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J. Biol. Chem., Vol. 278, Issue 36, 34373-34379, September 5, 2003
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From the Section of Molecular and Cellular Biology, University of California, Davis, California 95616
Received for publication, March 18, 2003 , and in revised form, June 4, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-propellers and has important implications for the
structure of Aip1 from other organisms and WD repeat-containing proteins in
general. These results were unexpected because a maximum of 10 WD repeats had
been reported in the literature for this protein using sequence data. The
surfaces of the -propellers formed by the D-A and B-C loops are
positioned adjacent to one another, giving Aip1p a shape that resembles an
open "clamshell." The mapping of conserved residues to the
structure of Aip1p reveals dense patches of conserved residues on the surface
of one -propeller and at the interface of the two -propellers.
These two patches of conserved residues suggest a potential binding site for
F-actin on Aip1p and that the orientation of the -propellers with
respect to one another plays a role in binding an actin-cofilin complex. In
addition, the conserved interface between the domains is mediated by a number
of interactions that appear to impart rigidity between the two domains of
Aip1p and may make a large substrate-induced conformational change
difficult. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Aip1p has also been shown to play an important role in the regulation of the actin cytoskeleton (13, 14) and is thought to be conserved in all of the eukaryotes. Previous studies have shown that Aip1p is involved in endocytosis and cytokinesis and that motility in Dictyostelium (13) is required for the normal localization of cofilin to cortical actin patches in Saccharomyces cerevisiae (10) and is essential for the organized assembly of muscle actin filaments in Caenorhabditis elegans (14). It has also been shown that in the absence of cofilin, Aip1p has little ability to depolymerize or bind to F-actin (12, 15). Additionally, a number of alanine-scanning mutations in yeast actin that should hinder the binding of cofilin (16) also prevent the interaction of actin with Aip1p (17), suggesting that the actual target of Aip1p in vivo is a binary complex of F-actin and cofilin. Initial studies on S. cerevisiae Aip1p suggested that the addition of substoichiometric amounts of Aip1p to F-actin in the presence of equimolar amounts of cofilin significantly improves the rate and extent of F-actin depolymerization in vitro (10, 11). More recent studies on Xenopus laevis Aip1p have indicated that Aip1p enhances the fragmentation of actin filaments by capping their barbed ends in the presence of cofilin (15), thus preventing the elongation of cofilin-severed actin filaments. These studies have indicated a likely binding site for Aip1p on F-actin (15); however, the specific nature of its interactions with cofilin and F-actin are still unknown.
Aip1p is a member of the WD repeat family of proteins and has been
predicted to contain as many as 10 WD repeats
(14). These repeats, also
known as WD40 repeats, are homologous sequences of 
40 amino acids
frequently bracketed by the amino acid pairs Gly-His on the amino end of the
repeat and Trp-Asp at the carboxyl end and are almost exclusive to eukaryotes
(18). Analyses of well
characterized genomes such as S. cerevisiae have shown that
approximately 1% codes for WD repeat-containing proteins. Domains constructed
from WD repeats are utilized by many proteins in the reversible binding and
regulation of specific protein targets in an extremely diverse set of cellular
systems (18). Structural
characterization of a number of WD repeat proteins has revealed that they form
multi-bladed -propeller structures
(19–22)
with nearly all of them containing seven blades. Each of the blades in the
-propeller is formed by a small anti-parallel -sheet consisting of
four -strands, labeled A, B, C, and D, beginning with the strand closest
to the center of the propeller. A single WD sequence repeat encompasses the D
strand of one blade and the A, B, and C strands of the following blade. Each
repeat in sequence therefore does not coincide with a single structural
repeat.
Aip1p has been predicted to contain more WD repeats than any other
structurally characterized protein bringing into question whether its WD
repeats form one very large -propeller or multiple smaller
-propeller domains. This general issue has also been raised for other
proteins with larger numbers of WD repeats
(23). To answer this question
and to help elucidate the nature of the interactions of Aip1p with actin and
cofilin, we have determined the crystal structure of S. cerevisiae
Aip1p to a 2.3-Å resolution, the first structure of a WD repeat protein
containing more than eight WD repeats.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-D-galactopyranoside for 14 h at 15 °C.
