(Received for publication, May 25, 1995; and in revised form, September 7, 1995)
From the
Binding sites of actin and thymosin 4 were investigated
using a set of bifunctional thiol-specific reagents, which allowed the
insertion of cross-linkers of defined lengths between cysteine residues
of the complexed proteins. After the cross-linkers were attached to
actin specifically at either Cys
, Cys
, or to
the sulfur atom of the ATP analog adenosine
5`-O-(thiotriphosphate) (ATP
S), the actin derivatives
were reacted with synthetic thymosin
4 analogs containing a
cysteine at one of the positions 6, 17, 28, 34, and 40. Immediate
cross-linking as followed by UV spectroscopy was found for Cys
of actin and Cys
of thymosin
4, indicating that
the N terminus of thymosin
4 is in close proximity (
9.2
Å) to the C terminus of actin. In contrast, only insignificant
reactivity was measured for all thymosin
4 analogs when the
cross-linkers were anchored at Cys
of actin. A second
contact site was identified by cross-linking of Cys
and
Cys
in thymosin
4 with the ATP
S derivative bound
to actin, indicating that the hexamotif of thymosin
4 (positions
17-22) is in close proximity (
9.2 Å) to the nucleotide.
The importance of the amino acids 17 and 28 in thymosin
4 for the
interaction with actin was emphasized by the finding that thymosin
analogs containing cysteine in these positions exhibited strongly
reduced abilities to inhibit actin polymerization.
The physiological conditions within nonmuscle cells favor the
assembly of actin monomers. Therefore, the pool of monomeric actin has
to be maintained by complexation with small G-actin binding proteins.
Particularly thymosin 4 (T
4), (
)which forms a 1:1
complex with actin monomers, is believed to be involved in preventing
actin
polymerization(1, 2, 3, 4, 5, 6) .
Dissociation constants of the actin
T
4 complex were found to
be in the range of 0.4-0.7 µM for platelet actin and
0.7-2.0 µM for muscle actin(4, 7) .
While complexed with T
4, the exchange of the bound nucleotide in
actin is retarded (8) . In a detailed study, Vancompernolle et al.(9) have shown that binding of T
4 to actin
is mainly mediated by the hexamotif
LKKTET(17, 18, 19, 20, 21, 22) ,
since loss of this sequence is paralleled by an almost complete loss of
inhibitory activity. Alterations in the N-terminal part (1-16) of
the peptide strongly influence the inhibitory activity of T
4,
whereas alterations in the C-terminal part (31-43) seem to be of
minor importance (9) . As shown by
H NMR
spectroscopy(10, 11) T
4 does not contain an
ordered conformation in aqueous solution but tends to form an
-helical conformation between residues 5 and 16 (11) . It
has been proposed that T
4 is likely to adopt a unique conformation
upon binding actin(12) .
One of the binding sites of T4
on the actin molecule seems to be located in subdomain 1 as suggested
by cross-linking studies(13, 14) . In order to gain
more knowledge about contact sites in the actin
T
4 complex,
we performed a structural analysis using bifunctional thiol-specific
reagents of the type alkylene-bis-[5-dithio-(2-nitrobenzoic
acid)] for intermolecular cross-linking of two cysteine residues.
Such reagents were successfully used for cross-linking two distinct
cysteines in muscle actin as well as for preparing a defined
disulfide-linked actin dimer(15, 16) . By varying the
length of the cross-linkers (as well as using Ellman's reagent
for zero-length cross-linking), information can be obtained about the
distance up to which two thiol groups in the complexed proteins can
approach. In a first reaction, the cross-linkers (9.2Å to
18.4Å) were anchored monovalently at one of three thiols in
monomeric actin. Since native T
4 does not contain any cysteine,
T
4 analogs were synthesized, each containing cysteine at one of
the positions 6, 17, 28, 34, and 40. Thus, the substitutions were
distributed over the whole protein but were restricted to hydrophobic
amino acids. After adding the T
4 analogs to the actin derivatives,
the kinetics and extents of cross-linking were followed by
spectrophotometric analysis of the 2-nitro-5-thiobenzoate released.
