From the Department of Pathology, Emory University,
Atlanta, Georgia 30322, the ¶ Department of Biological Sciences,
Purdue University, West Lafayette, Indiana 47907,
Medical
Research Council Laboratory of Molecular Biology,
Cambridge CB2 2QH, United Kingdom, ** Frederick Douglas High
School, Atlanta, Georgia 30030, the
Microchemical Facility, Winship Cancer
Institute, Emory University, Atlanta, Georgia 30322, and
§§ BIMCORE, Molecular Graphics, Emory University,
Atlanta, Georgia 30322
Received for publication, August 18, 2000, and in revised form, October 16, 2000
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ABSTRACT |
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Actin depolymerizing factor (ADF)/cofilin changes
the twist of actin filaments by binding two longitudinally associated
actin subunits. In the absence of an atomic model of the
ADF/cofilin-F-actin complex, we have identified residues in ADF/cofilin
that are essential for filament binding. Here, we have characterized
the C-terminal tail of UNC-60B (a nematode ADF/cofilin isoform) as a
novel determinant for its association with F-actin. Removal of the
C-terminal isoleucine (Ile152) by carboxypeptidase A
or truncation by mutagenesis eliminated F-actin binding activity but
strongly enhanced actin depolymerizing activity. Replacement of
Ile152 by Ala had a similar but less marked effect; F-actin
binding was weakened and depolymerizing activity slightly enhanced.
Truncation of both Arg151 and Ile152 or
replacement of Arg151 with Ala also abolished F-actin
binding and enhanced depolymerizing activity. Loss of F-actin binding
in these mutants was accompanied by loss or greatly decreased severing
activity. All of the variants of UNC-60B interacted with G-actin in an
indistinguishable manner from wild type. Cryoelectron microscopy showed
that UNC-60B changed the twist of F-actin to a similar extent to
vertebrate ADF/cofilins. Helical reconstruction and structural modeling
of UNC-60B-F-actin complex reveal how the C terminus of UNC-60B might
be involved in one of the two actin-binding sites.
Actin depolymerizing factor
(ADF)1/cofilins are a family
of actin-regulatory proteins ubiquitous among eukaryotes that are essential for rapid turnover of the actin cytoskeleton in
vivo (1-3). ADF/cofilin enhances the turnover of actin filaments
by both increasing the rate of depolymerization from their pointed ends
(4, 5) and by severing them thereby increasing the number of ends
(5-14). These two activities can be uncoupled by point mutations (15,
16). Binding of ADF/cofilin to F-actin changes the twist of the
filaments (17) and leads to destabilization of the lateral contacts of
actin monomers within the filaments (18). This ADF/cofilin-mediated
structural change in F-actin is important for cooperative binding of
ADF/cofilin to F-actin (17, 19), resulting in the coexistence of bare
and ADF/cofilin-decorated actin filaments, as observed in the
lamellipodia of cultured cells (20).
The structural fold of the ADF/cofilin family proteins (21-23) is
similar to that of the individual segments of the gelsolin family (24).
Although the structure of gelsolin segment-1-actin complex has been
solved (25), predicted models of ADF/cofilin-actin complex that were
based on the topology of this complex (21, 26) differed significantly
and can not be used to predict F-actin binding. When one of these
predicted structures was extended to develop models of
ADF/cofilin-F-actin complex (17), the C terminus of ADF/cofilin was
placed away from the actin surface. However, alanine-scanning
mutagenesis of yeast cofilin (27) revealed that helix Mutagenesis of yeast cofilin (27) revealed two actin-binding surfaces
as follows: one that is essential for G-actin binding and a second that
is required for F-actin. The essential G-actin-binding surface includes
the N terminus, a portion of helix There is little detailed information regarding the site on ADF/cofilin
that is required for F-actin binding. Two regions have been implicated
in filament binding as follows: (i) a cluster of charged residues (15,
27) in a loop connecting We previously identified a novel function for the C-terminal tail of
ADF/cofilin (30). The tail is predicted to lie outside helix Homology Modeling of the Structure of UNC-60B--
Homology
modeling of UNC-60B was based on the known structures of
Saccharomyces cerevisiae cofilin (Protein Data Bank code 1cfy) (23), Acanthamoeba castellanii actophorin (Protein Data Bank code 1ahq) (22), and Sus scrofa destrin (Protein Data Bank code 1ak7) (21). Coordinate files were obtained from Protein
Data Bank. The initial alignment, generated by pattern-induced (local) multiple alignment, showed 10% identical and 57% conserved residues between UNC-60B and cofilin, actophorin and destrin (Fig. 1a). A model was built based on this initial alignment using
the program Modeller by Sali and Blundel (39).
