 |
INTRODUCTION |
Arguably the most regulated cytoskeletal protein, filamentous
actin (F-actin) is critical for cellular motility and mechanical strength, protein sorting and secretion, signal transduction, and cell
division. An atomic resolution understanding of the structure of
F-actin is a tantalizing goal. The tendency of monomeric actin to form
polymers of varying lengths has prevented crystallization and atomic
resolution structural analysis of F-actin. To date, all but one of the
atomic resolution structures of actin are of the monomer bound to
proteins that prevent polymerization, such as DNaseI (2), GS-1 (3),
profilin (4, 5), and, most recently, vitamin D-binding
protein (6, 7). A structure of uncomplexed monomeric actin labeled with
the dye tetramethylrhodamine (8) reveals that actin-binding proteins
did not alter the structure of the actin monomer significantly. The
structure of an antiparallel actin dimer has been determined (9), but
how this structure relates to helical F-actin is unclear.
A model describing F-actin as a helical polymer has been proposed based
on fitting the crystal structure of monomeric actin into x-ray fiber
diffraction data from oriented actin gels (1). Schutt and co-workers
(4, 5, 10) have constructed a topologically different helical model for
F-actin by twisting the ribbon-like assembly of actin molecules found
in profilin-actin crystals. Transitioning between twisted and untwisted
forms of this ribbon was proposed to be a basis for contractile force
generation (11-13). To date, the ribbon actin assembly has been
observed only in the crystals of profilin-actin heterodimers (4, 5,
10).
A key component to the dynamic assembly and disassembly of actin
filaments is the intrinsic hydrolysis of bound ATP. This hydrolysis
promotes the dissociation of actin subunits from filament ends.
In vivo, actin assembly and disassembly are regulated
tightly by binding proteins that likely respond to changes in F-actin structure accompanying ATP hydrolysis (14, 15).
Our goal was to obtain F-actin fragments of defined length for
biochemical and high resolution structural analysis. We therefore linked successive actin protomers in F-actin. A specific covalent link
is formed between Cys-376 and Lys-193 (in this paper, amino acid
residue numbers correspond to chicken skeletal
-actin (16)) of
adjacent actin protomers in the filament by
N,N'-1,4-phenylenedismaleimide (pPDM)1 (17-19). The ability
of purified covalently cross-linked actin oligomers to nucleate actin
assembly has been described (20). However, these actin oligomers form
filaments under crystallization conditions and are therefore unsuitable
for crystallographic analysis.
To block assembly of the purified actin oligomers, we used recombinant
segment-1 of gelsolin (GS-1) (21-24) to generate GS-1-bound cross-linked actin oligomers. Because of its strong actin monomer binding activity (Kd = 5 nM) (21-24),
GS-1 was expected to facilitate depolymerization by both sequestering
monomeric G-actin and binding to the fast growing barbed end of newly
formed F-actin fragments. Here, we show that mixtures of the
cross-linked GS-1-bound actin oligomers can be fractionated readily
according to their size and crystallized. We report the crystal
structure of the GS-1-bound cross-linked actin trimer to 2.2-Å
resolution. Although this structure is incompatible with the filament
structure proposed by Schutt and co-workers (11-13), it is
topologically similar to the helical model of F-actin of Holmes
et al. (1). However, the GS-1-bound actin trimer has a
distorted symmetry compared with that of the helical model (1). Our
data show that this distortion results from intercalation of GS-1
between actin protomers in the filament.
An ability of GS-1 to intercalate between actin protomers and sever
actin filaments was suggested by earlier work with chymotryptic segment-1 of gelsolin (25). However, subsequent work (26) indicated
that the chymotryptic GS-1 samples might been contaminated with a small
amount of a longer fragment, containing segments-1-3 of gelsolin,
which was responsible for the observed severing activity. Therefore it
is commonly believed that GS-1 alone has no severing activity (3, 27).
However, the lack of F-actin severing activity of GS-1 has never been
verified using homogeneous samples of recombinant GS-1. Combined
structural and biochemical data presented and discussed in this work
show that segment-1 of gelsolin does indeed sever actin filaments,
although the severing activity of GS-1 is significantly lower than that
of full-length gelsolin (28).
 |
MATERIALS AND METHODS |
Protein Preparation--
GS-1 was purified by from
Escherichia coli by overnight growth and expression
in the absence of inducing agents employing pGS1.36 (a generous gift of
B. Pope and A. G. Weeds, MRC Laboratory of Molecular Biology,
Cambridge). Cells were lysed in Lysis Buffer A (4 mM
Tris-HCl, pH 8.0, 25% sucrose (w/v), 4 mM EDTA, 1 mM benzamidine, 0.5 mM phenylmethylsulfonyl
fluoride) by two passes through a French Press at 16,000 p.s.i.
The cell lysate was then cleared by centrifugation at 19,500 rpm in a
JA-20 rotor (Beckman) for 20 min. The supernatant was passed through a
20-ml DEAE column equilibrated in Buffer 1 (2 mM Tris-HCl,
pH 8.0, 0.4 mM EDTA, 0.4 mM CaCl2,
3 mM
-mercaptoethanol), and the flow-through was loaded
immediately onto a 100-ml Affi-Gel Blue column (Bio-Rad) equilibrated
in Buffer 1. This was then developed with a linear gradient of NaCl to
0.5 M in Buffer 1. GS-1 eluted from this column was ~95%
pure as judged by SDS-PAGE. GS-1 eluted from Affi-Gel Blue was
denatured in 6 M urea and dialyzed overnight into 2 mM Tris-HCl, pH 8.0, 0.5 mM CaCl2.
The renatured protein was then separated by Superdex-200 gel filtration
chromatography in Buffer 1 containing 20 mM NaCl. This was
stored at
80 °C in Buffer 1 with 20 mM NaCl without
concentration for several months without any loss in activity. We
typically obtained about 100 mg of purified GS-1 per liter of cell culture.
Actin was prepared from chicken muscle as described previously (29).
Pyrenyl-actin was prepared as outlined (30). Actin was polymerized and
cross-linked with pPDM (Aldrich) at a molar ratio of 1.5 pPDM:actin according to published methods (18) employing a 25 mM solution of pPDM in DMF. Cross-linked F-actin was then
harvested by centrifugation at 45,000 rpm for 60 min in a Ti60 rotor (Beckman).
