Document

doi:10.1006/jmbi.2001.5281 available online at http://www.idealibrary.com on
J. Mol. Biol. (2002) 315, 613±625
Polymerisation of Chemically Cross-linked
Actin:Thymosin b 4 Complex to Filamentous Actin:
Alteration in Helical Parameters and Visualisation of
Thymosin b 4 Binding on F-actin
Edda Ballweber1, Ewald Hannappel2, Thomas Huff2, Harald Stephan1
Markus Haener3, Nicole Taschner3, Daniel Stoffler3, Ueli Aebi3
and Hans Georg Mannherz1*
1
Department of Anatomy and
Cell Biology, Ruhr-University
Bochum, Germany
2
Institute of Biochemistry,
Friedrich-Alexander-University
Erlangen, Germany
3
Biozentrum, M.E. MuÈller
Institute for Structural Biology
University of Basel, Basel
Switzerland
The b-thymosins are intracellular monomeric (G-)actin sequestering proteins forming 1:1 complexes with G-actin. Here, we analysed the interaction of thymosin b4 with F-actin. Thymosin b4 at 200 mM was
chemically cross-linked to F-actin. In the presence of phalloidin, the
chemically cross-linked actin:thymosin b4 complex was incorporated into
F-actin. These mixed ®laments were of normal appearance when
inspected by conventional transmission electron microscopy after negative staining. We puri®ed the chemically cross-linked actin:thymosin b4
complex, which polymerised only when phalloidin and the gelsolin:2actin complex were present simultaneously. Using scanning transmission
electron microscopy, the mass-per-length of control and actin:thymosin b4
®laments was found to be 16.0(0.8) kDa/nm and 18.0(0.9) kDa/nm,
respectively, indicating an increase in subunit mass of 5.4 kDa. Analysis
of the helical parameters revealed an increase of the crossover spacing of
the two right-handed long-pitch helical strands from 36.0 to 40.5 nm.
Difference map analysis of 3-D helical reconstruction of control and
actin:thymosin b4 ®laments yielded an elongated extra mass. Qualitatively, the overall size and shape of the difference mass were compatible
with published data of the atomic structure of thymosin b4. The deduced
binding sites of thymosin b4 to actin were in agreement with those identi®ed previously. However, parts of the difference map might represent
subtle conformational changes of both proteins occurring upon complex
formation.
# 2002 Academic Press
*Corresponding author
Keywords: actin; DNase I; gelsolin; phalloidin; b-thymosins
Introduction
Abbreviations used: ADF, annular dark ®eld; A:Tb4,
actin:thymosin b4 complex; AwTb4, chemically crosslinked actin:Tb4; CTEM, conventional transmission
electron microscopy; Cc, critical concentration of actin
polymerisation; DNase I, deoxyribonuclease I
(EC 3.1.21.1.); EDC, 1-ethyl-3[3-(dimethylamino)propyl]
carbodiimide; EDC-actin, actin treated with EDC; EM,
electron microscope/microscopy; GA2, gelsolin in
complex with two actin molecules; PI-actin, pyrenylactin; RMS, root-mean-square; STEM, scanning
transmission EM; Tb4, thymosin b4.
E-mail address of the corresponding author:
[email protected]
0022-2836/02/040613±13 $35.00/0
Among the numerous actin-binding proteins
there is a limited number of proteins that interact
preferentially with monomeric (G-)actin. These
proteins have the capacity to depolymerise ®lamentous (F-)actin, stabilise it in its monomeric
form and/or to sequester it from the equilibrium
between G- and F-actin, thus preventing its incorporation into the polymer moiety. A prototype of
an actin-sequestering protein is thymosin b4 (Tb4)
and related peptides (the b-thymosins), which in
some cells are present in high enough concentration to stabilize about 50 % of the intracellular
actin in G-form.1 ± 4 The b-thymosins have a mol# 2002 Academic Press
614
ecular mass of roughly 5 kDa and are found in
almost every eukaryotic cell. They form a 1:1 complex with G-actin of intermediate binding af®nity
(KD 0.2 to 5 mM).
Very little is known about the binding site of
thymosin b4 on actin. Attempts have been made to
identify residues or regions on actin involved in
thymosin b4 binding by chemical cross-linking
using 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide (EDC)5,6 or by competition of thymosins with
other actin-binding proteins..7-9 Both approaches
have led to the proposal that thymosin b4 binds to
the N-terminus located on subdomain 1 and to
Glu167 of subdomain 3 of actin.6,8 It has been
suggested that thymosin b4 might exist as an
elongated molecule that stretches from the bottom
of subdomain 3 to the DNase I binding region on
subdomain 2 of actin.6
The b-thymosins are able to intracellularly shift
the G/F-equilibrium of actin towards the monomeric state by removing (sequestering) G-actin
from this equilibrium. It has been proposed that a
rapid increase in the number of free ends of F-actin
upon cell stimulation leads to the incorporation of
free monomeric actin into F-actin. Indeed, in vitro
experiments have shown that an increase in the
number of free actin ®lament ends results in dissociation of the actin:thymosin b4 complex in order
to maintain the equilibrium between free G-actin
and the actin:thymosin b4 complex.10
Within the framework of this model, thymosin
b4 is able to bind to only G-actin. However, it has
been demonstrated recently that Tb4 is able to
interact with ®lamentous actin,11,12 since it was
shown that addition of high concentrations of
thymosin b4 (>200 mM) to polymerising actin
decreased the critical concentration of free actin to
polymerise, indicating the participation of the
actin:Tb4 complex in the polymerisation reaction.
Similarly, it has been reported that over-expression
of thymosin b4 in ®broblasts induces increased formation of actin containing stress ®bres and, at the
same time, decreased the amount of monomeric
actin.12
We therefore analysed the interaction of thymosin b4 with preformed F-actin, and the ability of
the actin:thymosin b4 complex chemically crosslinked by EDC to copolymerise with native actin
or to form actin ®laments by itself. Our data
demonstrated that in the simultaneous presence of
phalloidin and gelsolin, the puri®ed cross-linked
actin:thymosin b4 complex can be induced to polymerise into ®lamentous actin. Structural analysis
of electron micrographs of negatively stained
actin:Tb4 ®laments revealed a 4.5 nm increase of
the crossover spacing of the two long-pitch helical
strands. Difference map analysis of a 3-D helical
reconstruction of control and actin:thymosin b4
®laments revealed the possible contact sites of thymosin b4 with actin, and suggested the occurrence
of subtle conformational changes in actin upon
thymosin b4 binding.