Evaluation of the initial model, superimposed with the known
structures, suggested changes in the alignment in the region of the
insertion at UNC-60B Lys58-Lys65. Multiple
cycles of realignment and rebuilding of the models were completed. Side
chain conformations, along the length of the UNC-60B sequence, were
optimized manually to satisfy individual residue structural functions
(i.e. van der Waals packing, ionic interactions, H bonds
between side chain and main chain, etc.) as seen in the known
structures. Additional refinement and minimization of the model are underway.
Proteins--
Recombinant wild-type UNC-60B was purified as
described (40). Rabbit muscle actin was purified as described (41), and G-actin was further purified by gel filtration with Sephacryl S-300 or
purchased from Cytoskeleton Inc. (Denver, CO). We obtained indistinguishable results using either actin preparation. C. elegans actin was purified from wild-type N2 strain (obtained from
the Caenorhabditis Genetics Center, St Paul, MN) as
described (42). Bovine pancreas carboxypeptidase A (CPA) (EC 3.4.17.1)
was purchased from Roche Molecular Biochemicals.
CPA Treatment of UNC-60B--
UNC-60B (1 mg/ml) was incubated
with CPA (20 µg/ml) in a buffer containing 0.1 M KCl, 20 mM HEPES-NaOH, 1 mM dithiothreitol, pH 7.5, at
25 °C for 3 h. The digestion was stopped by adding 1,10-phenanthroline (Fisher) at a final concentration of 0.5 mM.
Electrospray Ionization Mass Spectrometry--
The protein
solution was acidified to pH ~2 with 10% trifluoroacetic acid, and
the protein was chromatographed on a Jupiter-C4 column (1 × 150 mm) equilibrated in 0.1% aqueous trifluoroacetic acid and eluted using
a linear gradient of acetonitrile in 0.08% aqueous trifluoroacetic
acid. The column eluate was monitored at 210 nm, and the fractions
containing the protein (eluting as a major peak at ~30%
acetonitrile) were collected for further analysis. The fractions were
mixed 1:1 with isopropyl alcohol/water/acetic acid (50:50:1) and
directly infused at a flow rate of 15 µl/min into a MicroIon source
of model API3000 triple quadrupole mass spectrometer. Following spectra
deconvolution (using the BioSpec BioReconstruct routine), the mass of
the protein was determined.
N-terminal Sequence Analysis--
The proteins were subjected to
automated Edman degradation in a Procise-cLC sequencer (PE Biosystems)
using a standard manufacturer's protocol.
Mutant UNC-60B Proteins--
Mutations were introduced in
synthetic oligonucleotides that complement the 3'-end of the coding
sequence of the cDNA for UNC-60B. The cDNA fragments that carry
mutations were amplified by polymerase chain reaction using these
mutant primers and a common forward primer
(5'-GATCCCATGGCTTCCGGAGTCAAAGTTG). The amplified DNA fragments were
digested by NcoI and BamHI at the sites
introduced by the primers and cloned into pET-3d (Novagen, Madison,
WI). The sequences of the inserts were verified by DNA sequencing not to contain any polymerase chain reaction-induced errors. The mutant primers used are 5'-CTAGGGATCCTTATCTTTGGTTGGACATCAGGTC for Actin-binding Assays--
Copelleting assays of UNC-60B with
rabbit muscle F-actin were performed as described previously (40) in a
buffer containing 0.1 M KCl, 2 mM
MgCl2, 20 mM HEPES-NaOH, 1 mM
dithiothreitol, pH 7.5. All protein solutions except F-actin were
clarified by ultracentrifugation prior to the pelleting assays.
Ultracentrifugation was performed with a Beckmen Airfuge at 28 pounds/square inch for 20 min. Assay for nucleation of actin
polymerization was performed as described previously (30) except that
rabbit muscle actin was used as seeds in this study.
Nondenaturing Polyacrylamide Gel
Electrophoresis--
Nondenaturing polyacrylamide gel electrophoresis
was performed as described by Safer (43). CaATP-G-actin and UNC-60B
were incubated in G-buffer (2 mM Tris-HCl, 0.2 mM CaCl2, 0.2 mM dithiothreitol, 0.2 mM ATP, pH 7.5) for 30 min at room temperature. The
samples were then supplemented with 0.25 volume of a loading
buffer (50% glycerol, 0.05% bromphenol blue) and electrophoresed
using a Bicine/triethanolamine buffer system. The proteins were
visualized by staining with Coomassie Brilliant Blue R-250 (Sigma).