Purified GS-1 in Buffer 1 with 20 mM NaCl was added to the
cross-linked F-actin pellet at a 2:1 molar ratio and incubated for 30 min at 25 °C. Typically, we employed 100 mg of cross-linked F-actin
and depolymerized it with 30 ml of 2.5 mg ml
1 GS-1. The
reaction mixture was sedimented at 45,000 rpm for 60 min using a Ti60
rotor (Beckman), and the resulting 30-ml supernatant was applied to a
Mono-Q 10/10 column (Amersham Biosciences). GS-1 bound oligomers
of actin eluted from the Mono-Q according to size employing a gradient
(0.175 to 0.375 M) of NaCl in Buffer 1. To increase the
purity of each oligomer, the gradient was held when each oligomer began
to elute. The gradient was held until baseline was achieved after the
protein had eluted. Oligomers were characterized using SDS-PAGE and
mass spectrometry (Voyager-DETM; PerSeptive Biosystems).
For each oligomer, both an upper and lower cross-linked band is
observed with SDS-PAGE, as reported previously (18, 31, 32). The nature
of these two species is not clear; however, it is thought that the
upper band corresponds to oligomers that are cross-linked between
Lys-193 and Cys-376 whereas the lower band may represent cross-linking
between Cys-376 of one actin molecule and Cys-376 of another. The
GS-1-complexed actin oligomers were then concentrated to ~12 mg
ml
1 employing Centricon-10 concentrators (Amicon). The
GS-1-complexed actin trimer was used fresh for crystallization
experiments. Protein concentrations were determined with Bio-Rad
protein dye reagent employing G-actin as the standard.
Actin Polymerization and Depolymerization Assays--
G-actin
(15% pyrenyl-G-actin) was polymerized in 50 mM KCl, 2 mM MgCl2, 1 mM EGTA, and 0.1 mM ATP in 25 mM Tris-HCl, pH 8.0, overnight. To
measure the depolymerization of F-actin in the presence of GS-1,
various concentrations of GS-1 were added to 2.5 µM
F-actin (15% pyrenyl-F-actin), and the fluorescence was monitored in a Aminco Bowman Series 2 fluorimeter with an excitation wavelength of 347 nm and emission wavelength of 407 nm. To avoid photobleaching, a 0°
polarizing filter was used for the excitation beam with a 1-nm slit
width. All solutions and reagents were equilibrated to 25 °C before
assays were performed. 5 mM CaCl2 was added by removing the cuvette from the fluorimeter, mixing, and reinserting the
cuvette into the fluorimeter.
To measure F-actin polymerization in the presence of GS-1-bound
cross-linked actin oligomer, 5 µM G-actin (15%
pyrenyl-G-actin) was polymerized in 50 mM KCl, 2 mM MgCl2, 1 mM EGTA, and 0.1 mM ATP in 25 mM Tris-Cl, pH 8.0, and the change
in pyrene fluorescence was monitored as described above. All solutions
and reagents were equilibrated at 25 °C before assays were
performed. Varying concentrations of GS-1-bound cross-linked actin
oligomer with polymerization salts were added to the G-actin at the
beginning of the assay. This mixture was then placed in the
fluorimeter. As a result, the start time of the reaction and the start
time of data collection are offset slightly. This difference was taken
in account when computer simulations were performed. The concentration
of GS-1-complexed actin oligomers was determined by Bio-Rad protein
assay employing the molecular mass of the whole complex (118 kDa for
the dimer and 178 kDa for the trimer). The concentration of F-actin is
reported as the concentration of total actin monomer rather than the
concentration of F-actin ends.
Computer Simulation--
The experimentally measured pyrene
fluorescence was converted to F-actin concentration (µM)
using Equation 1.
|
(Eq. 1)
|
In the equation, Fmax was the
steady-state fluorescence obtained from control actin polymerization
reactions employing F-actin as nuclei, Fmin was
the minimum fluorescence measured in control reactions of spontaneous
nucleation, and F(t) was the fluorescence at any
time, t, of an experiment.
As discussed in the text, the kinetic and structural data are
consistent with actin assembly from the pointed ends of the cross-linked actin oligomers. Therefore, we employed the published polymerization rate constants for ATP-actin at the pointed end of 1 µM
1s
1 and 0.5 s
1 for the k+ and
k
, respectively (33).
Nucleation Model--
The model involves the formation of a
nucleus by the binding of an actin monomer to the pointed end of a
GS-1-bound cross-linked actin oligomer. In this case, active nuclei,
N*, are in equilibrium with cross-linked actin, N, and G-actin, A, with
a forward rate constant, k1, and a backwards
rate constant, k2.
|
(Eq. 2)
|
G-actin then assembles from the nuclei, N*, with the known
k+ and k
for the
pointed end, to form the mass of F-actin, M.
|
(Eq. 3)
|
We assume that spontaneous nucleation is insignificant when
X-actin is added to the assembly reactions. Because the total concentration of filaments assembled is equal to the concentration of
nuclei formed, assembly of filaments can be described as a function of
the concentration of N* (34).
We chose to include the contribution of the concentration of nuclei,
N*, into the concentration of actin filaments, M, because the addition
of actin monomer to X-actin may contribute to the increase in pyrene
signal accompanying F-actin assembly. However, the contribution of N*
to M is insignificant in the best-fit simulations, and almost identical
curves are generated without including N* in M. We fit the following
equations to the experimental data.
|
(Eq. 4)
|
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(Eq. 5)
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(Eq. 6)
|
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(Eq. 7)
|
Best-fit curves were generated using Berkeley Madonna software
(www.berkeleymadonna.com) by varying the initial G-actin concentration, A, the rate constants k1 and
k2 and by fitting the start time, t0. The average best-fit
k1 values were 1.6 × 10
4
µM
1s
1 ± 3.9% (S.E.;
n = 7) and 2.7 × 10
4
µM
1s
1 ± 3.6% (S.E.;
n = 8) for the GS-1-bound cross-linked actin dimer and
trimer, respectively. The average best-fit k2
values for the dimer and trimer were 0.026 s
1 ± 9.0%
(S.E.; n = 7) and 0.024 s
1 ± 3.7%
(S.E.; n = 8), respectively. The root mean square
deviations ranged between 0.0116 and 0.0320 for the calculated curves
fitting the GS-1-bound cross-linked actin dimer nucleated data set and 0.0126 and 0.0426 for the trimer curves. An additional round of simulation was performed employing the average
k1 and k2 values from the
initial simulations and fitting the start time,
t0, and initial G-actin concentration to obtain
the curves shown in Fig. 2. Initial G-actin concentration was fit to
account for possible pipetting errors that can influence the
steady-state pyrene fluorescence measured. The average best-fit
starting G-actin concentration for the calculated dimer-nucleated
assembly curves was 5.3 µM ± 1.09% (S.E.;
n = 7) and 5.2 µM ± 1.47% (S.E.;
n = 8) for the trimer curves.