Binding of Thymosin 4 to F-actin
Results
Thymosin b 4 can be cross-linked to F-actin
It has been shown that thymosin b4 can be crosslinked to F-actin.11 However, since kinetic analysis
demonstrated that F-actin depolymerising ability
by free thymosin b4 is most probably due to a shift
in the G/F-equilibrium and the formation of
actin:Tb4,11 we repeated these experiments in the
absence or presence of phalloidin, which stabilises
preformed F-actin in the presence of Tb4.10 After
incubation overnight (about 16 hours) of F-actin
and thymosin b4 added at 200 mM (14-fold excess
over actin), the mixture was treated with EDC (see
Materials and Methods). The samples were subjected to ultracentrifugation and the resulting
supernatants and pellets were analysed separately
by SDS-PAGE. As can be seen from Figure 1(a),
Tb4 was cross-linked to ®lamentous actin both in
the absence and the presence of phalloidin under
these conditions. When using phalloidin-stabilised
F-actin, the generated cross-linked actin:Tb4 was
only in the pellet, whereas in its absence a considerable amount of actin:Tb4 was found in the
supernatant, demonstrating that Tb4 induced depolymerisation of F-actin (Figure 1(a); see also
Figure 1(b), lanes 3 and 4). In contrast, when
native, i.e. not cross-linked actin:Tb4, obtained by
mixing actin and thymosin b4 at almost equimolar
ratio, was ®rst induced to polymerise by addition
of phalloidin and then treated with EDC, no crosslinked actin:Tb4 in the pellet after sedimentation
was detected by SDS-PAGE (Figure 1(b)), indicating that thymosin b4 had dissociated from actin
during the polymerisation reaction. In this particular experiment, we employed dansyl-labelled
thymosin b4, therefore dissociation of thymosin b4
from polymerising actin was veri®ed directly by
¯uorescence analysis of the gel (Figure 1(c)).
Dansylation of thymosin b4 has been shown not to
affect its actin-binding ability.13
Chemically cross-linked actin:Tb
b4
copolymerises with actin
Due to the G-actin-sequestering activity of thymosin b4, actin:Tb4 has been regarded not to be
able to copolymerise with native actin.2 We analysed this property of actin:Tb4 directly by using
chemically cross-linked actin:Tb4. Actin and thymosin b4 were cross-linked by EDC under mild
conditions (see Materials and Methods), yielding
about 40 % of total actin as the cross-linked adduct.
KCl has been shown to reduce the af®nity of thymosin b4 for G-actin.7 Therefore, the concentration
of the remaining non-cross-linked actin moiety was
reduced by inducing polymerisation in the presence of 2 mM MgCl2 and 0.1 M KCl, followed by
high-speed centrifugation, which led to an enrichment of the cross-linked actin:Tb4 in the supernatant to a ratio of approximately 1:1 to non-crosslinked actin:Tb4 (Figure 2, lane 1). In the absence of
Binding of Thymosin 4 to F-actin
615
Figure 2. AwTb4-complex is incorporated into actin
®laments. A solution of nearly equimolar amounts of
cross-linked and not cross-linked A:Tb4-complex (1.5 mM
each) was employed for sedimentation. In the absence
of salt no sedimentation of actin or cross-linked A:Tb4complex occurred (lanes 1 and 2). Sedimentation after
addition of 2 mM MgCl2 and 0.1 M KCl (lanes 3 and 4)
and in the additional presence of phalloidin (lanes 5 and
6). Supernatants (S) and pellets (P) were analysed on an
SDS/10 % (w/v) polyacrylamide gel.
Figure 1. Interaction of thymosin b4 with monomeric
and ®lamentous actin. (a) Preformed F-actin (14.2 mM)
was treated with a high molar excess of thymosin b4
(200 mM) under polymerising ionic conditions in the presence and absence of phalloidin (20.65 mM). The incubation was allowed to proceed overnight at room
temperature. After reaching the new equilibrium, Tb4
was cross-linked to actin, and monomeric and ®lamentous actin were separated by sedimentation. Supernatants (S) and pellets (P) were analysed on an SDS/
7.5 % (w/v) polyacrylamide gel. (b) In order to test
incorporation of the native actin:thymosin b4 complex
into F-actin, the actin:Tb4 complex (11.7 mM G-actin,
12.6 mM Tb4) was polymerised by the addition of phalloidin (18.5 mM), 100 mM KCl, and 2 mM MgCl2. After
an incubation period of about 15 hours at room temperature, G and F-actin were separated by centrifugation. Supernatants (S) and pellets (P) were analysed
on an SDS/7.5 % polyacrylamide gel. (c) The corresponding ¯uorescence pattern of the dansyl-labelled Tb4
as detected by a UV-illuminator.
salt, we observed no sedimentation of this material
(Figure 2, lanes 1 and 2). After addition of 2 mM
MgCl2 and 0.1 M KCl, only very little AwTb4 copolymerised with native actin, in agreement with the
sequestering function of thymosin b4. However, if
phalloidin was present, incorporation of AwTb4
into F-actin was observed, as veri®ed by sedimentation experiments (Figure 2, lanes 5 and 6).
Electron microscopy after negative staining
showed that these ®laments were of normal F-actin
appearance (Figure 3(a)). In order to check the
incorporation of cross-linked A:Tb4 into these ®laments, we treated them with a polyclonal antithymosin b4 antibody followed by a secondary
antibody attached to 10 nm gold beads (see
Materials and Methods). Immunogold labelling
revealed in many instances a ``beads-on-a-string''
like alignment of the gold particles (Figure 3(b)),
suggesting random incorporation along the entire
length of the ®lament rather than preferential binding to ®lament ends.