Cryoelectron Microscopy and Modeling of the Structure of
UNC-60B-F-actin Complex--
Decorated filaments were prepared in one
of two ways. In most cases, pre-formed actin filaments (3 µM) were incubated at room temperature for ~2 h with 10 µM UNC-60B in 20 mM MOPS, pH 7, 0.1 M KCl, 2 mM MgCl2, 1 mM
dithiothreitol, 0.5 mM EGTA. Although ATP was present in
the polymerized F-actin solution, no additional ATP was added upon
dilution into the UNC-60B-containing buffer, resulting in a final
concentration of <0.04 mM ATP. For partial decoration of
filaments, 10 µM F-actin was decorated on ice overnight in the presence of 10 µM UNC-60B using the same buffer
conditions and diluted to a final actin concentration of 2.5 µM just prior to plunging. Filament decoration was
confirmed by pelleting assays in a Beckman Airfuge at 20 pounds/square
inch for 15 min followed by SDS-polyacrylamide gel electrophoresis.
Decorated filaments were rapidly frozen on holey carbon films in ethane
slush cooled with liquid nitrogen and then imaged using a JEOL 1200EX
transmission electron cryomicroscope at the National Center for
Macromolecular Imaging at Baylor College of Medicine. Micrographs were
recorded at an accelerating voltage of 100 kV and a nominal
magnification of × 30,000.
Suitable micrographs were digitized using a Zeiss SCAI film scanner at
a final resolution of 4.7 Å per pixel. Tobacco mosaic virus particles
(0.05 mg/ml; kindly provided by Dr. Ruben Diaz, Florida State
University) were included in the samples to provide an internal
magnification standard. The defocus of each micrograph was determined
by incoherent averaging of regions of protein embedded in ice (44). The
micrographs used in this study were recorded at
For image reconstructions, layer line data were collected to a
radial resolution of 0.032 Å
Pseudo-atomic models of UNC-60B-F-actin were derived using the actin
subunits from the Lorenz filament model (twisted to match the
UNC-60B-induced actin structure) and the homology-based model of
UNC-60B. The UNC-60B model structure was positioned interactively using
the programs O (48) and Iris Explorer (Numerical Algorithms Group,
Downers Grove, IL) running on a Silicon Graphics Indigo R4000 work
station (Mountain View, CA). The goal was to produce a model that fit
inside the envelope of the electron microscopy reconstruction well and
also taking into account available genetic and biochemical data
concerning the cofilin residues involved in actin contacts (27, 28, 30,
31). Ribbon diagrams were generated using Ribbons 2.65 (49), saved as
Inventor format files, and displayed in IRIS Explorer.
Homology Modeling of the Structure of UNC-60B--
The structure
of UNC-60B was modeled based on the known structures of three
ADF/cofilin proteins. The model presented in Fig. 1c incorporated the tertiary
structures of the known ADF/cofilin proteins, a three-layer Ile152 of UNC-60B Is Required for Maintaining Stable
Association with F-actin--
The C-terminal isoleucine at position
152 of UNC-60B was removed by carboxypeptidase, and its effect on the
activity of UNC-60B was examined. We found that CPA removed only
Ile152 because Arg151 is not a substrate of
CPA. Removal of Ile152 was confirmed by electrospray mass
spectrometry. Molecular mass of control UNC-60B was determined as
16,919 atomic mass units that corresponded to residues 2-152 of
UNC-60B (the predicted mass, 16,915 atomic mass units). However, the
spectrum of CPA-treated UNC-60B yielded a major peak of 16,806 atomic
mass units and a minor one of 16,921 atomic mass units. These
correspond to residues 2-151 (the predicted mass, 16,802 atomic mass
units) and 2-152, respectively. The mass of the major peak was
consistent with that of UNC-60B-
Interaction of CPA-treated UNC-60B with F-actin was surprisingly
different from that of control UNC-60B. Whereas control UNC-60B cosedimented with rabbit muscle F-actin without significantly depolymerizing it (Fig. 2a, lane
2, compare with lane 1) (40, 42), CPA-treated UNC-60B
increased the amount of unpolymerized actin in the supernatant and did
not cosediment with F-actin (Fig. 2a, lane 3). Thus,
CPA-treated UNC-60B depolymerizes F-actin and prevents
re-polymerization. CPA itself did not cause actin depolymerization (Fig. 2a, lane 4). Furthermore, preincubation of CPA with
its inhibitor, 1,10-phenanthroline, abolished the ability of CPA to change the activity of UNC-60B (Fig. 2a, lane 5). These
results indicate that removal of the C-terminal Ile from UNC-60B
converts it from a filament-binding protein to a depolymerizing factor. Nearly complete depolymerization of F-actin was achieved at molar ratios at greater than 1:1 of CPA-treated UNC-60B to actin monomer (Fig. 2b). CPA-treated UNC-60B also showed enhanced actin
depolymerizing activity using C. elegans actin as compared
with control UNC-60B (data not shown). Since rabbit muscle actin
consists of a single isoform that has been very well characterized, we
used rabbit muscle actin in the biochemical experiments to correlate
better the biochemical data with the structural interpretation as
described below.
