Sec10p, suggesting that MBP was
solubilizing misfolded and/or aggregated Sec10p protein; indeed,
this problem has been previously observed with other proteins
. We also tried to co-express Sec10p and several truncations
with either its partner exocyst subunit Sec15p  or with the
chaperones GroEL/GroES . These strategies did not improve
the solubility of the Sec10p constructs (data not shown).
Based on the HMM structure predictions described above, we
identified N- and C-terminal ends of a putative Sec10p structural
domain. The HMM analyses predicted a domain with similarity in
the N-terminal region to the structure of Exo70p (in the range of
186–465) and in the C-terminal region to the structure of Sec6CT
(range of 563–860). Based on this structural assignment, we
designed a fragment that would encompass both domains.
Consideration of the secondary structure prediction for this region
(Figure 2A) led us to clone a construct from residues 145–827,
containing a Pro at the N-terminus and a Gly at the C-terminus.
This domain was predicted to contain four separate helical
bundles, similar to those found in Exo70p and Sec6CT. This
construct, Sec10(145–827), was cloned into the T7 expression
vector pET15b. We chose to use an N-terminal His6tag in order
to maximize the likelihood of obtaining properly folded protein.
Our previous experience using other constructs containing His6
tags is that this tag is not capable of solubilizing improperly folded
recombinant proteins, but is useful for affinity purification. Upon
overexpression in BL21(DE3) cells, this 145–827 Sec10p trunca-
tion construct was found to be quite soluble, compared to other
truncations, including 1–589, 590–871, 75–859, and 55–589
The Sec10(145–827) protein was expressed and purified using
Ni-NTA chromatography, followed by size exclusion chromatog-
raphy to remove several co-purifying contaminants (Figure 2B).
Circular dichroism (CD) studies on the purified Sec10(145–827)
protein indicate a predominantly helical protein (Figure 3A);
calculations of apparent a-helicity suggest that Sec10(145–827) is
approximately 60% helical , which is consistent with the
,60% helical content that we predicted. Moreover, purified
Sec10(145–827) displays a symmetrical monodisperse gel filtration
profile, which indicates a single well-folded protein species
Function of Sec10(145–827)
Because this construct of Sec10p appeared to be an indepen-
dently folded structural domain, we tested to see if it was
Table 2. Similarity between full-length exocyst subunits.
Exo70Exo84Sec6 Sec15Sec3 Sec5 Sec8 Sec10
YJL085W Exo70p0 4.40E-021.20E-01 2.30E-026.30E-051.30E-01 4.50E-02 6.70E-07
YBR102C Exo84p4.60E-020 2.00E-022.50E-021.00E-02 2.10E-06 3.80E-044.40E-03
YIL068C Sec6p 1.20E-011.90E-020 8.60E-025.70E-06 1.40E-025.80E-02 8.50E-03
YGL233W Sec15p 2.30E-022.40E-02 8.80E-020 1.40E-022.10E-07 1.00E-083.60E-04
YER008CSec3p 9.10E-051.10E-02 1.10E-05 1.60E-020 2.40E-036.60E-04 1.40E-05
YDR166C Sec5p1.40E-01 3.80E-061.60E-02 4.90E-072.40E-030 1.10E-05 5.20E-05
YPR055W Sec8p 4.10E-022.90E-04 5.40E-026.40E-093.60E-044.40E-060 9.30E-03
YLR166CSec10p 2.10E-06 6.90E-031.30E-02 7.70E-042.30E-058.60E-05 1.60E-020
HMM P-values of the comparisons are indicated for the full length proteins. SGD identifiers are indicated in the first column of the table.