Hannappel and Wartenberg have demonstrated
that thymosin b4 lacking the six N-terminal residues dissociated from actin under polymerising
conditions, indicating either reduced af®nity or
inability to sequester G-actin.14 We therefore
repeated the copolymerisation experiment using
the N-terminally truncated thymosin b4,7 ± 42 which
was cross-linked to G-actin by EDC as intact thymosin b4 with similar yield. The cross-linked
actin:thymosin b47 ± 42 complex was mixed, without
further enrichment, with native actin and subsequently allowed to polymerise for about 16
hours after addition of 2 mM MgCl2 and 0.1 M
KCl. The results obtained (Figure 4) showed that
the cross-linked actin:thymosin b47 ± 42 complex
616
Binding of Thymosin 4 to F-actin
Figure 4. Incorporation of actin in complex with truncated Tb47 ± 43 into F-actin. A mixture of cross-linked and
native complex of actin and truncated Tb47 ± 43 (12 mM)
was subjected to different actin polymerising conditions
as detailed (2 mM MgCl2, 100 mM KCl, 15.4 mM phalloidin). The samples were incubated overnight at room
temperature. After sedimentation of actin ®laments,
supernatants (S) and pellets (P) were separated and analysed on an SDS/10 % (w/v) polyacrylamide gel.
Figure 3. Electron microscopy of negatively stained
copolymerised ®laments. (a) Actin ®laments consisting
of actin and AwTb4 in a molar ratio of 2:1 were negatively stained and visualised at a magni®cation of
123,500. (b) Immunostaining on the grid using the
monoclonal anti-thymosin b4 antibody and gold-labelled
second antibody as detailed in Materials and Methods.
Particle size of gold granules, 10 nm.
copolymerised with native actin even in the
absence of phalloidin, thus demonstrating the loss
of its sequestering capacity.
Isolation and polymerisation of the chemically
cross-linked actin:thymosin b 4 complex
Since actinwTb4 copolymerised with native actin,
we wanted to test the ability of puri®ed actinwTb4
to form ®laments. Therefore, chemically crosslinked actin:Tb4 had to be puri®ed to homogeneity.
To achieve this, actin:Tb4 was cross-linked under
mild cross-linking conditions, i.e. by not affecting
the critical concentration of actin polymerisation,
Cc (see Materials and Methods). After puri®cation
as detailed in Materials and Methods, actinwTb4
was found to be about 98 % pure (Figure 5, ®rst
lane). The actinwTb4 contained tightly bound
nucleotide (not shown) and was able to inhibit the
enzymatic activity of DNase I, although to a lesser
extent than by native G-actin or G-actin treated
with EDC under identical conditions (not shown).
The ability of pure AwTb4 to form ®lamentous
actin was investigated under a number of different
conditions. Raising the ionic strength by addition
of 2 mM MgCl2 and 0.1 M KCl did not result in
polymerisation of AwTb4, not even in the presence
of an 8.7-fold molar excess of phalloidin (Figure 5).
However, when in addition to phalloidin, catalytic
amounts of gelsolin or a gelsolin:2-actin complex
(gelsolin/total actin molar ratio 1:150) were added,
AwTb4 polymerised in the absence of any native
actin, as veri®ed by sedimentation (Figure 5) and
electron microscopy (EM) in conventional transmission (CTEM) and scanning transmission
(STEM) mode after negative staining (Figure 6(b)
and inset, respectively). In fact, AwTb4 ®laments
were indistinguishable from control ®laments
(Figure 6(a) and inset), i.e. actin ®laments polymerised in the absence of thymosin b4 but otherwise
identically treated with EDC. Evidently, the gelsolin:2-actin (GA2) complex acted as seeds or nuclei
for AwTb4 to polymerise into ®laments. Most likely,
the GA2 complex was also generated upon
addition of gelsolin to unpolymerised AwTb4 by
complexation to remaining small amounts of free
actin present in the AwTb4 preparations. The fact
that GA2 was able to induce polymerisation of
AwTb4 indicated that AwTb4 was still able to bind
to the (ÿ)end of actin ®laments or nuclei.
EDC has been shown to cross-link Tb4 either via
Lys3 to Glu167 or via Lys18 to the N terminus of
actin.6 In agreement with these authors, we found
that puri®ed AwTb4 consisted predominantly of
Tb4 cross-linked to the actin N terminus. Only a
small fraction (less than 10 %) contained Tb4 crosslinked to Glu167 of actin, as veri®ed by treatment
with hydroxylamine (for details see Materials and
Methods, data not shown). When analysing the
formed AwTb4 ®laments by limited proteolysis,
both cross-linked species were found to be incorporated into the AwTb4 ®laments (data not shown).
617
Binding of Thymosin 4 to F-actin
Figure 5. Polymerisation of AwTb4 into ®laments in the simultaneous presence of GA2-complex and phalloidin.
Pure cross-linked A:Tb4-complex (1.5 mM) was treated with various combinations of actin-polymerising substances
(15.2 mM phalloidin, 10.13 nM GA2-complex or free gelsolin) as detailed. The polymerisation was allowed to continue
overnight at room temperature. The samples were centrifuged and the supernatants (S) and the pellets (P) were analysed on an SDS/10 % (w/v) polyacrylamide gel.
Mass-per-length determination of control and
Aw
w Tb
b 4 filaments
The mass-per-length (MPL) of unstained AwTb4
®laments was determined by STEM after freezedrying and compared to control F-actin ®laments
that had been produced after an identical treatment of G-actin with EDC and likewise polymerised in the presence of identical concentrations of
phalloidin and gelsolin (see also Figure 6(a) and (b)
for the ®lament morphologies). Figure 6 displays
selected STEM images of (c) control and (d) AwTb4
®laments, and Figure 6(e) reveals an MPL histogram computed from a mixture of control and
AwTb4 ®laments. As documented, the histogram
could be ®t by two Gaussian curves, one representing the control ®laments, i.e. peaking at an MPL of
16.0(0.8) kDa/nm, and the other representing the
AwTb4 ®laments, i.e. peaking at an MPL of
18.0(0.9) kDa/nm. Based on a crossover spacing
of 36.0 nm for the two long-pitch helical strands
and a helix geometry with 13 subunits per crossover for the control ®laments (see below), an MPL
of 15.8 kDa/nm is calculated (i.e. assuming an
actin:phalloidin monomer mass of 43.8 kDa). Similarly, taking a crossover spacing of 40.5 nm for the
two long-pitch helical strands and a helix geometry
with 15 subunits per crossover for the AwTb4 ®laments (see below), an MPL of 18.1 kDa/nm is calculated (i.e. assuming an actin:Tb4:phalloidin
monomer mass of 48.8 kDa).