In contrast to the effects on F-actin, CPA treatment of UNC-60B did not
affect its interaction with G-actin. Both control UNC-60B and
CPA-treated UNC-60B were resolved as a single band on nondenaturing
polyacrylamide gels (Fig. 3, lanes
2-5), whereas actin gave a doublet and smeared bands (Fig. 3,
lane 1). When actin and either control or CPA-treated
UNC-60B were mixed, a third band appeared (Fig. 3, lanes 6-9,
arrows). The intensity of this band increased as the concentration
of UNC-60B was increased (Fig. 3, lanes 6-9). This increase
was accompanied by a decrease in the intensities of both actin and
UNC-60B bands. These results indicate that the third band represents
the actin-UNC-60B complex. Results were similar with control or
CPA-treated UNC-60B (compare Fig. 3, upper and lower
panels), suggesting that CPA treatment of UNC-60B did not affect
its G-actin binding activity.
The role of Ile152 of UNC-60B was further investigated by
using recombinant mutant UNC-60B proteins. Mutant UNC-60B-
To investigate further the significance of the isoleucine residue at
position 152, we produced the mutant UNC-60B-I152A in which
Ile152 was replaced with Ala. UNC-60B-I152A showed weaker
binding to F-actin than wild-type UNC-60B but had increased
depolymerizing activity compared with the control (compare Fig. 4,
a and c). In the presence of 30 µM
UNC-60B-I152A, 6.6 ± 0.24 µM actin was precipitated, and with 30 µM wild-type, 8.0 ± 0.07 µM actin was precipitated (Fig. 4, c and
a, respectively). UNC-60B-I152A was indistinguishable from
wild type in its interactions with G-actin (data not shown). These
results suggest that Ala can partially substitute for Ile at position
152 but is not as efficient as Ile in maintaining stable association
with F-actin. Taken together, these data demonstrate the importance of
Ile152 for UNC-60B to make a stable complex with F-actin
and inhibit filament disassembly.
Arg151 of UNC-60B Is Required for F-actin Binding and
Efficient Actin Depolymerization--
We investigated the roles of
adjacent C-terminal amino acids of UNC-60B using bacterially expressed
mutant UNC-60B proteins with additional truncations at the C terminus.
Mutant UNC-60B-
We further characterized the role of Arg151 using
UNC-60B-R151A in which Arg151 was replaced with Ala.
UNC-60B-R151A cosedimented poorly with F-actin and showed significant
actin-depolymerizing activity despite the presence of
Ile152 (Fig. 4f). This suggests that
Arg151 is required for F-actin binding by UNC-60B, whereas
Ile152 has an accessory function. Interestingly, there were
quantitative differences in the depolymerizing activities of
UNC-60B-R151A and the truncated mutants. At molar ratios <1:1,
UNC-60B-R151A depolymerized actin to lesser extents than UNC-60B- Both Arg151 and Ile152 of UNC-60B Are
Required for F-actin Severing--
We explored the role of the
C-terminal tail of UNC-60B in severing actin filaments, and we found
that both Arg151 and Ile152 are required for
this activity. Wild-type UNC-60B increased the ability of F-actin seeds
to nucleate actin polymerization by 2.5-fold (Fig.
5), which is strong evidence that UNC-60B
increased the number of ends by severing filaments. However, this
activity was greatly reduced using UNC-60B-
Arg151 is also essential for the severing activity of
UNC-60B. Either truncation (UNC-60B- Structure of UNC-60B-F-actin Complex--
We first confirmed that
UNC-60B altered the F-actin twist by computing the Fourier transforms
of decorated filaments and measuring the height of the first layer line
that arises from the twist of the two-start actin helix. UNC-60B
produced a mean filament twist (expressed as rotation per subunit) of
161.6° for C. elegans F-actin. Filaments prepared using
rabbit muscle actin were twisted to a mean twist of 161.7° per
subunit. Thus, as was found with human cofilin and human ADF (17),
UNC-60B reduces the filament crossover length to about 270 Å irrespective of the source of the actin. Electron cryomicroscopy of
filaments decorated with sub-saturating amounts of UNC-60B confirmed
that the C. elegans protein shares the ability of the human
protein to bind filaments cooperatively.