Selenomethionine-labeled Aip1 (SeMet Aip1p) was produced for the
multiwavelength anomalous dispersion experiment by the addition of
Met-repressing amino acids (final concentrations: 0.1 mg/ml Lys, 0.1 mg/ml
Phe, 0.1 mg/ml Thr, 0.05 mg/ml Ile, 0.05 mg/ml Leu, 0.05 mg/ml Val) and 0.06
mg/ml selenomethionine to E. coli ER2566 cells in modified magnesium
medium at 37 °C 30 min before inducing with 0.5 mM
isopropyl-1-thio--D-galactopyranoside and lowering the
temperature to 15 °C. Cells were harvested by centrifugation 12 h after
induction and lysed using a microfluidizer at 15,000 p.s.i. Lysate was
clarified by centrifuging at 39,000 x g for 30 min.
The resulting cell extracts containing either wild-type or SeMet
Aip1p/intein/chitin-binding domain fusion protein were passed over a column of
chitin beads, which was washed with at least 20 column volumes of Buffer A (20
mM Tris, pH 8.0, 500 mM NaCl, 0.1 mM EDTA)
plus 0.1% Triton X-100 followed by 20 column volumes of Buffer A. The
Aip1p-intein fusion protein was then cleaved on the chitin column by
incubation for 12 h in Buffer A supplemented with 50 mM
-mercaptoethanol. Aip1p was eluted in Buffer A, dialyzed into 20
mM Tris, pH 8.0, and further purified by anion-exchange
chromatography on a quaternized polyethyleneimine HQ column using a BioCad
Sprint fast protein liquid chromatography. Aip1p was eluted with a 0–1
M NaCl gradient, eluting at a salt concentration of 450
mM. The purified protein was then exchanged into 20 mM
HEPES, pH 7.4, with 20 mM NaCl and concentrated to 15 mg/ml using
Millipore Ultrafree spin concentrators. Dynamic light-scattering experiments
using a Protein Solutions Dynapro 99 instrument on purified protein at 0.2
mg/ml concentration indicated that Aip1p exists as a monodisperse monomer with
a molecular mass of 61.3 kDa reasonably close to the predicted molecular mass
of 67 kDa.
Crystallization and Data Collection—Purified Aip1p and SeMet
Aip1p were crystallized at room temperature by the hanging drop vapor
diffusion method. Drops containing 1 µl of protein solution at a
concentration of 15 mg/ml were mixed with 1 µl of the precipitant solution
(100 mM HEPES, pH 7.5, 200 mM NaCl, 13% w/v polyethylene
glycol 4000) and suspended on siliconized glass coverslips over a 1-ml
reservoir of the precipitant solution. Crystals used in data collection were
transferred to a cryoprotectant solution consisting of 80% v/v precipitant
solution and 20% v/v ethylene glycol, picked up in rayon loops, and
flash-cooled in a stream of liquid nitrogen vapor at 110 K. Native crystals of
Aip1p belonged to the space group P21212 with
unit cell dimensions of a = 69.1 Å, b = 154.4 Å,
and c = 62.1 Å. Crystals of selenomethionine-containing Aip1p
belonged to the space group P21 with unit cell dimensions
of a = 60.8 Å, b = 154.5 Å, c = 69.0
Å, and = 90.65°. Multiple wavelength anomalous dispersion
data were collected on SeMet Aip1p crystals at beamline 9-2 at the Stanford
Synchrotron Radiation Laboratory (SSRL) at 110 K using an ADSC CCD detector
(Table I). Data were collected
on native crystals at beamline 9-1 at SSRL at 110 K using a Mar 345 imaging
plate detector. All of the data were processed using the programs Denzo and
Scalepack (24).
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Structure Determination, Model Building, and Refinement—The
positions of 12 selenium sites were located, and multiple wavelength anomalous
dispersion phases were calculated to a 2.7-Å resolution using SOLVE
(25). Density-modified phases
using the program RESOLVE (26)
yielded interpretable electron density maps with a figure of merit of 0.61.