Figure 1:
Reaction of the
cross-linking reagent alkylene-bis-[5-dithio-(2-nitrobenzoic
acid)], (ArSS-(CH)
-SSAr), with
cysteine 374 of actin. Yield of the actin derivative
(actin
SS-(CH
)
-SSAr) was
followed by the release of the yellow 2-nitro-5-thiobenzoate
(ArS
) monitored at 412
nm.
To introduce
the cross-linkers into position 10 of actin, the cysteine residue in
position 374 was blocked by incubating G-actin in buffer G with a
100-fold excess of N-ethylmaleimide (NEM) at 4 °C for 30
min. The reaction was quenched with excess DTT, and the protein was
separated on a Bio-Rad P2 column equilibrated with buffer G. NEM-actin
was polymerized by the addition of 0.2 mM EGTA, 1 mM
MgCl, and removal of ATP was achieved by incubation with
hexokinase (5 units/ml actin solution, Sigma) and 0.4 mM glucose for 90 min at room temperature(19) . After
centrifugation at 100,000
g, the pellets were allowed
to soften on ice in ADP buffer (2 mM Tris, 1 mM ADP,
0.02% NaN
, pH 7.8) for 30 h. After that time, Cys
was completely accessible (20) and could be reacted with
one end of the cross-linking reagent. The
NEM-actin
SS-(CH
)
-SSAr
was purified as described above. Yield of the labeling reaction was
80-90%.
For labeling of ATPS (Sigma), the nucleotide
was reacted with 1.5 equivalents of reagent (n = 3 or
9) in 1 M imidazol, pH 6.5, at 4 °C overnight. Excess of
reagent was removed on a Sephadex LH20 column (2
45 cm)
(Pharmacia Biotech Inc.), equilibrated with 10% 2 mM Tris, pH
7.0, and 90% methanol. After removal of methanol in vacuo at 4
°C, the fractions containing the labeled nucleotide were used as
softening buffer (
0.1 mM labeled ATP
S, 2 mM
Tris, 0.1 mM CaCl
, 0.02% NaN
, pH 8) as
described above.
Incorporation of
ATPS-S-(CH
)
-SSAr was performed only with
actin blocked at Cys
and Cys
in order to
exclude any unspecific reaction. For this, ADP
G-actin with both
cysteines exposed was prepared as described above. Reaction with NEM
and removal of excess reagent were achieved as described for
NEM-actin. The resulting
(NEM)
-actin was polymerized, and ATP was
removed by the hexokinase reaction. The
(NEM)
-actin pellet was allowed to soften
on ice in the preformed softening buffer in order to incorporate the
labeled ATP
S. Directly before use, excess of
ATP
S-S-(CH
)
-SSAr was removed on a Bio-Rad
P2 column (1
18 cm) equilibrated with ADP buffer 2 (2 mM Tris, 0.2 mM ADP, 0.1 mM CaCl
, 0.02%
NaN
, pH 7.8) yielding a fraction of actin that contained
nearly one equivalent (95%) of the labeled nucleotide.
All steps were performed in an argon atmosphere in order to minimize oxidation of the unprotected cysteine residue in the thymosin analogs.
For measuring the effects of the
different thymosins on the nucleotide exchange rate, the T4
analogs (8 µM) were added to the
-ATP buffer. For
comparing the nucleotide exchange rates of
(NEM)
-actin containing either the
ATP
S derivative or ATP as the bound nucleotide, both actin
derivatives were purified on a Bio-Rad P2 column (1
18 cm)
equilibrated with ADP buffer 2, assayed for concentration, and applied
to the nucleotide exchange measurements just after elution from the
column.
A method was developed that allowed the investigation of
contact sites between actin and thymosin 4 by assessing whether
two thiol groups in the protein complex could approach sufficiently
close to allow cross-linking by thiol-specific cross-linkers of
different lengths.