Crystallization of GS-1-complexed Actin Trimer--
A solution
at ~12 mg ml
1 of the GS-1-complexed actin trimer was
used for crystallization by the vapor diffusion method. For crystals of
the complex that were grown in the presence of ATP, 2 µl of protein
solution were mixed with 2 µl of reservoir buffer containing 15%
(w/v) polyethylene glycol 1000, 25 mM Tris-HCl, pH 8.0, 2 mM ATP, 100 mM MgCl2, 2 mM CaCl2 and equilibrated against this buffer
for 5-7 days at 4 °C. Crystals of the GS-1-complexed actin trimer
in the presence of ADP were grown under the same conditions using 8%
(w/v) polyethylene glycol 3350, 25 mM Tris-HCl, pH 8.0, 2 mM ADP, 100 mM CaCl2, and 2 mM MgCl2 as reservoir buffer. Before data
collection, both crystal forms were cryoprotected by adding glycerol to
a final concentration of 15% and then flash-frozen in liquid nitrogen.
Identification of Molecules in Crystals--
To confirm the
presence of GS-1-bound actin trimers in crystals, crystals grown in the
presence of either ATP or ADP were harvested, washed, and analyzed
using SDS-PAGE and mass spectrometry (Voyager-DETM;
PerSeptive Biosystems). Results of SDS-PAGE and mass spectrometry analysis confirmed the presence of GS-1 and actin trimer in both crystal forms. Neither actin dimers nor monomers were detected in the crystals.
Data Collection, Model Building, and Refinement--
For both
crystal forms, x-ray diffraction data were measured at
180 °C. The
data in both cases were collected to 2.2 Å at Advanced Light Source
(Lawrence Berkeley National Laboratory) beamline 5.0.2 (
= 1.1 Å). Both data sets were integrated using DENZO and scaled with
SCALEPACK (35). Both crystal forms were of the monoclinic space group
P21 with two GS-1-complexed actin molecules in the
asymmetric unit and cell dimensions of a = 67.2 Å,
b = 75.9 Å, c = 96.8 Å,
= 91.9° and a = 66.7 Å, b = 75.9 Å,
c = 97.9 Å,
= 90.1° for the crystals grown
in the presence of ADP and ATP, respectively.
Both structures were determined by the molecular replacement method
(CNS; see Ref. 36) using atomic coordinates for the GS-1-complexed rabbit actin (3). Electron density maps based on
coefficients 2Fo - Fc
were calculated from the phases of the initial models. Subsequent
rounds of model building and refinement were performed using programs
QUANTA (Molecular Simulations, Inc.) and CNS (36), respectively.
Electron density corresponding to the bound nucleotide was well defined
in all maps calculated for both structures. In both cases, ATP was
bound in the nucleotide pocket. Both structures were virtually
identical. However, because both the quality of x-ray data and the
current refinement statistics are better for the structure obtained
from the crystals grown in the presence of ADP, the latter structure is
described in this paper. The current structure is refined to
R/Rfree values of 19.3/23.3 using all
data in the resolution range of 25.0 to 2.2 Å (F > 0, a bulk solvent correction was applied; see Table I). Both
GS-1-complexed actin protomers present in the crystal asymmetric unit
include Ca2+-ATP and two additional Ca2+ ions.
Residues 2-125 and 3-125 are built for two GS-1 segments, and
residues 7-41, 50-375 and 7-44, 51-374 are built for two actin molecules, respectively. There are 471 water molecules in the asymmetric unit of the current model.
F-actin Severing Assays--
5 µM F-actin (15%
pyrenyl-actin) was incubated with 1.5 µM GS-1 in 50 mM KCl, 2 mM MgCl2, 1 mM EGTA, and 0.1 mM ATP in 25 mM
Tris-HCl, pH 8.0, in 100 µl total in the presence or absence of 5 mM CaCl2 for 10 min at 25 °C. 20 µl of
stock 20 µM G-actin (15% pyrenyl-actin) in 10 mM Tris-HCl, pH 8.0, 0.3 mM CaCl2,
0.1 mM EDTA, 0.2 mM
-mercaptoethanol, and
0.2 mM ATP was then added to the reaction (final
concentration 3.3 µM G-actin), and the change in
fluorescence was monitored. The initial rate of elongation for each
experiment was employed to determine the concentration of free pointed
ends in the assay, assuming that GS-1 remains bound to the barbed ends
of filaments, employing the known on-rate for ATP-actin at the
pointed end of 1 µM
1s
1 and
the relationship d(F)/dt = kPon[ends][G-actin].
 |
RESULTS |
Purification of GS-1-bound Actin Oligomers--
We polymerized
F-actin purified from chicken and treated it with pPDM, which
cross-links specifically between Cys-376 and Lys-193 of two adjacent
actin protomers in a filament (17-19). This F-actin was then
depolymerized in the presence of excess recombinant GS-1 to generate
GS-1-bound cross-linked actin oligomers of varying lengths (see
"Materials and Methods"). The heterogeneous mixture of GS-1-bound
actin oligomers was resolved by a combination of gel filtration and
anion exchange chromatography to yield purified GS-1-bound cross-linked
actin oligomers of defined length (Fig. 1). The identification of purified
cross-linked actin oligomers was confirmed by mass spectrometry (data
not shown).

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Fig. 1.
Analysis of the GS-1-bound X-actin oligomers.
10% SDS-PAGE analysis of the soluble products of a reaction
between GS-1 and cross-linked F-actin (lane 1). Purified
GS-1-bound cross-linked actin monomer (lane 2), dimer
(lane 3), and trimer (lane 4).
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Cross-linked Actin Oligomers Nucleate Actin
Polymerization--
Both GS-1-bound cross-linked actin dimers and
trimers nucleated actin assembly (Fig. 2,
B and C), whereas GS-1-bound actin monomer
purified from the same cross-linking reaction inhibited even
spontaneous nucleation (Fig. 2A). No free GS-1 was detected in these polymerization reactions (data not shown), suggesting that
GS-1 remains bound to each subunit of the purified actin oligomer
during the assembly reaction.

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Fig. 2.