Phases were extended to 2.5 Å using the program CNS
(27). Two molecules of SeMet
Aip1p were then built into the asymmetric unit using the program O
(28). The molecular model was
refined by iterative cycles of rigid body refinement, energy minimization,
simulated torsional annealing, and individual B-factor refinement in the
program CNS (27) followed by
model rebuilding in program O. Non-crystallographic symmetry restraints were
used in the initial rounds of refinement and released once the model of the
protein molecules was completed. The Rfree value was used
to monitor the progress of the refinement and was calculated using 7% of the
data. Solvent molecules were added in peaks >3
in
Fo – Fc difference electron
density maps and retained if their B-values remained <60
Å2 after a full round of refinement. Two peaks >8
in Fo – Fc difference electron
density maps that were each surrounded by a tetrahedral arrangement of two
histidines and two glutamate residues were modeled as zinc ions and refined
with B-values similar to those of the surrounding residues. The observed metal
ions were bound in pockets created by the crystal packing of the two
SeMet-Aip1p molecules present in the asymmetric unit and are probably not
bound to Aip1p outside of the crystal lattice. The final SeMet Aip1p molecular
model was refined to a 2.3-Å resolution with Rfree =
0.257 and Rcryst = 0.204. The final model of SeMet-Aip1p
comprises residues 2–543 and 550–613 in one Aip1p molecule and
residues 2–543 and 550–615 in the other. Superposition of the two
SeMet-Aip1p molecules in the asymmetric unit reveals no significant
differences with a root mean square difference of 0.31 Å over
606-C
positions. The structure of native Aip1p was determined by
molecular replacement in CNS using the finished model of SeMet Aip1p without
solvent molecules or zinc ions as the search model. The molecular model of
native Aip1p was refined to a 2.5-Å resolution in the same manner as
SeMet Aip1p. Superposition of the final models of native Aip1p and molecule A
of SeMet Aip1p in program O shows that the two structures are virtually
identical with a root mean square difference of only 0.30 Å over
604-C positions. Figures were made in MOLSCRIPT
(29), BOBSCRIPT
(30), RASTER3D, and Grasp
(31).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-propellers
domains. These domains are both regular and do not contain any significant
structural excursions. These are oriented such that the top surfaces of the
propellers, those formed by the D-A and B-C loops of each blade, face toward
one another (Fig. 1). The
resulting overall structure of Aip1p resembles a clamshell opened at an angle
of 110° with the two doughnut-shaped -propeller domains of
Aip1p twisted 15° askew relative to one another. The two
-propeller domains are similar in size, each roughly 45 Å in
diameter and 25 Å high. The first -propeller domain (propeller 1)
is formed by blades 1–7 (residues 20–335), and the second
-propeller domain (propeller 2) is formed from blades 8–14
(residues 4–10 and 340–613). The only significant difference in
the makeup of the two individual domains is that propeller 1 consists of a
continuous string of residues, while the NH2-terminal 10 residues
of Aip1p form the final -strand of blade 14 in propeller 2. This
structural motif, sometimes referred to as "molecular velcro," has
been observed in the structures of other WD repeat proteins and other
-propeller proteins as well
(18,
32). These interactions help
to keep propeller 2 tightly folded and are believed to be a major factor in
the stability of the WD fold. Therefore, there are two separate segments
involved in the region connecting the two domains of Aip1p. These regions are
conserved and are composed of residues 10–20 and 337–341.
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The Interdomain Interface—There is an extensive interface
mediated by numerous contacts between the two -propeller domains of
Aip1p. This surface is formed by the D-strands of blades 1, 7, and
14, various residues from blade 8, and the loop connecting blades 7 and 8
(residues 337–341). A hydrogen-bonding network links the side chains of
Ser-352 and Ser-354 on strand 8B with the imidazole ring of His-338 from the
loop connecting blades 7 and 8 (Fig.
2). Additionally, the hydroxyl group of Ser-352 forms a hydrogen
bond with the indole ring nitrogen of Trp-362, possibly helping to orient its
hydrophobic side chain. Significant interactions are also observed in the
three hydrophobic pockets formed by: (a) Leu-6, Ile-9, Pro-11,
Phe-42, and Val-56; (b) Ile-10, Leu-350, Trp-362, and Ile-607; and
(c) Pro-14, Leu-315, Leu-321, Phe-323, and Ile-335. Other notable
interactions are hydrogen bonds between the side chain amino group of Lys-584
and the carbonyl oxygen of Gly-63; the guanidinium group of Arg-358 and the
carbonyl oxygens of Asp-318 and Gly-337; and the phenolic hydroxyl group of
Tyr-35, the guanidinium group of Arg-44, and the carbonyl oxygen of Pro-14.
These interactions may all contribute to the formation of a relatively
rigid-connecting region between the two -propellers of Aip1p that allows
very limited movement of its two domains in the absence of its binding
partners.
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Location of Conserved Residues in the Structure of Aip1p—
Actin, one of the substrates of Aip1p, is highly conserved, which suggests
that the residues mediating the Aip1p-actin interface will also be conserved.