A three-dimensional reconstruction was calculated from electron
micrographs of frozen-hydrated UNC-60B-F-actin filaments essentially as
described previously (17) (Fig.
6a). This reconstruction was
used to provide a molecular envelope into which the UNC-60B and actin
subunit could be positioned (Fig. 6b). In addition to using
the reconstruction as a constraint, available biochemical and genetic
data implicating the long helix
The resulting orientation is similar to an earlier model of human ADF
in terms of the long helix involvement and its "polarity" relative
to the filament. Residues Val104-Ser113 of the
long helix are still in proximity to actin, but the remaining 7 residues (Val114-Ser120) are rotated away from
the filament. Interestingly, the portion of helix In this study, we show that alterations of UNC-60B at the C
terminus inhibit F-actin binding and severing without changing G-actin
binding. We found that F-actin binding of UNC-60B (Fig. 4) was
correlated with the extent of filament severing (Fig. 5). This suggests
that severing occurs as a consequence of physical distortions in the
helix that arise as a result of the change in twist induced when these
proteins bind to the side of filaments (Fig. 6). This is consistent
with previously suggested models (17, 18).
Cryoelectron microscopy and structural modeling of UNC-60B-F-actin
complex suggest that the C terminus is involved in the second
actin-binding site. The alterations at the C terminus are not likely to
cause major disruption of the structural fold, because we were unable
to detect any changes in far UV circular dichroism or in their
interactions with G-actin. However, we cannot exclude the possibility
of minor conformational changes caused by these alterations. The
structures of the C-terminal regions of yeast cofilin (23) and
actophorin (22) are disordered in the crystals, which suggests that the
C terminus is not well ordered although it may be stabilized upon
binding to F-actin. The fact that Ile152 is a substrate of
CPA indicates that this residue is exposed on the surface of the
molecule and readily accessible to its active site.
Our reconstructions and structural modeling suggest that both the N and
C termini are positioned near the actin interface, but the predicted
interactions they make with the filament are very different. The N
terminus is positioned near the subdomain 1 of the upper actin subunit,
whereas the C terminus falls near subdomain 2 of the lower actin. Since
cofilin and profilin compete for binding to monomeric actin (8, 50),
and profilin binds on the "bottom" of actin between subdomains 1 and 3, this implies that the N terminus, but not the C terminus, is
involved in G-actin interactions. The C terminus, on the other hand,
primarily influences F-actin interactions. Mutational analysis of human
cofilin suggested that F-actin binding occurs initially through this
site with the lower actin, followed by interaction with the upper actin
subunit via the G-actin site (15). Interestingly, subdomain 2 is
generally accepted as a molecular "sensor" for the nucleotide state
(51, 52). The finding that F-actin binding by actophorin increases the
rate of phosphate release (12) raises the possibility that the C
terminus is directly involved in the nucleotide sensitivity of cofilin
for actin. It will be interesting to find out whether the C terminus is
involved in the activation of phosphate release. If the C-terminal tail
of UNC-60B is flexible, preliminary molecular graphic simulations
suggested that it could extend into the ATP-binding pocket of actin and
interact with the nucleotide-binding site.
By using these mutants, we have also shown enhanced depolymerizing
activity. The mechanism by which this occurs is uncertain, but our
preliminary experiments using gelsolin-capped filaments show that these
mutants activate subunit dissociation from the pointed ends of
filaments. The extent of depolymerization differed between different
mutants even in the absence of any severing. This supports recent
reports that depolymerization and severing activities of ADF/cofilin
can be uncoupled (15, 16). Detailed kinetic analysis of
depolymerization by our mutant versions of UNC-60B should provide
further insight into this mechanism.
Our data suggest that the C terminus of ADF/cofilin is the structural
determinant of the quantitative differences in F-actin binding and
actin-depolymerizing activities that have been observed for different
ADF/cofilin family members (5, 7, 19, 40, 53). The C-terminal region is
highly divergent in both sequence and length (54). For example, human
ADF is a much more potent depolymerizing agent than human cofilin (55),
and the largest region of sequence divergence is located in the
C-terminal 20% of the protein. Moreover, two C. elegans
isoforms, UNC-60A and UNC-60B, show far greater sequence divergence and
functional difference than the two human isoforms, with the biggest
differences in sequence seen near the C terminus (40, 56). It will be
interesting to examine the structural basis of these differences and
the extent to which various C-terminal sequences are involved in
functional diversity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4 near the C
terminus (Fig. 1b) is a part of the actin-binding surface
that confers filament binding.