Polymerization of actin in the presence of
varying concentrations of X-actin oligomers. 5 µM G-actin (15% pyrenyl-G-actin) was polymerized as
described under "Materials and Methods" with varying concentrations
of purified X-actin oligomers. Raw fluorescence measurements were
converted to µM F-actin according to the increase in
pyrene fluorescence over time. A, addition of 2 µM F-actin seeds (red) results in rapid
elongation without an initial lag phase, whereas spontaneous nucleation
(blue) results in very slow polymerization. The addition of
GS-1-bound actin monomer at various concentrations (2 µM,
black; 1 µM, green; and 0.1 µM, pink) inhibits even spontaneous
nucleation. B, addition of GS-1-bound cross-linked actin
dimer results in an increased rate of polymerization dependent on the
concentration of dimer added (1.5 µM, red; 1.0 µM, blue; 0.5 µM,
green; 0.35 µM, black; 0.25 µM, pink; 0.15 µM,
cyan; 0.1 µM, yellow). Plotting
symbols show 16.6% of the data points for
clarity. Drawn curves show the best fit to the experimental
data employing the model and the average kinetic constants outlined
under "Materials and Methods." C, addition of GS-1-bound
cross-linked actin trimer results in an increased rate of
polymerization dependent on the concentration of trimer added (1.0 µM, red; 0.75 µM,
blue; 0.5 µM, green; 0.35 µM, black; 0.25 µM,
pink; 0.2 µM, cyan; 0.15 µM, yellow; 0.1 µM,
orange). Plotting symbols show 16.6% of the
data points for clarity. Drawn curves show the
best fit to the experimental data employing the model and the average
kinetic constants outlined under "Materials and Methods."
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The average steady-state concentration of F-actin formed when
GS-1-bound cross-linked actin oligomers were employed as nuclei was
only 4.4 µM ± 0.77% (±S.E.; n = 11).
Given that 5 µM G-actin was included in these actin
assembly reactions, this F-actin steady-state concentration is
consistent with pointed end growth, where the critical concentration is
known to be about 0.6 µM (33).
The concentration of GS-1-bound cross-linked actin oligomers required
for a substantial increase in assembly rate is in the micromolar range,
whereas nanomolar concentrations of pPDM cross-linked actin trimer
purified with no GS-1 bound nucleates rapid assembly (20). The presence
of a lag phase at the beginning of elongation assays with added
GS-1-bound cross-linked actin oligomers indicates that the rate of
nucleation is slower than the rate of elongation. This suggests that
the added GS-1-bound cross-linked actin oligomer undergoes a transition
from a nucleation-incompetent form to a nucleation-competent form. This
transition may occur through a conformational change in the actin
oligomer itself or when the first actin monomer binds to the pointed
end of the cross-linked actin oligomer. Modeling of the assembly data
(see "Materials and Methods," and the calculated curves in Fig. 2,
B and C) are consistent with the latter.
The average on-rates, k1, for the first actin
monomer binding to either the cross-linked actin dimer or the trimer
are similar (1.6 × 10
4 and 2.7 × 10
4 µM
1s
1,
respectively), as are the average best-fit rate constants for that
monomer dissociation, k2 (0.026 and 0.024 s
1, for the dimer and trimer, respectively). These
results are consistent with the cross-linked actin oligomers providing
a dimer interface for an incoming actin monomer to bind, thereby
forming a nucleus for assembly.
Atomic Resolution Structural Studies of the GS-1-bound Cross-linked
Actin Trimer--
The nucleation activity of the GS-1-bound actin
oligomers suggested that these oligomers might retain important
elements of their initial F-actin structure. To determine the
three-dimensional structure of the actin oligomers, the GS-1-complexed
actin trimer was crystallized in the presence of either ATP or ADP, and
the structures of both complexes were solved by molecular
replacement and refined to 2.2 Å (see Table
I and "Materials and Methods").
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Table I
Data collection and refinement statistics
Data is shown for the GS1-complexed actin trimer crystallized in the
presence of ADP (see "Materials and Methods").
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Preferential Binding of ATP to the GS-1-complexed Actin
Oligomers--
Both structures were found to be essentially identical,
with ATP present in the nucleotide binding pocket of the crystalline GS-1-bound actin formed in the presence of either ATP or ADP (Fig. 3). Given the kinetics of ATP hydrolysis
in F-actin (33), the cross-linked F-actin must have contained ADP or
ADP and inorganic phosphate prior to depolymerization by excess
GS-1 (see "Materials and Methods"). The binding of GS-1 to the
cross-linked actin likely promoted the exchange of ADP for ATP, which
was present in the actin polymerization buffer (see "Materials and
Methods"). This ATP remained tightly bound throughout the subsequent
purification procedure, which was carried out in the absence of added
nucleotide. Furthermore, the bound ATP was not exchanged for ADP during
a week-long crystallization process in which 2 mM ADP was
present (see "Materials and Methods").

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Fig. 3.
Stereo view of electron density corresponding
to the bound Ca2+-ATP in GS-1-bound actin trimer
crystallized in the presence of ADP. Simulated annealing composite
omit map was calculated for the refined model and is displayed at 1.0 in gray. The bound ATP and Ca2+ are shown as
a stick model and yellow sphere, respectively.
Molecules of water coordinating the bound cation are shown as red
spheres.
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The Crystalline GS-1-bound Actin Trimer Is an Untwisted and
Extended F-actin--
The structures of GS-1-complexed actin trimer
consisted of one molecule of GS-1 bound to each actin monomer in the
trimer complex with a molecular mass of 178,000 Da, with
the GS-1-bound protomers related by a 180° rotation and a ~37.9-Å
translation along the rotation axis (Fig.
4).

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Fig. 4.
Structure of the GS-1-complexed actin
trimer. Actin subunits in the trimer complex are colored in
dark green, orange, and blue. The
corresponding segments of gelsolin are light green,
yellow, and cyan. The structure of each
GS-1-bound protomer in the trimer is similar to the structures of
GS-1-complexed actin solved previously. In each protomer, the bound
nucleotide, Ca2+-ATP, is shown as a gray
space-filling model. Coordinated ions of Ca2+ are
drawn as red spheres. The pPDM cross-link is between Lys-193
of one actin subunit and Cys-376 of its neighbor. In each actin
subunit, the C-terminal residues 375-377 and residues corresponding to
DNase I binding loop are disordered and are not included in the model.
The dashed lines, therefore, connect the cross-linked
Lys-193 of subunits 1 and 2 to the last visible C-terminal residue,
Arg-374, of subunits 2 and 3, respectively.
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When the unit cell of the GS-1-complexed actin trimer crystal is
repeated and translated along the 2-fold rotation axis, an extended
filamentous actin structure emerges (Fig.
5B). This filament, although
untwisted and more extended than F-actin (rotational symmetries are 180 and 167 degrees, and axial translations per subunit are 75.9 and 55.0 Å in crystalline GS-1-bound actin trimer and F-actin, respectively;
see Fig. 5, B and C), possesses the general
topological hallmarks of the helical model of F-actin (1).