Amino acid homology and identity between the yeast Aip1p sequence and that of
other eukaryotes is 50 and 30%, respectively. Residues in the amino acid
sequence of S. cerevisiae Aip1p were considered to be strongly
conserved if unchanged or replaced by a homologous residue (Arg 
Lys or
Asp Glu) in at least four of six representative Aip1p proteins from
Schizosaccharomyces pombe, Physarum polycephalum, Dictyostelium
discoideum, C. elegans, X. laevis, and Homo sapiens. Dense
patches of conserved residues occur in the structure of Aip1p in two areas:
neighboring regions in blades 2–5 and at the interface between the two
-propellers (Fig. 3, B and
D). The residues that form the C-D loops at the bottom of
blades 3, 4, and 5 are highly conserved and are part of a contiguous surface
of conserved residues on the side and bottom of propeller 1. At the
-propeller interface, conserved residues involved in the formation of
hydrophobic patches include Ile-335 and Trp-362 from blades 7 and 8. All four
of the residues involved in the hydrogen-bonding network linking blades 7 and
8 are highly conserved in other organisms as well. Additionally, two glycines
at the -propeller interface, Gly-319 in blade 7 and Gly-357 in blade 8,
are highly conserved. Notably, mutation of these glycines in C.
elegans Aip1p have been shown to disrupt actin filament organization in
body wall muscle (14).
Finally, a few conserved residues are scattered across the solvent-exposed
surface on top of the two -propellers. These residues, which include
Arg-18, Phe-208, Lys-340, and Gln-408, may be very important if Aip1p binds
part of an actin-cofilin complex between its two -propeller domains.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-propeller domains in an open clamshell-shaped molecule, an
architecture that is almost certainly conserved among species. This
arrangement of the -propeller domains in Aip1p allows for the existence
of multiple adjacent protein binding surfaces oriented at specific angles with
respect to one another. In related proteins with multiple WD repeat-containing
domains, it may be particularly advantageous for binding large substrates,
such as protein complexes, or the simultaneous binding of multiple target
proteins. Another interesting aspect of the structure is that both of the
-propellers in Aip1p contain seven blades, which supports the idea that
a WD repeat structure constructed from multiple seven-bladed -propellers
is more favorable than a single -propeller containing a large number of
blades. A number of other proteins are predicted to contain more than seven WD
repeats, and their structures may also be composed of multiple seven-bladed
-propellers as opposed to a single -propeller constructed from
more than eight blades. The structure of Aip1p may serve as a good model for
the WD repeat regions of these proteins.
One well studied example is human apoptotic
protease-activating factor 1 (APAF1), a
protein that is 24% identical to homologous regions of Aip1p. APAF1 is a
component in a 7-fold symmetrical, 1.4-MDa complex dubbed the apoptosome, a
key complex in the activation of an initiator caspase. A substantial portion
of APAF1 is predicted to be composed of as many as 12 or 13 WD repeats
(33). Cryo-electron
microscrope images of the apoptosome, a heptameric complex containing APAF1
and cytochrome c, reveal that the WD repeat domains of APAF1 form
spokes radiating from the caspase-binding hub of this complex
(34). Although the structure
has been determined at a relatively low resolution (27 Å), the
cryo-electron microscrope electron density of the domains in the spoke regions
is consistent with a clamshell-shaped molecule formed by two -propeller
domains with one or two cytochrome c molecules bound between the
-propeller domains
(34).
Hidden WD Repeats—Because of the diversity found in these motifs, identifying WD repeats by sequence alone appears to be difficult. Varying numbers of repeats have been reported in the Aip1p proteins, ranging from 7 in Xenopus (12) and 9 in Dictyostelium (13) and chick (35) to 10 in C. elegans (14). Based upon sequence and functional conservation among these related proteins, it seems clear that all will fold into a homologous two-domain structure.
Moreover, it is clear from the Aip1p structure that all of the
"extra" blades are derived from hidden WD repeats. Structure-based
sequence alignments of these repeats within Aip1p
(Fig. 4) suggest plausible
albeit weaker homology with the repeat consensus. Structural overlap of these
repeats within the Aip1p model (Fig.
5) confirms that they are authentic with root mean square
deviations for the C positions in the A, B, and C strands of each WD
repeat relative to those in the sixth WD repeat (residues 236–280)
ranging from 0.49 to 1.89 Å. These results collectively suggest that
there may be many of these repeats that have yet to be identified in the
sequence data.