3, and the turn connecting strand
6 and helix
4 (27) (Fig. 1b). Helix
3 includes two
highly conserved basic residues that have been implicated in monomer
binding in several ADF/cofilin proteins by chemical cross-linking (28),
mutagenesis (16, 29, 30), and peptide competition (28, 31). The N
terminus is also involved in actin binding (32), and this region
includes the phosphorylation site Ser-3 (Ser-6 in plant ADF) that is
responsible for phosphorylation-dependent inactivation of
actin binding by ADF/cofilin (33-36). These regions of the molecule
are close together in the three-dimensional structures and probably
form a binding surface that interacts with subdomains 1 and 3 of
G-actin (21-23).
2 and
3 in destrin and at a similar
position at the beginning of
5 in yeast cofilin, and (ii) a group of
charged residues in helix
4 in yeast cofilin (27). In addition,
mutations of conserved tyrosine residues (Tyr67 and
Tyr70 in maize ADF3) in the apolar core of the molecule
results in uncoupling of monomer and filament binding activities
(37).
4 (Fig.
1c) and is not included in the crystal structures of either
yeast cofilin (23) or Acanthamoeba actophorin (38), suggesting structural flexibility in this region. A mutant form of
UNC-60B (a Caenorhabditis elegans ADF/cofilin isoform) that lacks three C-terminal amino acids (Fig. 1c) shows loss of
both F-actin binding and severing activity but still depolymerizes F-actin, possibly through its monomer sequestering activity (30). Importantly, a C. elegans strain that expresses this mutant
is defective in proper assembly of actin into myofibrils (30), indicating that the C-terminal tail of UNC-60B is functionally significant in vivo. Therefore, in this study, we
characterized the role of the C-terminal tail of UNC-60B in its actin
regulating activity.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
I, 5'-CTAGGGATCCTTATTGGTTGGACATCAGGTCGC for
RI,
5'-CTAGGGATCCTTAGATTCTTTAGTTGGACATC for
QRI,
5'-CTAGGGATCCTTAGGCTCTTTGGTTGGACATC for I152A, and
5'-CTAGGGATCCTTAGATTGCTTGGTTGGACATC for R151A. The mutant UNC-60B
proteins were expressed and purified as described previously for
wild-type UNC-60B (40).
2.1- and
2.6-µm
defocus. Filament images were analyzed using the helical image
processing package PHOELIX (45, 46). Mean crossover lengths were
measured from positions of the first layer line in computed diffraction
patterns of computationally straightened filaments.
1 (within the
first node of the contrast transfer function) and separated into
near and far side data sets. Filaments that best conformed to the 20:9
selection rule and that showed good symmetry between the near and far
sides of the Fourier transform were selected for further processing. A
total of 22 data sets were averaged, and a three-dimensional structure
was calculated by Fourier-Bessel inversion (47) to a resolution limit
of 0.032 Å
1 radially using the following
layer lines (l, n): 0, 0; 2, 2; 4, 4; 5,
5; 7,
3; 9, -1;
11, 1; 13, 3; 15, 5. The mean phase residuals during the last round of
alignment of the 22 data sets were 31.8° and 47.6° for the correct
and incorrect polarity, respectively. The resulting reconstruction was
aligned to a previously calculated F-actin reconstruction (17) in
Fourier space to aid in model building.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
-
sandwich, with an internal five-stranded
-sheet. Significant
differences in the model resulted from insertions in the UNC-60B
sequence between helix
2 and strand
3 (UNC-60B Lys58-Lys65 inserted between Pro58
and Asn60 of cofilin) and between strands
3 and
4
(UNC-60B Arg80-Thr86 inserted between
Gly75 and Gly78 of cofilin). There is also an
addition of 4 residues at the C terminus (UNC-60B
Asn149-Ile152), the structure of which is
modeled after the destrin C-terminal structure (residues
Leu156-Ala159).
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Fig. 1.