Determination of the helical parameters of
Aw
w Tb
b 4 filaments
STEM annular dark ®eld (ADF) images of 30
well-preserved, evenly negatively stained and relatively straight 100-200 nm ®lament segments each
of control and AwTb4 ®laments were selected by
visual inspection and computationally unbent
(Figure 6(f), ÿ and ). Next, the optimal crossover
spacing of the two long-pitch helical strands was
determined for each ®lament segment by varying
it so that upon averaging over three crossovers the
power loss became minimal and the image
enhancement maximal (Figure 6(g), ÿ and ).
Accordingly, the average crossover spacing for the
30 control ®laments amounted to 36.0 nm and that
for the 30 AwTb4 ®laments to 40.5 nm. Based on
these crossover spacings, the following integer helical selection rules were determined for the ``genetic'' one-start helix: 13 subunits in six left-handed
turns (i.e. a screw angle of ÿ166.2 and an axial
rise of 2.77 nm per subunit) for the control ®laments, and 15 subunits in seven left-handed turns
(i.e. a screw angle of ÿ168.0 and an axial raise of
2.70 nm per subunit) for the AwTb4 ®laments.
Finally, the averaged three-crossover long ®lament
segments (Figure 6(g), ÿ and ) were D(Z,k)®ltered according to the above integer helical selection rules (Figure 6(h), ÿ and ).15 This change of
the helical parameters might have been caused
either by a direct steric effect of thymosin b4 on the
®lament geometry and/or a thymosin b4-induced
conformational change on actin.
3-D helical reconstruction of control and
Aw
w Tb
b 4 filaments
The formation of stable ®laments composed of
AwTb4 with an apparently typical F-actin appearance (Figure 6) indicated that, even in the presence
of chemically cross-linked thymosin b4, actin was
still able to form ®lamentous contacts similar to
those of native F-actin. Therefore the structure of
the control and AwTb4 ®laments was further analysed by 3-D helical reconstruction of the 12 best
three-crossover long ®lament segments each. The
individual 3-D reconstruction of both the control
and AwTb4 ®laments (Figure 7(a), 1 and 2, respectively) were aligned relative to a reference and averaged. Moreover, a difference map was computed
by subtracting the averaged control reconstruction
reconstruction
from
the
averaged
AwTb4
618
Binding of Thymosin 4 to F-actin
Figure 6. (a) and (b) Conventional and scanning transmission electron microscopy (CTEM and STEM) of negatively
stained control and AwTb4 ®laments. (a) CTEM of F-actin polymerised from EDC-treated actin by addition of phalloidin and GA2 complex. The inset shows identically prepared control ®laments viewed by STEM in annular dark ®eld
mode. (b) CTEM of actin ®laments polymerised from AwTb4 as detailed in Materials and Methods. The inset displays
identically obtained AwTb4 ®laments viewed by STEM. The scale bars represent 100 nm. (c) to (e) Mass-per-length
determination by STEM of control and AwTb4 ®laments. The mass-per-length (MPL) was determined on stretches of
an equimolar mixture of control and AwTb4 ®laments. For this purpose, ®laments were prepared using identical conditions. (c) A selected control ®lament; (d) a selected AwTb4 ®lament; and (e) an MPL (kDa/nm) histogram of the
equimolar mixture of control and AwTb4 ®laments. Gaussian curve ®tting of the MPL histogram yielded two wellresolved curves, one peaking at 16.0 kDa/nm (peak 1), and the second peaking at 18.0 kDa/nm (peak 2). (c) and (d)
The scale bar represents 100 nm. (f) to (h) Comparison of the helical parameters of control and AwTb4 ®laments. (f)
STEM dark-®eld images of three-crossover long (i.e. 130 nm) segments of a selected control (ÿ) and AwTb4 () ®lament, prepared using identical conditions. (g) Linear averages over exactly three crossovers followed by periodic continuation to the original length of the control (ÿ) and AwTb4 () ®lament segments displayed in (f). (h) Helically
®ltered (i.e. D(Z,k)-®ltered; see Materials and Methods) images of exactly three-crossover long segments of the control
(ÿ) and AwTb4 () ®laments shown in (f), employing the integer helical selection rule l ÿ 6n 13 m (i.e. for the
control actin ®laments) and l ÿ 7n 15 m (i.e. for the AwTb4 ®laments), respectively. Note the increase in the crossover spacing from 36.0 nm in the case of the control ®lament (ÿ) to 40.5 nm in the case of the AwTb4 ®lament ().
(Figure 7(a), 3). To achieve subtraction of two 3-D
reconstructions with different helical selection
rules, the averaged AwTb4 reconstruction was
mapped onto the genetic helix of the control ®laments (i.e. 13 subunits in six left-handed turns).16
Furthermore, surface renderings were contoured to
include 100 % (Figure 7(a), 1 and 2) and 30 % mass
(Figure 7(a), 5 and 6) of the aligned and averaged
reconstructions computed from 12 control and 12
AwTb4 ®laments. Taken together, the reconstructions demonstrate clearly that the AwTb4 ®laments
are of a ``more compact'' appearance than the control ®laments, in the sense that the inter-subunit
contacts along the two long-pitch helical strands
Binding of Thymosin 4 to F-actin
619
Figure 7. 3-D helical reconstruction of control and AwTb4 ®laments, together with difference map and its location
relative to the Holmes-Lorenz atomic model of the F-actin ®lament. (a) The 3-D helical reconstruction of control (1, 5)
and AwTb4 (2, 6) ®laments, contoured at a mass density level so as to include 100 % (1, 2) and 30 % (5, 6) mass,
respectively. Both the control and the AwTb4 ®lament reconstruction represent and average over 12 individual, threecrossover long ®lament segments. Difference map (3), i.e. computed as AwTb4 (2) minus control (1) ®lament, reconstruction and its mapping onto the control ®lament contoured at 100 % (4) and 30 % (7) mass, respectively. (b) Modelling of the molecular F-actin ®lament backbone (i.e. as a ribbon representation of just one long-pitch helical strand of
the Holmes-Lorenz atomic model of the F-actin ®lament; cf. Lorenz et al.18) into the control ®lament plus the difference map, exactly as in (a)4 except with the F-actin mass envelope being semi-transparent. (c) Same as (b) but with
the control ®lament mass envelope removed.
are signi®cantly more massive. The extra mass of
the AwTb4 ®laments relative to the control ®laments can be much better appreciated and delineated in the difference map (Figure 7(a), 3), and
by overlaying the difference map onto the control
®lament 3-D reconstruction (Figure 7(a), 4 and 7).