Specifically, subdomain 2 and the N terminus (Fig. 5, B and
C, red and yellow, respectively) in
each actin subunit are positioned at higher radius whereas subdomain 3 (Fig. 5, B and C, blue) is located at
lower radius in both actin filaments. The hydrophobic loop (Fig. 5,
B and C, cyan) is positioned in the center in both cases. Because of the topological similarity between the
helical model of F-actin and the filament observed in the GS-1-complexed actin trimer crystal, we term the GS-1-complexed actin
trimer structure an "eXtended" F-actin (X-actin; see
Fig. 5B). The observed distorted symmetry of X-actin
compared with that of F-actin appears to result from the binding of
GS-1.

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Fig. 5.
Structural similarity between the X-actin and
F-actin filaments. A, topological features of an actin
monomer. Subdomain 2 (amino acids 35-71) is shown in
red, subdomain 3 (amino acids 152-182, 276-339) is
dark blue, the N terminus is yellow, and the
hydrophobic loop (amino acids 263-275) is cyan. Topological
positions of these features are compared in B and
C. The coloring scheme is identical in A,
B, and C. B, arrangement of actin
subunits and subdomains in each actin subunit within the X-actin
filament structure. C, relative position of actin subunits
and subdomains in each actin subunit in the helical model of the
F-actin filament.
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The normal contacts between actin protomers of helical F-actin are also
distorted in the X-actin arrangement to accommodate the bound GS-1. The
interposition of GS-1 results in the loss of longitudinal contacts in
X-actin (between subunits 1-3, 2-4, 3-5, etc.; see Fig.
6, A and B).
Moreover, rotation of actin protomers relative to their position in
helical F-actin results in a partial loss and rearrangement of the
intersubunit contacts in the core of the filament. The resulting
interface between actin protomers in the X-actin filament is less
extensive (~1100 Å2 of buried surface area) compared
with that predicted to be in F-actin (1). Seven structural elements
forming the intersubunit interface in the X-actin are shown in Fig. 6,
A and C. Although the same structural elements
form a subset of the intersubunit interactions in the core of the
F-actin (1), their positions relative to each other are different in
undistorted actin filaments (Fig. 6, B and
D).

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Fig. 6.
Comparative analysis of the intersubunit
contacts in the X-actin and F-actin filaments. A,
structural elements forming the interface in the X-actin. Amino acid
residues 197-199 (yellow), 225-238 (red), and
253-258 (orange) from an actin subunit N and residues
112-116 (light green), 174-180 (dark green),
271-274 (blue), and 285-289 (cyan) from an
adjacent subunit N+1 are highlighted, and their positions
are indicated in the F-actin filament (B). Magnified view of
the structural elements buried at the X-actin interface (C)
and their positions at the interface of the F-actin (D) are
shown. Coloring scheme is consistent for all panels.
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Lateral Interactions between Two X-actin Filaments Form a
Ribbon-like Structure--
More than one actin assembly is formed in
the GS-1-complexed actin trimer crystals. Lateral contacts between two
neighboring filaments of X-actin in the crystal create a second
assembly of actin protomers. This assembly is strikingly similar to the
ribbon structure observed in profilin-actin crystals, as
described by Schutt and co-workers (4, 5) (Fig.
7, A and B, in
blue; see figure legend for details). However, the actin
protomers in the ribbon created by two neighboring X-actin filaments
are rotated (by ~5 degrees) and moved further from each other (by
~2 Å) compared with the protomers in profilin-actin crystals (4, 5).
The contacts at the ribbon interface between the neighboring X-actin filaments do not exceed 700 Å2 and involve mostly poorly
structured regions on the surface of actin. Notably, the arrangement of
actin protomers within the X-actin filament positions one of the
cross-linked residues (Lys-193; see Fig. 7A,
green) in each protomer ~16 Å away from the last visible
C-terminal residue (Arg-374; see Fig. 7A, red) of
the neighboring actin protomer. Given the flexibility of the following stretch of C-terminal residues of actin, this arrangement places the
second, Cys-376, residue of the cross-linked pair within the physical
length (~12 Å) (37) of the cross-linker. In contrast, the distance
between Lys-193 and Cys-376 in the ribbon arrangement (Fig.
7B, green and red, respectively)
approaches 40 Å.

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Fig. 7.
Filamentous actin assemblies observed in the
crystalline GS-1-bound actin trimer. A, filamentous
structure of X-actin. Actin subunits are shown in yellow and
purple. The complexed GS-1 is in gray. The
side-chain of Lys-193 and C atom of the last visible C-terminal
residue, Arg-374, are shown as a green space-filling model
and a red sphere, respectively, in each actin subunit.
B, the ribbon-like actin assembly formed by crystal packing.
This ribbon-like structure (shown in purple and
blue) is formed at the interface between two (I
and II) filaments of X-actin. Filament I in
B is rotated about 90° relative to that in A.
The coloring scheme is similar to that of panel A, except
that the purple and blue actin subunits, which
form the ribbon-like arrangement, are highlighted and the
yellow subunits are de-emphasized in B.
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GS-1 Severs Actin Filaments in the Presence of
Ca2+--
The observed stoichiometry and symmetry of the
GS-1-bound actin trimer suggested that segment-1 of gelsolin might
intercalate between actin protomers in F-actin and possibly sever actin
filaments. Additional experiments (Fig.
8) aimed to verify this hypothesis showed
that the rate of F-actin depolymerization in the presence of
Ca2+ and excess GS-1 varies with different GS-1
concentrations (Fig. 8A). These data argue against simple
monomer sequestration as the mechanism of GS-1-mediated F-actin
depolymerization, which would rely solely on the off-rate of actin
monomers from F-actin. Furthermore, the concentration of free pointed
ends in F-actin that were incubated with a substoichiometric
concentration of GS-1 was calculated employing the initial rate of
polymerization after additional G-actin was added to the reaction (Fig.
8B). This concentration is higher in F-actin-GS-1 reactions
in the presence of Ca2+ (3.4 nM) than that in
the absence of Ca2+ (0.6 nM). The latter data
suggest the production of new ends as a result binding of GS-1 and
support the hypothesis that GS-1 is able to sever F-actin.

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Fig. 8.