Exocyst Helical Bundles
PLoS ONE | www.plosone.org4 February 2009 | Volume 4 | Issue 2 | e4443
functional. We performed in vitro binding experiments with other
purified exocyst subunits that bind Sec10p: Sec6p, Exo70p and
Exo84p. Sec6p and Exo70p interactions with full-length Sec10p
were previously shown by qualitative pull-down binding assays
using partially purified proteins [12,15], while binding of Exo84p
had only been observed in yeast two-hybrid assays . For
Exo70p (a.a. 63–623) and Exo84CT (a.a. 523–753), we used
soluble truncations that had been determined by limited
proteolysis and whose structures had previously been determined
. Sec6p, Exo70p and Exo84CT were N-terminally tagged with
Maltose Binding Protein (MBP) for use in qualitative pull-down
experiments. When the purified Sec10(145–827) protein was
incubated for 1 h with MBP alone, or with the MBP-tagged
proteins, it was found to bind specifically to MBP-Sec6p, -Exo70p
and -Exo84CT, above background binding to MBP alone
(Figure 4). Therefore, we conclude that Sec10(145–827) is properly
folded and contains the binding domain(s) for Sec6p, Exo70p and
the C-terminal domain of Exo84p.
Remarkably, under our conditions, only about 10–20% of each
recombinant exocyst subunit appears to interact with each other in
in vitro binding experiments. Similar results have also been
observed for the full-length proteins [12,15]. One possibility for
Figure 2. Recombinant Sec10(145–827) is soluble. Several Sec10p truncation constructs designed using secondary structure predictions are
not generally soluble. (A) Secondary structure prediction  and schematic of several representative N- and C-terminal truncations tested. The
secondary structure prediction is schematically depicted as in Figure 1. Truncations 1–589 and 590–871 were derived from dominant negative
constructs described previously . (B) E. coli cells were transformed with Sec10p truncation variants cloned with an N-terminal His6-tag in the
vector pET15b (Novagen). Expression was induced by addition of IPTG to 0.1 mM, and growth was continued at 15uC for 14–18 h. Cells were pelleted,
lysed and the insoluble (P) material was separated from the soluble material (S) by centrifugation; these were run on a 10% SDS-PAGE gel and stained
with Coomassie blue dye. Asterisks indicate the migration of each construct. For each construct except Sec10(145–827), very little of the His6-tagged
protein was in the soluble fraction. Although the Sec10(75–859) construct initially appeared promising, it was sticky and aggregated after partial
purification on Ni-NTA resin. The right hand lane contains Sec10(145–827) after purification by Ni-NTA resin and gel filtration chromatography.
Exocyst Helical Bundles
PLoS ONE | www.plosone.org5 February 2009 | Volume 4 | Issue 2 | e4443
Figure 3. Sec10(145–827) is folded and a-helical. (A) The far-UV CD wavelength spectrum of Sec10(145–827) was measured between 200 and
270 nm at 4uC. The characteristic minimum at 222 nm is indicative of ,60% helicity. (B) Gel filtration profile of Sec10(145–827). Purified Sec10(145–
827) was applied to a Superdex 200 gel filtration column and the absorbance was monitored at 280 nm. The retention volume of molecular weight
standards (in kD) are indicated at the top. Sec10(145–827) elutes in a single, monodisperse peak, although the apparent molecular weight of
Sec10(145–827), based on the MW standards, indicates that it elutes slightly smaller than expected, suggesting deviation from a spherical shape, or a
small amount of reversible non-specific interaction with the column.
Figure 4. Sec10(145–827) is functional for protein-protein interactions in vitro. Sec10(145–827) binds to MBP-Sec6p, MBP-Exo70p (residues
63–623) and MBP-Exo84p (residues 523–753), but not to MBP alone. The MBP, MBP-tagged Sec6p, Exo70p and Exo84p proteins were immobilized on
amylose resin and incubated with Sec10(145–827). Equivalent volumes of the bound fractions [10% of the input of Sec10(145–827) is shown in the
first lane as a control for the amount of Sec10(145–827) bound] were analyzed on denaturing SDS-PAGE gels. His6-tagged Sec10(145–827) and MBP-
tagged partners were detected by Western blot analyses using a-His5and a-MBP antibodies, respectively.