Discussion
Kinetic experiments analysing the effect of high
concentrations of thymosin b4 on the Cc or the G/
F-equilibrium had demonstrated that thymosin b4
was able to interact with ®lamentous actin.11,12
Therefore, we analysed the ability of thymosin b4
to interact with ®lamentous actin and of chemically
cross-linked actin:thymosin b4 complex to incorporate into or to even form actin polymers by
itself. Using chemical cross-linking, we showed
that thymosin b4 was able to bind to F-actin and
when employing the chemically cross-linked
actin:thymosin b4 complex, we documented that
this complex can copolymerise with native actin.
The heterogeneous ®laments were of normal
appearance as judged by electron microscopy after
negative staining. Immunogold labelling suggested
that cross-linked A:Tb4 was incorporated randomly
along the entire ®lament length, excluding the
possibility of its preferential binding to ®lament
ends.
Surprisingly, it was possible to induce polymerisation of the puri®ed AwTb4 complex to ®lamentous actin, although it occurred only under
``strong'' polymerising conditions, i.e. in the simultaneous presence of catalytic amounts of the gelso-
620
lin:2-actin complex (molar ratio of GA2 to actin:
1:150) and phalloidin, which strongly decreases the
Cc. Hence, short ®laments consisting of only AwTb4
were formed at low protein concentration. These
®laments were also of normal appearance and,
probably due to the presence of phalloidin, we did
not observe unravelling of the two long-pitch
strands.11 The ability of catalytic amounts of the
gelsolin:2-actin complex to induce ®lament formation might be due to its ability to act as competent nuclei for puri®ed AwTb4 complex, suggesting
that an additional in vivo function of thymosin b4
may be the inhibition of the formation of polymerisation-competent actin nuclei.
EDC has been shown to cross-link Lys3 and
Lys18 of thymosin b4 to Glu167 (i.e. on subdomain
3) and the N terminus (i.e. on subdomain 1) of
actin.6,17 Our data indicated that both cross-linked
species were incorporated into the AwTb4 ®laments
(data not shown). However, the complex crosslinked via Lys3 of thymosin b4 to Glu167 of actin is
produced only in small yield (our own
observations).6 Incorporation of this complex is
surprising, since Glu167 is believed to reside
within the long-pitch helix actin:actin interface,
suggesting thymosin b4 binding or cross-linking to
actin should result in steric hindrance of the formation of this actin:actin contact.18 In contrast, polymerisation of the complex cross-linked via Lys18
of Tb4 to the N terminus of actin might not be surprising, since the N terminus of actin is located on
the surface of the atomic model built by Holmes &
Lorenz.18 During polymerisation of the AwTb4
complex, the N-terminal half of thymosin b4 might
have been pushed ``out of the way''. Evidently,
this hypothesis is supported by the data showing
that actin cross-linked to the truncated thymosin
b74 ± 42 polymerised even in the absence of phalloidin
and gelsolin. Hence, we have to assume that the
presence of the six N-terminal residues of thymosin
b4 has a decisive effect on its sequestering activity
either by a steric blocking type of mechanism or by
inducing a subtle conformational change(s) in the
actin molecule.
Polymerisation of AwTb4 under our conditions
was accompanied by alterations of the helical parameters of the resulting AwTb4 ®laments relative to
the control F-actin ®laments. Most strikingly, an
increase of the crossover spacing of the two righthanded long-pitch helical strands from 36.0 nm for
the control ®laments to 40.5 nm for the AwTb4 ®laments was observed, originating from an increase
of the screw angle of the left-handed genetic helix
from ÿ166.15 to ÿ168.0 . Since phalloidin preferentially stabilises the intersubunit contacts along
the genetic helix,19 it is conceivable that AwTb4 polymerisation was largely driven by these actin:actin
contacts rather than by those along the two longpitch helical strands that might have been modi®ed
by the thymosin b4 binding (see above). The fact
that phalloidin was necessary to induce polymerisation of AwTb4 indicated that the geometry of its
binding site as determined previously19 was not
Binding of Thymosin 4 to F-actin
affected severely by the observed ®lament untwisting or by thymosin b4 binding (see also Figure 7).
A decrease of the crossover spacing of the two
right-handed long-pitch helical strands from 36 nm
to 28 nm has been reported to occur upon binding
of co®lin to F-actin ®laments.16 Hence, induced
changes of the helical parameters of F-actin by
actin-binding proteins may be a more general
phenomenon. In fact, such induced structural
changes of the actin ®lament geometry may be
employed intracellularly to modify its mechanical
properties or its dynamic behaviour, or to generate
subtle modi®cations of exposed interaction sites on
the ®lament surface related to the observed cooperative binding of a number of F-actin-binding proteins. Thus, it was observed that co®lin binding
excludes phalloidin binding, although their binding sites are distinct.16 It is conceivable that actin
®laments containing bound b-thymosins might
exist in vivo; for example, in regions of high local
concentrations of actin and b-thymosins, or in the
presence of high concentrations of polymerisation
nuclei and ®lament-stabilising proteins such as
tropomyosins or myosins. Such ®laments might
possess slightly altered biological properties,
resulting, for instance, in a modi®ed ®lament stability and/or interaction modes with other actinbinding proteins. Although cosedimentation data
indicated that the AwTb4 ®laments were able to
bind myosin subfragment 1 in an ATP-dependent
manner similar to control ®laments, this method
may be too crude to detect more subtle changes.