Analysis of F-actin depolymerizing activity
of GS-1. A, different rates of depolymerization with
different excess GS-1 concentrations. Various concentrations of GS-1
were added in excess to 2.5 µM F-actin (15%
pyrenyl-F-actin) in the presence of Ca2+, and the
depolymerizing activity was monitored as a function of pyrene
fluorescence over time. The concentrations of GS-1 added to each assay
were 5 µM (purple), 12.5 µM
(green), 25 µM (blue), and 50 µM (red). Plotting symbols show
30% of the data points for clarity. B,
GS-1-mediated depolymerization generates more pointed ends for actin
assembly. 5 µM F-actin (15% pyrenyl-F-actin) was
incubated with 1.5 µM GS-1 in the presence
(red) or absence (blue) of 5 mM
CaCl2 for 10 min. 4 µM G-actin (15%
pyrenyl-G-actin) was then added to the reaction. The increase in
fluorescence during this phase because of the polymerization of the
added G-actin is shown. In the presence of Ca2+, the rate
of polymerization of the added G-actin is higher than that in the
absence of Ca2+. Only 10% of the data points
are shown for clarity, and the lines are the smoothed
average of each data set. C, phalloidin inhibits the F-actin
depolymerizing activity of GS-1 in the presence of Ca2+. 5 µM F-actin (15% pyrenyl-F-actin) in the absence
(red) or presence (blue) of phalloidin was
incubated with 5 µM GS-1 in the absence of
Ca2+ for 100 s. At 100 s, 5 µM
CaCl2 was added, and the pyrene fluorescence was monitored
as described "Materials and Methods." The decrease in pyrene
fluorescence indicates depolymerization of F-actin. Only 10% of the
data points are shown for clarity, and the lines
are the smoothed average of each data set.
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GS-1-mediated Depolymerization of F-actin in the Presence of
Ca2+ Is Inhibited by Phalloidin--
Analogous to
full-length gelsolin (38, 39), the severing activity of GS-1 is
Ca2+-dependent (Fig. 8C,
red). Previous work showed that the F-actin binding and
severing activity of both full-length gelsolin (40) and its N-terminal
half (segments-1-3, GS-1-3) (41) could be separated in the presence
of phalloidin. Bound to F-actin, phalloidin still permits binding of
the full-length gelsolin or GS-1-3 but inhibits their severing
activity. Consistent with these data, our experiments showed that
F-actin decorated with phalloidin was not depolymerized in the presence
of excess GS-1 and Ca2+ (Fig. 8C,
blue). Unlike full-length gelsolin or GS-1-3 (41), however,
GS-1 did not co-sediment with phalloidin-decorated F-actin in the
presence of Ca2+ (data not shown). The latter result could
be explained by the absence of segment-2 (GS-2) whose interactions with
F-actin along the side of the filament are not affected by binding of
phalloidin (27, 39, 42).
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DISCUSSION |
Combined results of our structural and biochemical studies clarify
several important aspects of actin dynamics and its regulation.
Segment-1 of Gelsolin Severs F-actin--
Actin assembly is
tightly regulated by specialized actin binding proteins. Among these is
gelsolin, an 84-kDa protein that remodels the actin cytoskeleton during
cell movement, apoptosis, and cytokinesis (reviewed in Refs. 23, 24,
and 28). To accomplish this, gelsolin binds, severs, and caps actin
filaments in a Ca2+-regulated manner (23, 24, 28, 39).
Gelsolin consists of six segments that share significant sequence
similarity (38). Numerous studies have focused on attributing the
activities of gelsolin to its segments (3, 21, 39, 42, 43). Initially,
proteolytic fragments of gelsolin were tested. The first work with
chymotryptic segment-1 of gelsolin (GS-1) suggested it might sever
actin filaments (25). However, subsequent work suggested that
chymotryptic GS-1 preparations were contaminated with a small amount of
a longer fragment of gelsolin (GS-1-3), which was responsible for the
observed severing activity (26). Therefore, it is now commonly held
that GS-1 alone has no severing activity (3, 27).
Recombinant techniques permit purification of homogeneous preparations
of specific segments of gelsolin where contamination with full-length
gelsolin is not possible. Because the actin monomer binding activity of
recombinant GS-1 has been studied extensively (3, 22), in our
experiments on preparation of F-actin fragments (Fig. 1), recombinant
GS-1 was expected to facilitate depolymerization by both sequestering
monomeric G-actin and binding to the fast growing barbed end of newly
formed F-actin fragments.
However, atomic resolution structural analysis of GS-1-bound actin
trimer (Fig. 4) suggested that, in the presence of Ca2+,
GS-1 intercalates between actin subunits in the filament, perturbing both the arrangement of the protomers in F-actin (Fig. 5) and the
intersubunit contacts responsible for stability of the actin filament
(Fig. 6). Consistent with the structural data, results of our
biochemical experiments (Fig. 8) showed that GS-1 mediates depolymerization of F-actin in a Ca2+-dependent
manner by severing the actin filaments. The different rates of
depolymerization of F-actin at different excess concentrations of GS-1
argue against a simple monomer sequestration model, which would rely
solely on the off-rate of actin monomers from F-actin, and suggests
that GS-1 plays a severing role in the depolymerization of F-actin. Our
biochemical data also shows that GS-1 produces new actin filament
pointed ends for nucleation, again consistent with a severing mechanism.
The concentration of free pointed ends (3.4 nM) in the
presence of Ca2+ was much less than the concentration of
GS-1 employed (1.5 µM). This is consistent with a very
inefficient severing protein, compared with full-length gelsolin, which
produces one break for every molecule (28). The observed difference
could be explained by the absence of segments-2-3, which may play the
role of binding to the side of F-actin and correctly position GS-1 on
the F-actin surface (3, 27, 39, 42). Without GS-2-3 segments, correct positioning of the GS-1 along the actin filament for severing may rely
on random interactions with the filament and may therefore be inefficient.
GS-1-bound Cross-linked Actin Oligomers Retain Important Structural
Features of F-actin and Nucleate Actin Polymerization--
GS-1
present at a 2:1 ratio depolymerizes cross-linked F-actin completely,
resulting in a mixture of GS-1-bound cross-linked actin oligomers of
varying lengths than can be separated according to their size (Fig. 1)
and analyzed. Crystallographic analysis of GS-1-bound actin trimer
showed that, although perturbed by the binding of GS-1, it retained the
general topological features of helical F-actin (1) (see Figs. 4 and
5).
Modeling of GS 1-mediated actin filament assembly data (Fig.
2C) suggests that the cross-linked actin trimer undergoes a
conformational change when the first actin monomer binds to its pointed
end, providing a proper interface for an incoming actin monomer to bind, thereby forming a nucleus for assembly. These data indicate that,
although arranged by the 21 symmetry in the crystal (Fig. 4), the protomers of the actin trimer may be flexible in solution. Retaining their close association, the protomers within the actin trimer in solution are most likely free to rotate and adopt a structure
that is able to bind an actin monomer and form an actin nucleus.