Exocyst Helical Bundles
PLoS ONE | www.plosone.org6 February 2009 | Volume 4 | Issue 2 | e4443
such weak interactions is that only 10–20% of the recombinant
proteins are properly folded, although this idea is not supported by
our CD and gel filtration results (Figure 3). Alternatively, these
experiments were performed at protein concentrations (1–5 mM)
that may be substantially lower than the binding constants for the
protein-protein interactions. We suggest that the exocyst complex
is formed from a combination of many low affinity interactions,
which would lead to cooperative assembly and disassembly of the
complex at sites of secretion in vivo. Regulation of this process by
Rho/Rab-GTP binding partners on the vesicle and plasma
membranes may trigger conformational changes to activate
assembly of the subunits .
We used a profile HMM prediction algorithm to predict that all
of the exocyst subunits will have similar helical bundle structures.
This approach has been shown to be generally useful to examine
other families of proteins without high sequence similarity .
The HMM predictions have also proven to be a sensitive
computational tool for defining structural domains. It ultimately
allowed us to express and purify a soluble domain of the yeast
exocyst protein Sec10p that will enable further biochemical and
structural analyses. Similar analyses are being used for the other
exocyst subunits, which will significantly contribute to our ability
to elucidate the structure of the entire exocyst complex and its
function in exocytosis.
Materials and Methods
Prediction of protein structure homology
We built hidden Markov models for each exocyst component
starting from the yeast proteins. Homologous proteins were
collected using PSI-Blast for a maximum of 2 iterations with
default parameters. Secondary structure was predicted by
PSIPRED  and added to the profiles. HMMs were built
using the HHmake function of the HHpred suite . HMMs
were compared to each other and to a PDB HMM database using
the HHSearch function of the HHpred suite. Models for each
sequence were built using MODELLER , and evaluated for
the general trend of atomic interaction by statistical potential 
and a composite model evaluation criterion .
To create a soluble construct of Sec10p, the HMM profile of
Sec10p was directly compared with the profiles for the known
structures of Exo70p, Sec15CT, Sec6CT and Exo84CT. A
structural domain in the range of residues 148–867 was predicted.
Using this information and secondary structure predictions
(http://npsa-pbil.ibcp.fr/ and , we chose the N- and C-
terminal ends of the structural domain to be residues 145 and 827,
residues predicted to be at the ends of helices.
Protein Expression and Purification
Genes encoding full-length Sec10p (residues 1–872), the various
truncations [Sec10(1–589); Sec10(590–871); Sec10(75–859), and
the predicted structural domain Sec10(145–827)] were amplified
by polymerase chain reaction and cloned into the NdeI and BamHI
restriction sites of the vector pET15b (Novagen), which introduces
a 6-histidine tag (His6) at the N termini. All constructs were
confirmed by sequencing. All proteins were expressed in E. coli
BL21(DE3) cells. To maximize protein solubility, cells were grown
in LB to an OD at 600 nm of ,0.4 at 37uC. Cells were shifted to
15uC until the OD600reached between 0.6–0.9. Protein expression
was induced with 0.1 mM isopropyl-b-D-thiogalactoside (IPTG),
and cells were grown for an additional 14–18 h at 15uC. Cells
were harvested and frozen at 280uC until lysis. His6-tagged
Sec10(145–827) protein was purified using nickel-NTA resin
(Qiagen). b-Mercaptoethanol (5 mM) or dithiothreitol (DTT,
1 mM) was used in all buffers. Sec10(145–827) was purified using
a Superdex 200 16/60 gel filtration column (GE) in KPhos buffer
(10 mM potassium phosphate at pH 7.4 containing 140 mM KCl
and 1 mM DTT) plus 10% glycerol. Fractions were analyzed by
SDS-PAGE and stained with Coomassie blue. Those fractions
containing .90% pure protein were pooled. Protein was
concentrated using a stirred cell concentrator (Millipore) to
,1 mg/ml, and the protein concentration was determined by
measuring the absorbance at 280 nm and by a quantitative
ninhydrin protein assay . Protein was flash frozen in liquid
nitrogen in KPhos buffer containing 10% glycerol and stored at
Circular Dichroism Spectroscopy
CD spectra were recorded on a J810 spectropolarimeter (Jasco)
fitted with a Peltier-type temperature controller set to 4uC. The
Sec10(145–827) protein was at a concentration of 0.8 mM in
KPhos buffer containing 1 mM DTT and 10% glycerol. The
spectra were recorded as an average of three scans from 200 to
270 nm, in a 1 mm path-length quartz cuvette (Hellma). For each
spectrum, the minimum at 222 nm was used to estimate the mean
residue ellipticity and percent helicity .