Based on the observation that the C terminus of
thymosin b4 can be enzymatically cross-linked to
Gln41 of actin (subdomain 2) by the action of
transglutaminase, although with low yield, it has
been suggested that the C-terminal half of thymosin b4 extends to subdomain 2 of actin.6,}20 To test
this hypothesis, we evaluated our AwTb4 ®lament
3-D reconstruction by ®tting an atomic model of
the F-actin ®lament18 into the reconstruction
shown in Figure 7(a) 4 (Figure 7(b); for simplicity,
only one long-pitch helical strand of the atomic
model has been drawn). As illustrated in
Figure 7(c), to better appreciate the placement of
the difference map (in pink) relative to the atomic
model, the EM-based F-actin ®lament envelope
present in Figure 7(b) was removed. Accordingly,
the elongated, tripartite extra mass was seated
with one of its legs resting on subdomain 1 and
the other on subdomain 3, and with its third leg
lining subdomain 2 so as to make contact with the
bottom of the actin subunit above. More accurately, whereas the longer of the two legs is ``padding the cavity'' separating subdomains 1 and 3
from subdomains 2 and 4, the shorter leg is partially ``®lling the gap'' between subdomains 1 and
2.
To evaluate the interaction of the difference map
(Figure 8(a), left) with actin at atomic detail, we
tried to ®t the atomic model of thymosin b4 that
was determined by NMR spectroscopy in
Ê restri¯uoroethanol21 and surface-rendered at 25 A
Binding of Thymosin 4 to F-actin
621
Figure 8. Molecular modelling of
the interaction of thymosin b4 (Tb4)
with the F-actin ®lament. (a)
Attempt to align (right) an NMR
structure of Tb4 (middle) within the
difference map (left) computed
between the AwTb4 and the control
®lament 3-D helical reconstruction
(see Figure 7a3). The atomic structure of Tb4 is ``embedded'' in a
semi-transparent envelope representing the electron density map
Ê resolution from
computed at 25 A
its NMR structure and surface-rendered so as to include 100 % of its
nominal mass. (b) Stereo pair of the
best ®t of the Tb4 backbone (i.e. as
shown in (a), middle) onto the
F-actin monomer backbone using
mapped cross-links between Tb4
and actin (i.e. Lys3-Glu167, Lys18Asp1, and Lys38-Gln41; cf. Safer
et al.6) as spatial constrains. For
optimising the ®t, both the Tb4 and
the F-actin monomer backbone
were kept rigid, i.e. no structural
changes of Tb4 or actin were
allowed. (c) Stereo pair yielding the
relative alignment of the Tb4 ®t
onto the F-actin monomer as
shown in (b) with the difference
map plus 3-D helical reconstruction
of the control ®lament (i.e. as
shown in Figure 7(b)). In this montage, the atomic structure of Tb4 is
shown as a space-®lling CPK representation.
olution (Figure 8(a), middle) into the extra mass
(Figure 8(a), right). Whereas this ®t, which minimised the root-mean-square (RMS) difference
between the two density maps, is far from perfect,
it represents a unique solution, so that thymosin b4
can be oriented unambiguously (i.e. with regard to
its N and C terminus; see Figure 8(a), middle)
within the difference map. Structural studies of
thymosin b4 have demonstrated that it is a highly
mobile molecule that lacks a uniquely de®ned conformation in water at temperatures above 1 C,
although a propensity to form an N-terminal
a-helix was noted.22 However, in aequeous
solutions containing ¯uorinated alcohols thymosin
b4 adopts a well-de®ned fold containing an N and
a C-terminal a-helix comprising residues 5 to 19
and 30 to 40, respectively, that are joined by a loop
extending from residue 24 to residue 29 so that a
bending angle of about 60 is formed.21 Based on
this ``qualitative'', yet unique ®t (see Figure 8(a),
right) it is tempting to speculate that the two long
legs of the difference map (see Figure 8(a), left) represent the N and C-terminal a-helices of thymosin
b4, whereas the short leg corresponds to the loop
joining the two a-helices.
622
As a next step, we tried to ®t the NMR-based
atomic model of thymosin b4 (see Figure 8(a),
middle) into the atomic model of the F-actin monomer employing the known chemical cross-links
between thymosin b4 and actin as constraints.6
As may be gathered from Figure 8(b), the ``best''
solution is given by an elongated thymosin b4
molecule and an F-actin monomer without conformational changes. Even then, the distances
between the side-chains of the participating residues on thymosin b4 and actin were too far apart
Ê ; Lys18-Asp1, 18.85 A
Ê;
(i.e. Lys3-Glu167, 10.45 A
Ê ) to ful®l the spatial
and Lys38-Gln41, 10.12 A
requirements for being zero-length cross-links.
As illustrated in Figure 8(c), the ``best'' qualitative ®t of thymosin b4 onto the F-actin monomer
(see Figure 8(b)) departed both in relative position
and orientation from the extra mass representing
the difference map computed between AwTb4 and
the control ®lament. Accordingly, the largest difference occurred in the orientation of the N-terminal
a-helix of thymosin b4, whereas the C terminus of
thymosin b4, located on subdomain 2 of F-actin,
®ts well into the difference map. Evidently, the C
terminus of thymosin b4 behaved like a ®xed
hinge, about which the rest of the molecule was
rotated so that its N terminus moved upwards into
the vicinity of the longer leg of the difference map.
As detailed in Results, this ``upward'' movement
of the N terminus might have occurred during the
polymerisation reaction. Nevertheless, a signi®cant
part of these differences might be due to conformational changes taking place in both proteins
upon interacting with each other as demonstrated
recently for actin.20 For instance, the angle between
the N and C-terminal a-helices of thymosin b4
appeared to have become more acute, most likely
due to a structural change of the connecting loop.
Nevertheless, our data suggest that upon binding
to actin, thymosin b4 adopted a structural fold
similar to that it assumes in aqueous solution in
the presence of ¯uorinated alcohols (see Figure 8(a),
middle).21 Final proof, however, will have to await
the availability of co-crystals of actin and thymosin
b4.