Of interest is the finding that low concentrations of GS-1-bound actin
monomer suppress spontaneous F-actin assembly (Fig. 2A).
GS-1-bound actin monomer most likely binds the fast growing barbed end
of rare actin dimers or trimers formed spontaneously, thereby
inhibiting their potential to nucleate polymerization (44). Because the
concentration of dimers and trimers formed spontaneously is very low
compared with free monomer, added GS-1-bound actin dimer or trimer is
far more likely to interact with monomer. According to the best-fit
kinetic model, these more frequent interactions of GS-1-bound
cross-linked actin oligomers with free actin monomer lead to nucleation.
Rotational Freedom of F-actin Is Important Functionally--
Cell
biology experiments have demonstrated beautifully the dynamic features
of actin fibers (reviewed in Ref. 45). An emerging theme of F-actin
structure is the importance of rotational freedom of actin protomers in
the filament. The ability of F-actin to tolerate such rotational
freedom is demonstrated most dramatically by the structure of
cofilin-decorated F-actin, where the filament is super-twisted by 5 degrees and still exists as a polymer (46). Changing the twist of
F-actin is most likely a major component to the severing activity of
many proteins including gelsolin. Recently, structural analysis of
F-actin has shown that it can exist as a super-twisted polymer in the
absence of other proteins (47, 48). This mode of F-actin is thought to
involve a change in the way that the subunits of F-actin interact with
each other (48).
Rather than being super-twisted, our X-actin structure is under-twisted
by 13° relative to normal F-actin because of the GS-1 binding (see
Figs. 4 and 5). With GS-1 removed, the X-actin filament can be
transformed into the helical model of F-actin by combination of
translational (~10.4 Å) and rotational (13° twist with additional tilt) movements of each of the actin protomers within the filament. These movements would result in the filament twisting and shortening (an animation of this transition is available as Supplemental Material). Interestingly, like the super-twisted polymer observed by
Egelman and co-workers (48), under-twisted X-actin employs an
alternative arrangement of the interacting elements compared with that
found at the interface in F-actin (Fig. 6). The ability of the F-actin
interface to accommodate rotational movements of the subunits within
the filament and to adjust radically in different actin
self-associations may be inherent in the evolved design of the actin
surface. Furthermore, this property of actin might provide a structural
basis for the variable twist of the F-actin helix observed
experimentally (49, 50) and could be important for the dynamics of the
actin filament. It should be noted that, in contrast to extensive data
on variable twist within F-actin, there are no observations of
significant extension of F-actin structure along its axis. It is
therefore possible that the described extended actin trimer structure
caused by the intercalation of GS-1 might exist only within crystals.
Rotational freedom of the actin subunits also explains a paradoxical
structural feature of the X-actin trimer (Fig. 4). Because of the
non-equivalent nature of the cross-linked residues, we anticipated all
three actin protomers of the complex bound covalently to be in the same
crystal asymmetric unit. Surprisingly, we found each GS-1-bound actin
protomer in a different asymmetric unit with the first and the third
protomers being in neighboring unit cells. This apparent paradox was
resolved by the realization that the covalent linkages tethering the
protomers in the trimer complex are flexible. This flexibility results
in both pPDM and the last three C-terminal residues of each actin
protomer (including Cys-376 to which the cross-linker is attached)
being disordered and not visible in our electron density maps. This
flexibility also explains the fact that the assembly of the three actin
protomers in the complex shows a strict rotational and translational
symmetry corresponding to a crystallographic 2-fold screw axis (Fig.
4). As we already discussed, although arranged by the 21
symmetry in the crystal lattice, the protomers of the actin trimer most
likely are free to rotate in solution.
Structural Transitions between Helical F-actin- and
Profilin-Actin-like Ribbons Are Not Possible--
Our analysis of the
crystal lattice revealed an additional arrangement of the GS-1-bound
actin subunits. This arrangement (Fig. 7B, blue),
which is found at the interface between two strands of actin trimers
coming from different X-actin oligomers, is strikingly similar to that
previously found exclusively in profilin-actin crystals and called the
ribbon structure (4, 5). The distance between Lys-193 (Fig.
7B, green) of one actin subunit and the C
terminus (Fig. 7B, red) of the next subunit in
the ribbon between X-actin trimers approaches 40 Å. This is too far to
be cross-linked by pPDM, which spans 11.4 Å (37). In contrast, the
distance between Lys-193 and the last visible C-terminal residue of the neighboring subunits in the X-actin arrangement is about ~18 Å (Fig.
7A, green and red, respectively). This
arrangement accommodates the covalent cross-link between the Lys-193
and Cys-376, given some flexibility of the C-terminal tail. Our
analysis shows that the X-actin trimer is derived from a cross-linked
helical filament whereas the profilin-actin-like ribbon arrangement
cannot result from untwisting a single F-actin filament. Like other
experimental data, the actin assemblies found in the GS-1-bound actin
trimer crystals rule out the models both for F-actin and for muscle
contraction based on a single F-actin filament transitioning between
the helical and ribbon forms described by Schutt et al.
(11-13).
Nucleotide and Actin Dynamics--
It is intriguing to note that
Ca2+-ATP was found in the nucleotide binding pocket of the
X-actin trimer structure (Fig. 3) even though the trimer was created
from F-actin that presumably contained ADP, and the crystals were
formed in the presence of ADP (see "Materials and Methods"). It
would appear that the ATP present in solution at the time that the GS-1
was used to disassemble the actin filaments exchanged for ADP in the
nucleotide pocket and then remained there bound tightly. Although the
higher affinity of uncomplexed monomeric actin for ATP is well
documented (Kd for ATP is 70 pM and for
ADP is 12 nM (33)), the absence of the nucleotide exchange
in the case of GS-1-bound actin trimer was not expected. In the
presence of 1 mM ADP and in the absence of any ATP in
crystallization mother liquor, the reported off-rate of ATP for G-actin
(0.015 s
1) (33) would allow the bound ATP to exchange for
ADP. Under similar conditions, two structures of monomeric actin have
been crystallized with ADP bound in the active site (2, 7). In the case
of GS-1-bound actin trimer, however, electron density maps reveal that
highly ordered ATP occupies the nucleotide pocket of actin subunits
fully (Fig. 3). This result suggests that the binding of GS-1 limits
the exchange of nucleotide in the complex. Tight binding of ATP in
actin-gelsolin complexes has been reported previously (51). The
following structural analysis offers an explanation for the
preferential binding of ATP by the GS-1-complexed actin.