Analytical Gel Filtration
Sec10(145–827) was chromatographed on a Superdex 200 10/
30 column (GE) at a protein concentration of 1 mM. The column
was pre-equilibrated in sodium phosphate buffer containing
300 mM NaCl, 10% glyercol and 1 mM DTT; eluted peaks were
observed by monitoring the absorbance at 280 nm. The gel
filtration column was calibrated using standards (thyroglobulin,
670 kD; c-globulin, 158 kD; ovalbumin, 44 kD; myoglobin,
17 kD; Bio-Rad).
MBP pull-down assays and Western blot analyses
Sec6p (residues 1–805), Exo70p (residues 63–623; , and
Exo84CT (residues 523–753;  were subcloned into pMALc2X
(New England Biolabs). Maltose binding protein (MBP) was
expressed at 37uC and MBP-tagged Sec6p, Exo70p, and
Exo84CT were expressed at 20uC for 3 h after induction with
0.1 mM IPTG. They were purified using amylose resin affinity
chromatography (New England Biolabs). The binding reactions
contained purified MBP-tagged proteins immobilized on amylose
resin in binding buffer (10 mM HEPES pH 7, 100 mM NaCl,
0.1% NP-40, and 1 mM DTT), and an equimolar amount of the
purified Sec10(145–827) protein (1 mM) was added. The reactions
were incubated for 1 h at 4uC with mixing to allow binding. Beads
were centrifuged and washed three times in binding buffer and
analyzed by SDS-PAGE. Proteins were transferred to nitrocellu-
lose and probed with a-His5 (Qiagen) or a-MBP antibodies
(Invitrogen). Western blots were developed using horseradish
peroxidase-conjugated a-mouse IgG (Roche), followed by chemi-
luminescent detection (ECL; Amersham) and luminescent image
analysis (FUJIFILM LAS-3000). The blots shown are representa-
tive of at least three separate experiments.
We are grateful to A. Malaby for technical assistance and to Dr. C. R.
Matthews and his lab for use of the CD and advice. We thank members of
the Munson lab for critical reading of this manuscript and suggestions. We
dedicate this to Jen Songer (1979–2008), who inspired us all, in so many
Exocyst Helical Bundles
PLoS ONE | www.plosone.org7 February 2009 | Volume 4 | Issue 2 | e4443
Author Contributions Download full-text
Conceived and designed the experiments: NJC MLMF MM. Performed
the experiments: NJC DD MM. Analyzed the data: NJC DD MM.
Contributed reagents/materials/analysis tools: MLMF. Wrote the paper:
NJC MLMF DD MM.
1. TerBush DR, Maurice T, Roth D, Novick P (1996) The Exocyst is a
multiprotein complex required for exocytosis in Saccharomyces cerevisiae.
Embo J 15: 6483–6494.
2. Guo W, Roth D, Walch-Solimena C, Novick P (1999) The exocyst is an effector
for Sec4p, targeting secretory vesicles to sites of exocytosis. Embo J 18:
3. Munson M, Novick P (2006) The exocyst defrocked, a framework of rods
revealed. Nat Struct Mol Biol 13: 577–581.