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Aip1p-Actin Interactions—Recent studies have shown that the interactions of Aip1p with F-actin strongly depend on the presence of cofilin (15), implying that Aip1p binds to newly formed barbed ends of actin while cofilin is still bound. However, the specific manner in which Aip1p interacts with F-actin and cofilin is still unclear, as the residues on Aip1p that directly interact with F-actin have not yet been identified. The structures and amino acid sequences of actins are highly conserved, and it seems likely that any residues in Aip1p that directly interact with F-actin would also be conserved. A potential interaction site is the large patch of conserved residues along the circumference and bottom loops of blades 2–5 (Fig. 3). This surface of Aip1p includes the residues Arg-130, Arg-172, Lys-194, and Lys-237, which form a positively charged area that could interact strongly with residues on the surface of F-actin.
A second potential binding area for an actin-cofilin complex is the region
between the two -propellers within the Aip1p clamshell. This space
between the -propeller domains measures 50 Å across the
clamshell and maximally 15 Å deep and, by inspection, appears to be
large enough to accommodate at least part of such a complex. This would
implicate the top surfaces of the -propellers of Aip1p in binding, a
region commonly used by WD repeat proteins to bind partner proteins
(18). The interface between
the two domains of Aip1p consists of hydrophobic pockets formed partly by
residues on loops connecting the two propellers and a number of
hydrogen-bonding interactions that link blades 7 and 8. Due to these
interactions, the hinge region connecting the two -propellers is likely
to be inflexible, preventing significant changes in the orientation of the two
domains relative to one another in the absence of binding partners. Many of
the residues involved in these interactions are conserved in other organisms
as well, implying that any rigidity they impart on the Aip1p interface is
probably also conserved.
A loss of function was observed C. elegans Aip1p mutants
(14) corresponding to G319E
and G357E in S. cerevisiae Aip1p. Based upon the structure of S.
cerevisiae Aip1p, the G319E mutation would cause significant steric
clashes between the mutated residue and either Gln-13, Asn-339, or Arg-358. At
the very least, this mutation would probably result in the displacement of the
B-C loop in blade 7 and the disruption of the hydrogen bonds between Asp-318
and Arg-358 at the -propeller interface. Similarly, the G357E mutation
would cause a steric clash between the mutated residue and the D-A loop
connecting blades 8 and 9. This mutation would probably cause the displacement
of the B-C loop of blade 8, disrupting the interfacial hydrogen-bonding
network formed by Arg-358 and possibly disrupting the interactions formed by
Trp-362. These observations support the hypothesis that disruption of the
-propeller interface and displacement of the -propellers from
their proper orientation prevents Aip1p from recognizing its substrate. A lack
in flexibility may actually aid Aip1p function, possibly making it more
specific for particular actin-cofilin complexes, such as those found at the
barbed ends of actin filaments
(15).
The interaction of Aip1p with F-actin appears to be dependent upon the binding of cofilin, as most of the mutations in actin that should hinder cofilin binding also interrupt the interaction of actin with Aip1p (10). However, a double mutation has been made in actin (E253A, R254A) that disrupts interactions with Aip1p but does not interfere with cofilin-actin interactions (10). These findings suggest that Aip1p interacts with actin directly in subdomain 4 and through cofilin-mediated interactions, and that all of these interactions are crucial for proper Aip1p function (36). It seems likely that one of more of the patches of conserved residues on the surface of Aip1p are involved in these interactions, and more work on the Aip1p-cofilin-actin system is necessary to elucidate their specific nature. In these efforts, the structure of Aip1p can be used as a tool in the identification of key residues that interact with F-actin and cofilin.
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FOOTNOTES |
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* This work was supported by Grant GM66135 from the National Institutes of
Health (to D. K. W.) and the Keck Foundation. The data collection facilities
at Stanford Synchrotron Radiation Laboratory are funded by the U. S.
Department of Energy and the National Institutes of Health. The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
To whom correspondence should be addressed: Section of Molecular and Cellular
Biology, One Shields Ave., University of California, Davis, CA 95616. Tel.:
530-752-1136; Fax: 530-752-3085; E-mail:
dave{at}alanine.ucdavis.edu.
1 The abbreviations used are: Aip1p, actin-interacting protein; Se-Met-Aip1p:
selenomethionine-labeled Aip1p; APAF1, apoptosis protease-activating factor
1.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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