Materials and Methods
Materials
The chemical cross-linker 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide (EDC) was obtained from Pierce
(Rockford, IL, USA). Monodansylcadaverine, tissue type
transglutaminase (guinea pig liver) and subtilisin were
commercial products of Sigma Corp., MuÈnchen,
Germany. All other reagents were of analytical grade.
Protein preparations
Rabbit skeletal muscle actin was puri®ed as
described23 as modi®ed by Ballweber7 and taken up in
G-buffer (5 mM Hepes-OH (pH 7.4), 0.1 mM CaCl2,
0.5 mM NaN3, 0.2 mM ATP). Human plasma gelsolin
was expressed in Escherichia coli and further puri®ed.24
Binding of Thymosin 4 to F-actin
Bovine pancreatic deoxyribonuclease I (DNase I; EC
3.1.21.1.) was a commercial product (Paesel and Lorei,
Frankfurt, Germany) and further puri®ed by ionexchange chromatography on hydroxylapaptite.25
Thymosin b4 was isolated from bovine spleen.26
Gel electrophoretic procedures
Polyacrylamide gel electrophoresis was performed in
the presence of sodium dodecylsulfate (SDS-PAGE).27
For evaluation of the molecular mass, we employed prestained marker proteins (180 kDa, a2-macroglobulin;
116 kDa, b-galactosidase; 84 kDa, fructose-6-phosphate
kinase; 58 kDa, pyruvate kinase; 48.5 kDa, fumarase;
36.5 kDa, lactate dehydrogenase; and 26.5 kDa, triosephosphate isomerase) obtained from Sigma Corp.,
MuÈnchen, Germany. For densitometric analysis of
Coomassie blue-stained gels, we used an Intas gel
scanner (Intas, GoÈttingen, Germany) equipped with the
Intas program Cream-1D for quantitative analysis.
Chemical cross-linking of thymosin b 4 and F-actin
Thymosin b4 and ®lamentous actin were incubated
overnight at room temperature. Thereafter, cross-linking
was performed with 5 mM EDC in 25.7 mM Mes buffer
(pH 6.0) at room temperature. After two hours the reaction was stopped by the addition of 22.5 mM Tris.
Chemical cross-linking of thymosin b4 to G-actin for
copolymerisation assays
Native actin:thymosin b4 complex (11 mM) in G-buffer
was cross-linked by addition of 0.5 mM EDC in 5 mM
Mes (pH 6.0) at room temperature. After two hours the
reaction was stopped by adding 5.7 mM Tris. Under
these ``mild'' cross-linking conditions, no detectable
alteration of the polymerisation behaviour of uncomplexed control actin had occurred, since the critical concentration of actin polymerisation (Cc) of both native and
EDC-treated control actin were found to be identical
(data not shown).
Dansylation of thymosin b 4
Thymosin b4 contains glutamine residues at positions
23, 36 and 39. Dansylation of glutamine residues 23
and/or 36 was achieved by incubating 50 mM thymosin
b4with 250 mM mono-dansylcadaverin and 2 mg transglutaminase. The reaction was performed overnight at
room temperature in 10 mM Tris-HCl (pH 8.0), 1 mM
CaCl2, 5 mM dithioerythritol, 0.5 mM ATP, 0.2 mM
NaN3. Dansylation of thymosin b4 was veri®ed after
SDS-PAGE by placing the gels on a UV-illuminator.
Under these conditions, no cross-linking of actin to Tb4
was observed.13
Identification of the Tb
b 4 cross-linking sites on actin
In order to identify the region on actin to which Tb4
was linked by the chemical cross-linking reaction, the
actin of the cross-linked actin:Tb4 complex was either
proteolytically treated with subtilisin or chemically
cleaved by hydroxylamine. Subtilisin was shown to
cleave native monomeric actin after residue Met47 and
Gly48 and in addition after Glu42 and Val43.28,29 Subtilisin treatment of the chemically cross-linked actin:Tb4
complex was performed as described for actin.28 In some
623
Binding of Thymosin 4 to F-actin
of these experiments, dansylated thymosin b4 was
employed.13 For cleavage of actin of actin cross-linked to
Tb4, the polymerised complex was treated with 2 M guanidinium hydrochloride and 1 M hydroxylamine in the
presence of 1 mM ATP and 0.2 M K2CO3 (pH 9.0), in
order to achieve selective cleavage at the asparaginylglycine bond at position 12 of monomeric actin.30,31
Enrichment and purification of the cross-linked
A:Tb
b 4 complex
For copolymerisation assays, the cross-linked A:Tb4
complex was enriched by inducing polymerisation of the
actin moiety that was not cross-linked to Tb4 in the presence of 2 mM MgCl2 and 0.1 M KCl at room temperature. After about 15 hours, F-actin was separated from
globular A:Tb4 by ultracentrifugation for 1.5 hours at
100,000 g. The supernatant contained cross-linked and
native A:Tb4 in about equimolar amounts (about 1.5 mM
each) as veri®ed by SDS-PAGE.
The cross-linked A:Tb4 complex was further puri®ed
to almost homogeneity (about 95 % pure) by the following procedure. The volume of the equimolar mixture of
native and cross-linked A:Tb4 (about 1.5 mM each, see
above) was reduced tenfold by the use of Centricon 30
concentration capsules (Millipore, Witten, Germany),
which allowed the passage of uncomplexed thymosin b4.
Consequently, the native A:Tb4 complex dissociated into
the free components actin and thymosin b4. In the presence of 2 mM MgCl2 and 0.1 M KCl, the free actin
polymerised to F-actin, which was removed by ultracentrifugation for 1.5 hours at 100,000 g. Subsequently, the
supernatant was subjected to a number of cycles of
dialysis against F-buffer (G-buffer plus 2 mM MgCl2 and
0.1 M KCl) to remove free thymosin b4 and to induce
polymerisation of free actin. The dialysis/polymerisation/ultracentrifugation cycle was repeated several
times until the cross-linked A:Tb4 complex was almost
completely devoid of free actin and thymosin b4 as
judged by SDS-PAGE.