4. Wu H, Rossi G, Brennwald P (2008) The ghost in the machine: small GTPases
as spatial regulators of exocytosis. Trends Cell Biol 18: 397–404.
5. Wiederkehr A, De Craene JO, Ferro-Novick S, Novick P (2004) Functional
specialization within a vesicle tethering complex: bypass of a subset of exocyst
deletion mutants by Sec1p or Sec4p. J Cell Biol 167: 875–887.
6. Sivaram MV, Saporita JA, Furgason ML, Boettcher AJ, Munson M (2005)
Dimerization of the exocyst protein Sec6p and its interaction with the t-SNARE
Sec9p. Biochemistry 44: 6302–6311.
7. Whyte JR, Munro S (2002) Vesicle tethering complexes in membrane traffic.
J Cell Sci 115: 2627–2637.
8. Lupashin V, Sztul E (2005) Golgi tethering factors. Biochim Biophys Acta 1744:
9. Cavanaugh LF, Chen X, Richardson BC, Ungar D, Pelczer I, et al. (2007)
Structural analysis of conserved oligomeric Golgi complex subunit 2. J Biol
Chem 282: 23418–23426.
10. Kummel D, Heinemann U (2008) Diversity in structure and function of
tethering complexes: evidence for different mechanisms in vesicular transport
regulation. Curr Protein Pept Sci 9: 197–209.
11. Koumandou VL, Dacks JB, Coulson RM, Field MC (2007) Control systems for
membrane fusion in the ancestral eukaryote; evolution of tethering complexes
and SM proteins. BMC Evol Biol 7: 29.
12. Dong G, Hutagalung AH, Fu C, Novick P, Reinisch KM (2005) The structures
of exocyst subunit Exo70p and the Exo84p C-terminal domains reveal a
common motif. Nat Struct Mol Biol 12: 1094–1100.
13. Hamburger ZA, Hamburger AE, West AP, Weis WI (2006) Crystal Structure of
the S. cerevisiae Exocyst Component Exo70p. J Mol Biol 356: 9–21.
14. Moore BA, Robinson HH, Xu Z (2007) The crystal structure of mouse Exo70
reveals unique features of the mammalian exocyst. J Mol Biol 371: 410–421.
15. Sivaram MV, Furgason ML, Brewer DN, Munson M (2006) The structure of the
exocyst subunit Sec6p defines a conserved architecture with diverse roles. Nat
Struct Mol Biol 13: 555–556.
16. Wu S, Mehta SQ, Pichaud F, Bellen HJ, Quiocho FA (2005) Sec15 interacts
with Rab11 via a novel domain and affects Rab11 localization in vivo. Nat
Struct Mol Biol 12: 879–885.
17. Derewenda ZS (2004) The use of recombinant methods and molecular
engineering in protein crystallization. Methods 34: 354–363.
18. Dokudovskaya S, Williams R, Devos D, Sali A, Chait BT, et al. (2006) Protease
accessibility laddering: a proteomic tool for probing protein structure. Structure
19. Esposito D, Chatterjee DK (2006) Enhancement of soluble protein expression
through the use of fusion tags. Curr Opin Biotechnol 17: 353–358.
20. Peti W, Page R (2007) Strategies to maximize heterologous protein expression in
Escherichia coli with minimal cost. Protein Expr Purif 51: 1–10.
21. Graslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, et al. (2008) Protein
production and purification. Nat Methods 5: 135–146.
22. Yoon J, Kang Y, Kim K, Park J, Kim Y (2005) Identification and purification of
a soluble region of BubR1: a critical component of the mitotic checkpoint
complex. Protein Expr Purif 44: 1–9.
23. Chen Y, Qiu S, Luan CH, Luo M (2007) Domain selection combined with
improved cloning strategy for high throughput expression of higher eukaryotic
proteins. BMC Biotechnol 7: 45.