Homology modeling of the structure of
UNC-60B. a, sequence alignment of UNC-60B, yeast
cofilin (Y-cof), actophorin (Actoph), and porcine
destrin. Locations of helices 1-4 are indicated by bold
and underlined letters. Colored residues correspond to the
ones in b and c and are explained below.
b, crystal structure of yeast cofilin (Protein Data Bank
code 1cfy). Essential residues for actin-binding are shown in
red, and residues that confer F-actin binding are shown in
yellow (27). Note that residues 1-5 and 140-143 are not
visible in the crystal structure. Therefore, Val6 is
colored red instead of the essential residues 1-5.
c, a predicted structure of UNC-60B. Missense mutations that
have been reported by Ono et al. (30) are shown in
blue. Residues in the C-terminal tail (C-tail) that are
truncated in the unc-60(r398) mutation are shown in
green. The coordinates of this model (unc60b_model) are
available as Supplemental Material.
I (the experimentally determined
mass, 16,803 atomic mass units), a mutant lacking Ile152.
Area calculations of the peaks from the mass spectra suggested that
~17% of the total UNC-60B was not processed with CPA assuming that
both forms were ionized to the same extent. N-terminal sequencing of
CPA-treated UNC-60B yielded the identical sequence to control UNC-60B
starting from Ala2, indicating that there was no detectable
amino- or endopeptidase activity in the CPA preparation. In addition,
there was no evidence of major alteration in secondary structure as
examined by circular dichroism (plots not shown), providing evidence
that removal of Ile152 does not significantly disrupt the
structure of the C-terminal helix
4.
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[in a new window]
Fig. 2.
Actin-regulating activity of CPA-treated
UNC-60B. a, copelleting assay of F-actin with UNC-60B.
F-actin suspension (10 µM) was incubated with buffer
(lane 1), control UNC-60B (10 µM) (lane
2), CPA-treated UNC-60B (10 µM) (lane 3),
CPA (lane 4), or phenanthroline/CPA-treated UNC-60B (10 µM) (lane 5). The mixtures were
ultracentrifuged, and the supernatants (s) and pellets
(p) were analyzed by SDS-polyacrylamide gel electrophoresis.
The positions of actin and UNC-60B are indicated by A and
U, respectively. Molecular mass markers in kDa are indicated
on the left. b, quantitative analysis of the
pelleting assay of F-actin (10 µM) with varied
concentrations of control (triangles) or CPA-treated UNC-60B
(circles). On the vertical axis are plotted the
sedimented portions (expressed as concentrations assuming that the
pellets were reconstituted in the original volumes) of actin
(open symbols) and control or CPA-treated UNC-60B
(filled symbols).
View larger version (83K):
[in a new window]
Fig. 3.
G-actin binding of control and CPA-treated
UNC-60B. Interactions of G-actin with control UNC-60B or
CPA-treated UNC-60B at indicated concentrations were analyzed by
nondenaturing polyacrylamide gel electrophoresis. In the presence of
both proteins, the complex of the two proteins emerged as the third
bands (arrows). The positions of actin and UNC-60B are
indicated by A and U, respectively.
I, which lacks Ile152, showed substantial actin-depolymerizing
activity and cosedimented poorly with F-actin (Fig.
4b). This is in marked
contrast to wild-type which binds F-actin but does not significantly
depolymerize it (Fig. 4a). These results are consistent with
those using CPA treatment, except that CPA-treated UNC-60B exhibited
stronger actin-depolymerizing activity than UNC-60B-
I.
UNC-60B-
I was indistinguishable from CPA-treated UNC-60B in its
interaction with G-actin, molecular mass, and N-terminal sequence (data
not shown). Treatment of UNC-60B-
I with CPA did not change its
behavior (data not shown), suggesting that the quantitative difference
may reflect slightly different folding of the bacterially expressed
I mutant compared with CPA-treated UNC-60B. Incomplete processing of
UNC-60B with CPA (17% of UNC-60B remained intact) is unlikely to cause
this difference because a mixture of control UNC-60B and UNC-60B-
I
at the equivalent ratio showed no difference in actin depolymerizing
activity compared with UNC-60B-
I alone (data not shown).
View larger version (18K):
[in a new window]
Fig. 4.
Interactions of mutant UNC-60B with
F-actin. F-actin (10 µM) was incubated with various
concentrations of wild-type (a), I (b), I152A
(c),
RI (d),
QRI (e), or R151A
(f) UNC-60B variants. The mixtures then were examined by
copelleting assays. On the vertical axes are plotted the
sedimented portions (expressed as concentrations assuming that the
pellets were reconstituted in the original volumes) of actin
(closed circles) and wild-type or mutant UNC-60B (open
circles). Alterations of UNC-60B at the C terminus (the C-terminal
sequences are shown at the top of each panel) cause variable
F-actin binding and depolymerizing activities, although all the
variants similarly interact with G-actin in nondenaturing
polyacrylamide gel electrophoresis (data not shown). Data shown are
means ± S.D. of three experiments.