A superposition of the actin structure from the GS-1-complexed actin
trimer with available actin structures revealed that all of them,
although differing in details, form two distinct categories. The first
group, which includes the structure of actin from the GS-1-complexed
actin trimer (as well as all structures of actin complexed with
GS-1 reported previously; thin gray lines in Fig.
9A), can be defined as the
"closed" category (Fig. 9A, red and
pink) as it contains the closed structure of actin found in
one actin-profilin complex (4). The second category includes the
"open" conformation of actin from a different actin-profilin complex (5) and is, therefore, defined as open (Fig.
9A, cyan). Detailed structural analysis of the
closed and open structures of actin suggested at the major difference
between these two conformations, closing or opening of the nucleotide
binding cleft, could be explained by combination of two rotational
movements (52). The first rotation would involve semi-rigid subdomain 2 moving around a hinge at the base of subdomain 2. The second rotation
would involve two domains of actin (subdomains 1 plus 2 and 3 plus 4, respectively) moving relative to each other around the extended hinge
(or shear, amino acid residues 137-152; see Fig. 9A,
yellow and orange) area of actin (52).
Comparative structural analysis, performed in this and related work
(15) on twelve actin structures (Fig. 9A), supports this
proposal. This analysis, as well as crystallographic study of
actin-related proteins Arp2/3 (53), suggest that the closed and open
structures of actin could be relevant physiologically and might
represent, respectively, the ATP/ADP and inorganic phosphate and
ADP/nucleotide free-like conformational states of actin (15).

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Fig. 9.
Comparative analysis of actin
structures. A, superposition of available actin
structures. The superposition was done using coordinates of C atoms
of residues from subdomains 3 (amino acids 152-182 and 276-339) and 4 (amino acids 183-275) in each case. The structures of the actin
subunit and the bound GS-1 from the GS-1-complexed actin trimer are
shown in red and light gray, respectively. The
closed (2BTF) and open (1HLU) actin structures from the actin-profilin
complexes (4, 5) are shown in pink and cyan with
their shear region (amino acids 137-152) in orange and
yellow, respectively. The polypeptide chains of the rest of
available actin structures (1ATN, 1C0F, 1DB0, 1J6Z, 1D4X, 1DGA, 1EQY,
1ESV, 1YAG) are drawn as thin gray lines. The position of
bound nucleotide (ATP) is indicated by a space-filling model
in cream. Subdomains 1, 2, 3, and 4 of actin are indicated,
and their movements between the open and closed conformations are shown
by red and cyan arrows, respectively. The
black arrows indicate steric clashes, which would occur
between the open actin structure and the bound GS-1. B,
conformations of the nucleotide binding loops in closed
(red) and open (cyan) structures of actin. The
closed conformations of the phosphate binding loops are coordinated by
the -phosphate of the bound nucleotide (in cream), which
forms hydrogen bonds with two conserved residues, Ser-16 and Gly-160
(marked in bold). The closed conformation of the nucleotide
cleft is stabilized further by a network of interactions that involve
residues from all four subdomains of actin. Several of these residues
in the vicinity of the nucleotide are marked. Hydrogen bonds are
indicated by dashed lines.
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Based on this analysis, we propose that the binding of GS-1 stabilizes
the closed ATP-like conformation of actin. We also suggest a mechanism
for the preferential binding of ATP by the GS-1-complexed actin. Fig.
9A shows that GS-1 intercalates between subdomains 1 and 3 in the shear area, requiring the two actin domains to rotate with
respect to each other (Fig. 9A, black arrows) and
to adopt the closed conformation as GS-1 can not fit into the open
structure. This rotational movement brings the nucleotide binding loops
of actin (Gln-14-Lys-20 and Asp-156-Gly-160; see Fig.
9B, red) closer in space. By forming hydrogen
bonds with two conserved residues (Ser-16 and Gly-160), the
-phosphate of ATP bridges the nucleotide binding loops stabilizing
their closed conformation. Formation of an extensive related network of
side- and main-chain interactions (Fig. 9B) stabilizes the
closed conformation of actin further, making strong binding of ATP by
the GS-1-actin complex favorable. Locked rigidly in the closed
conformation, the ATP-bound GS-1-complexed actin likely has a limited
possibility of "breathing," which would be required for
nucleotide exchange to occur.
Implications for the Mechanism of Severing by Gelsolin--
The
observed preferential binding of ATP and the absence of nucleotide
exchange in actin complexed with GS-1 might be relevant functionally
and could constitute a part of the gelsolin mechanism (23, 24, 28, 39).
Barbed ends containing ADP-bound actin subunits grow more slowly than
barbed ends containing ATP-bound actin (33). The proposed gelsolin
mechanism could involve recreating an ATP cap at the barbed ends of
severed F-actin fragments. After dissociation of gelsolin, the newly
formed F-actin fragments with ATP-bound actin subunits at the barbed
ends would regain their ability for the fast growth.
According to the most recent models of gelsolin action (3, 39)
(reviewed in Refs. 23, 24 and 28), segments-2 and -3 of gelsolin bind
to the side of an actin filament first. Bound to F-actin, these
segments position GS-1 to intercalate between actin subunits in the
filament in a calcium-dependent manner. The severing model
of McLaughlin et al. (3) suggests that the binding of GS-1
to a subunit of F-actin produces steric clashes between the GS-1-bound
barbed end of one subunit and the pointed end of the adjacent subunit.
It was predicted that only these longitudinal contacts are affected
directly by the intercalation of GS-1 but that enough of the stronger
bonds along the actin strand are broken to fragment the filament
(3).
The crystal structure of GS-1-bound cross-linked actin trimer in the
X-actin arrangement provides the first direct picture of the result of
gelsolin severing activity on F-actin. In support of the model proposed
by McLaughlin et al. (3), we find that the longitudinal
actin-actin contacts along the filament are broken to accommodate GS-1
(Fig. 6A). As a result, across-strand bonds are also
distorted (Fig. 6C), and the actin protomers are able to
rotate with respect to each other along the helix axis. These distortions allow the normal actin filament helical structure to be
relaxed. With the longitudinal contacts broken, the distorted across-strand bonds are not strong enough to maintain the helical integrity of the filament, and it breaks at the point of the GS-1 insertion. Our structure suggests that these atomic-scale distortions translate into an untwisting and eventual breakage of the actin helix
where the GS-1 is bound. An animation (available as Supplemental Material) showing the hypothetical transition between F-actin fragment
and GS-1-bound actin oligomer is made to show the relationship between
actin subunits before and after the GS-1 binding.