24. Graslund S, Sagemark J, Berglund H, Dahlgren LG, Flores A, et al. (2008) The
use of systematic N- and C-terminal deletions to promote production and
structural studies of recombinant proteins. Protein Expr Purif 58: 210–221.
25. Soding J, Biegert A, Lupas AN (2005) The HHpred interactive server for protein
homology detection and structure prediction. Nucleic Acids Res 33: W244–248.
26. Soding J (2005) Protein homology detection by HMM-HMM comparison.
Bioinformatics 21: 951–960.
27. Roth D, Guo W, Novick P (1998) Dominant negative alleles of SEC10 reveal
distinct domains involved in secretion and morphogenesis in yeast. Mol Biol Cell
28. Guo W, Grant A, Novick P (1999) Exo84p is an exocyst protein essential for
secretion. J Biol Chem 274: 23558–23564.
29. Hsu SC, Hazuka CD, Roth R, Foletti DL, Heuser J, et al. (1998) Subunit
composition, protein interactions, and structures of the mammalian brain sec6/8
complex and septin filaments. Neuron 20: 1111–1122.
30. Whyte JR, Munro S (2001) The Sec34/35 Golgi transport complex is related to
the exocyst, defining a family of complexes involved in multiple steps of
membrane traffic. Dev Cell 1: 527–537.
31. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, et al. (2000) The
Protein Data Bank. Nucleic Acids Res 28: 235–242.
32. Pashkova N, Jin Y, Ramaswamy S, Weisman LS (2006) Structural basis for
myosin V discrimination between distinct cargoes. Embo J 25: 693–700.
33. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped
BLAST and PSI-BLAST: a new generation of protein database search
programs. Nucleic Acids Res 25: 3389–3402.
34. Mott HR, Nietlispach D, Hopkins LJ, Mirey G, Camonis JH, et al. (2003)
Structure of the GTPase-binding domain of Sec5 and elucidation of its Ral
binding site. J Biol Chem 278: 17053–17059.
35. Fukai S, Matern HT, Jagath JR, Scheller RH, Brunger AT (2003) Structural
basis of the interaction between RalA and Sec5, a subunit of the sec6/8
complex. Embo J 22: 3267–3278.
36. Jin R, Junutula JR, Matern HT, Ervin KE, Scheller RH, et al. (2005) Exo84 and
Sec5 are competitive regulatory Sec6/8 effectors to the RalA GTPase. Embo J
37. Nallamsetty S, Waugh DS (2006) Solubility-enhancing proteins MBP and NusA
play a passive role in the folding of their fusion partners. Protein Expr Purif 45:
38. Thomas JG, Ayling A, Baneyx F (1997) Molecular chaperones, folding catalysts,
and the recovery of active recombinant proteins from E. coli. To fold or to
refold. Appl Biochem Biotechnol 66: 197–238.
39. Scholtz JM, Barrick D, York EJ, Stewart JM, Baldwin RL (1995) Urea unfolding
of peptide helices as a model for interpreting protein unfolding. Proc Natl Acad
Sci U S A 92: 185–189.
40. Devos D, Dokudovskaya S, Williams R, Alber F, Eswar N, et al. (2006) Simple
fold composition and modular architecture of the nuclear pore complex. Proc
Natl Acad Sci U S A 103: 2172–2177.
41. Jones DT (1999) Protein secondary structure prediction based on position-
specific scoring matrices. J Mol Biol 292: 195–202.
42. Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of
spatial restraints. J Mol Biol 234: 779–815.
43. Shen MY, Sali A (2006) Statistical potential for assessment and prediction of
protein structures. Protein Sci 15: 2507–2524.
44. Melo F, Sanchez R, Sali A (2002) Statistical potentials for fold assessment.
Protein Sci 11: 430–448.
45. Rosen H (1957) A modified ninhydrin colorimetric analysis for amino acids.
Arch Biochem Biophys 67: 10–15.
Exocyst Helical Bundles
PLoS ONE | www.plosone.org8 February 2009 | Volume 4 | Issue 2 | e4443