Cosedimentation
Cosedimentation experiments were performed using
either a Beckman Airfuge run at 20 psi (1 psi 6.9 kPa)
or a Beckman TL 100 refrigerated ultracentrifuge operated at 100,000 g for one hour. The sample size subjected
to ultracentrifugation was usually 30 ml. After centrifugation, the supernatants and pellets were collected separately and suspended to identical ®nal volumes in boiling
sample buffer. Identical volumes were analysed by SDSPAGE.
DNase I inhibition assay
DNase I activity was determined by using the optical
hyperchromicity assay.32,33 Puri®ed bovine pancreatic
DNase I was pre-incubated in G-buffer with native or
EDC-actin, or cross-linked actin:Tb4. The absorbance
density change was determined at 260 nm and room
temperature using a Beckman DU 640 spectrophotometer.
Electron microscopy
For conventional transmission electron microscopy
(CTEM), F-actin and actin:Tb4 ®laments were negatively
stained with 1 % (w/v) uranyl acetate as described10 and
visualised in a Philips 420 microscope operated at 80 kV.
For immunogold detection of thymosin b4, carbon-coated
grids were ®rst loaded with the actin sample. Then
unspeci®c binding sites were blocked with 0.1 mg/ml of
bovine serum albumin (this step made negative staining
and visualisation of F-actin impossible), followed by
treatment with a monoclonal anti-thymosin b4 antibody
(kindly provided by Professor B. Jockusch, Braunschweig, Germany) to label Tb4. Finally, the grids were
overlaid with gold-tagged anti-mouse IgG.
F-actin and actin:Tb4 ®lament preparations intended
for structural analysis and 3-D helical reconstruction
were negatively stained with 0.75 % (w/v) uranyl
formate and imaged by an annular dark-®eld (ADF)
detector in a dedicated scanning transmission electron
microscope (STEM; HB5, Vacuum Generators, East Grinstead, UK) at 100 kV acceleration voltage. For recording
images at 500,000 magni®cation the e ÿ dose was kept
at 5 103 e ÿ/nm2. STEM ADF images were digitally
recorded by a custom-built data acquisition system.35
The frame size of the images was 512 512 pixels
corresponding to a pixel size of 0.28 nm at the 500,000
magni®cation setting.
STEM mass measurements of control and
actin:Tb
b 4 filaments
For their mass-per-length (MPL) determination, an
equimolar mixture of control and actin:Tb4 ®laments
were adsorbed to glow-discharged ultrathin carbon ®lms
mounted on fenestrated carbon supports that were
adhered to gold-plated 200-mesh copper grids. The grids
were washed ®ve times in double quartz-distilled water
before being freeze-dried overnight in the pre-treatment
chamber connected directly to the specimen chamber of
the STEM. The MPL and full-width-half-maximum
(FWHM) of the resulting unstained freeze-dried ®laments were determined by quantitative STEM using an
HB5 STEM (Vacuum Generators, East Grinstead, UK),
operated at 80 kV.34,}35 STEM dark-®eld images consisting of 512 512 pixels were recorded at 200,000 nominal magni®cation. Air-dried tobacco mosaic virus (TMV;
kindly provided by Dr J. Witz, Institut de Biologie MoleÂculaire et Cellulaire, Strasbourg, France) prepared on
ultrathin carbon ®lms was imaged in the same way as
the ®laments, and used as an instrumental control. The
processing of STEM images was carried out using the
IMPSYS software package.34 For image analysis, ®lament
segments approximately 60 nm long were de®ned and
boxed. MPL and FWHM analysis was carried out on
each boxed ®lament segment using IMPSYS. The data
from all ®lament segments were then pooled and presented as a histogram that was ®t with Gaussian curves.
A series of pictures of the same 512 x 512 pixel area were
taken where the sample was irradiated sequentially with
increasing doses of electrons. From these images,
the mass-loss was calculated for a normal dose of
300 e ÿ/nm2. This mass loss was found to be <4 %, so
that no mass-loss correction of the data was performed.
Digital image processing and 3-D
helical reconstruction
For structural analysis and 3-D helical reconstruction,
well preserved, evenly negatively stained and relatively
straight ®lament segments were chosen by visual inspection of STEM ADF images and processed.36 Brie¯y,
about 30 ®lament stretches, 100-200 nm long, were digi-
624
tally straightened using the SEMPER6 image processing
package.37 The straightened ®lament stretches were subjected to 3-D helical reconstruction using the Micrograph
Data Processing Program MDPP.38 The three-crossover
long ®lament segments (i.e. three crossover spacings of
the two long-pitch helical strands) were D(Z,k)-®ltered,
employing either the integer helical selection rule
l ÿ6n 13 m (i.e. for the control actin ®laments) or
l ÿ7n 15 m (i.e. for the actin:Tb4 ®laments), whereby
the helical repeat length and radial position of the ®lament axis were optimised via a 3-D helical parameter
search.16,39,40 The 12 ®laments with the highest transmitted power upon D(Z,k)-®ltration in each case were
Fourier-transformed and their equator and the 1st, 2nd,
5th, 6th, 7th, 8th, 13th and 14th (i.e. for the control Factin ®laments), or their 1st, 2nd, 6th, 7th, 8th, 9th, 13th,
14th, 15th and 16th (i.e. for the actin:Tb4 ®laments) layerlines extracted. Layer-line correlation with a reference
was used to align and average a set of D(Z,k)-®ltered
and layer-line-extracted ®lament segments.39 The 3-D
reconstructions were computed and surface-rendered by
isocontouring them with a program implemented on a
Silicon Graphics (Mountain View, CA) computer.39,41,42
Contouring levels were selected so as to include the
nominal molecular volume of the 43 kDa actin subunit
or fractions of it, taking into consideration the calculated
cylindrical radius of gyration of the respective reconstructions.
Acknowledgements
It is a pleasure for us to thank Mrs Ulrike Ritenberg
for expert technical assistance and Dr T. Holak (Martinsried, Germany) for supplying the coordinates of thymosin b4; the Deutsche Forschungsgemeinschaft, the Swiss
National Science Foundation, the M.E. MuÈller Foundation of Switzerland, and the Canton Basel-Stadt for
®nancial support.
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Edited by W. Baumeister
(Received 27 July 2001; received in revised form 9 November 2001; accepted 13 November 2001)

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