RI cosedimented poorly with F-actin but showed
considerable depolymerizing activity. This mutant was not as effective
as UNC-60B-
I (compare Fig. 4, b and d),
suggesting that Arg151 is needed for efficient
depolymerizing activity. Additional truncation of Gln-150
(UNC-60B-
QRI, formerly designated as the r398 mutant (30)) did not cause any detectable difference in the depolymerizing activity compared with UNC-60B-
RI (Fig. 4e).
UNC-60B-
RI and UNC-60B-
QRI were indistinguishable from wild type
in their interaction with G-actin (data not shown).
I,
-
RI, or -
QRI. However, at higher molar ratios (>2:1), the extent
of depolymerization by UNC-60B-R151A was greater than that of
UNC-60B-
RI, or -
QRI, but slightly weaker than UNC-60B-
I. Thus,
it is possible that these mutants affect actin depolymerization in
different ways.
I or UNC-60B-I152A (Fig.
5). The severing activity of these mutants correlates with their weaker F-actin binding activity (Fig. 4c). CPA-treated UNC-60B has
no effect on the nucleation rate (Fig. 5), which again suggests
quantitative differences between CPA-treated UNC-60B and recombinant
UNC-60B-
I. Thus, Ile152 of UNC-60B is required for both
filament binding and efficient severing.
View larger version (18K):
[in a new window]
Fig. 5.
Effects of mutant UNC-60B on the nucleating
activity of F-actin seeds. F-actin (10 µM) was mixed
with the UNC-60B variants at various molar ratios and used as nuclei at
1.25 µM to induce polymerization of pyrene-labeled
G-actin. Nucleation rates were determined as the initial rates of the
increase in the pyrene fluorescence, which is proportional to the
number of free barbed ends. The data are expressed as relative
nucleation rates to that of F-actin alone and are means of three
experiments. Error bars are not included in the figure because they
make the graph difficult to read. Standard deviations of these data are
less than 0.27.
RI and -
QRI) or replacement
with Ala (UNC-60B-R151A) abolished this activity (Fig. 5). These
results suggest that both Arg151 and Ile152 are
essential for actin severing activity by UNC-60B.
3, the N terminus, and the
C-terminal residues (27, 28, 30, 31) were used to help orient the
UNC-60B molecule on the filament.
View larger version (63K):
[in a new window]
Fig. 6.
Structural model for UNC-60B binding
to F-actin. a, reconstruction of UNC-60B-F-actin
filament showing four filament crossovers (40 subunits). b,
model of UNC-60B binding to F-actin. Two actin subunits (red
and white ribbons) from the filament are shown. The
asterisks mark the positions of Ser112
(red), Ser113 (blue), and
Arg151 (black). The coordinates of this model
are available as Supplemental Material (actin_up, the upper actin;
actin_low, the lower actin; unc60b_comp, UNC-60B.
3 that falls near
actin includes Ser112 and Ser113; replacement
of either of these residues by apolar groups alters F-actin binding
(30). Significantly, both the N and C termini are positioned near the
actin interface in the new orientation, consistent with a number of
biochemical and genetic studies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Sharon Langley for technical assistance in DNA sequencing. Instrumentation Grants RR12878 and RR13948 were from the National Center for Research Resources of the National Institutes of Health to Emory Microchemical Facility. The electron microscopy facilities at Baylor College of Medicine are supported by a grant (to Wah Chiu) from the National Center for Research Resources of the National Institutes of Health.
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FOOTNOTES |
---|
* This work was supported by the American Heart Association Southeast Affiliate Grant 9960146V (to S. O.), the National Science Foundation Grant MCB-9728762 (to G. M. B.), and National Institutes of Health Grant GM59677 (to A. M.).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 on-line version of this article (available at
http://www.jbc.org) contains coordinates and structures. To view these
PDB files, you may use Rasmol software (http://www.rasmol.org).
§ To whom correspondence should be addressed: Dept. of Pathology, Emory University, 1639 Pierce Dr., Woodruff Memorial Bldg., Rm. 7109C, Atlanta, GA 30322. Tel.: 404-727-3916; Fax: 404-727-8540; E-mail: ono@bimcore.emory.edu.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M007563200
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ABBREVIATIONS |
---|
The abbreviations used are: ADF, actin depolymerizing factor; CPA, carboxypeptidase A; Bicine, N,N-bis(2-hydroxyethyl)glycine; MOPS, 4-morpholinepropanesulfonic acid.
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