Figure 2:
a,
SDS-PAGE of actins monovalently linked with the cross-linking reagents
at different positions. The gel shows that the actin derivatives are
indistinguishable from G-actin. 1, G-actin; 2,
actinSS-(CH
)
-SSAr; 3,
(NEM)
-actin
ATP
SS-(CH
)
-SSAr; 4,
NEM-actin
SS-(CH
)
-SSAr. b, SDS-PAGE of native actin and several actin derivatives
cross-linked with native T
4 using EDC/NHS. The presence of similar
amounts of the 47 kDa band representing the covalently linked
actin
T
4 complex indicates that none of the cross-linking
reagents attached to actin inhibited binding of T
4. Yield of
cross-linking was 25 ± 1% for G-actin and all actin derivatives
as determined by densitometric measurements. 1, G-actin; 2, G-actin cross-linked with native T
4; 3,
actin
SS-(CH
)
-SSAr cross-linked
with native T
4; 4,
NEM-actin
SS-(CH
)
-SSAr
cross-linked with native T
4.
For specific
labeling of Cys, ATP in
NEM-actin was
exchanged for ADP, a reaction that initiates a slow unfolding reaction
and results in selective and quantitative exposure of this cysteine
residue(20) . After reaction with a 3-fold excess of reagent,
the resulting
NEM-actin
SS-(CH
)
-SSAr (n = 3, 6, 9) was purified and shown to contain
0.8-0.9 equivalents of cross-linker as assessed by
spectrophotometry in the presence of DTT. The actin derivatives of this
type were again indistinguishable from G-actin in SDS-PAGE (Fig. 2a) as well as with respect to their binding
capacities for thymosin
4 (Fig. 2b).
In order
to prepare the actin derivative with the cross-linker anchored at the
actin-bound nucleotide, the cross-linking reagent had first to be
attached to ATPS. The modified ATP
S was identified by its
H NMR spectrum (
)as well as by UV spectrometry (Fig. 3a). The presence of a 1:1 adduct of ATP
S
and nonylene-5-dithio-2-nitrobenzoate was proved by evaluating the
amount of ArS
(
= 14,150 M
cm
(25)) released after
treatment with DTT (Fig. 3b), which corresponds to the
amount of cross-linker present in the modified nucleotide. (The molar
extinction coefficient of the cross-linker part is
= 9400 ± 50 M
cm
, a value that agrees with the extinction
coefficient previously reported for n-octyl-5-dithio-2-nitrobenzoate (
=
9050 M
cm
(26) )). Considering the contribution of
the cross-linking part to the absorbance at 259 nm (0.4
E
(26) ) the absorption of the adenosine
part at that wavelength (
of ATP = 16,415 M
cm
(27)) (Fig. 3a) reveals a ratio of 1:0.97 for the ATP
S
part and the cross-linking part. The modified nucleotide was exchanged
for ADP in
(NEM)
-actin, which was
prepared in order to avoid intramolecular cross-linking of the modified
nucleotide with the two potentially reactive thiol groups in actin,
yielding
(NEM)
-actin
ATP
SS-(CH
)
-SSAr (n = 3, 9). Since the affinity of the modified
ATP
S for actin is lower than that of ATP (see below), loading with
the labeled nucleotide was optimized by separating the excess of
unbound, labeled ATP
S just before use. Incorporation of the
labeled nucleotide into actin at the time of the experiment was then as
high as 95%.
Figure 3:
UV-spectrum of
ATPSS-(CH
)
-SSAr before (a) and
after treatment with DTT (b) showing the absorptions of
ArS
at 412 nm, of the alkyl-5-dithio-2-nitrobenzoate
part at 338 nm, and of the adenosine part at 259 nm (plus the
contribution of the alkyl-5-dithio-2-nitrobenzoate at that
wavelength).
For making sure that ATPS could indeed be used as
an anchoring point in actin, the affinity of the modified nucleotide to
actin was assayed by determining the exchange rate of the modified
ATP
S bound to
(NEM)
-actin for
-ATP. This exchange rate was found to be accelerated 5-fold in
comparison with normal ATP bound to
(NEM)
-actin (k = 2.8
10
± 0.2
10
s
in comparison with k = 5.8
10
± 0.3
10
s
).
Figure 4:
Thymosin 4 does not contain any
cysteine (a). Therefore five different T
4 analogs were
synthesized, each containing one cysteine in distinct positions (b-f). Substitutions were distributed over the
whole molecule and were restricted to hydrophobic amino
acids.
Figure 5:
SDS-PAGE of G-actin cross-linked to the
five T4 analogs using EDC/NHS. Comparable amounts of the 47 kDa
band representing the covalently linked actin
T
4 complex were
formed from all thiol-protected analogs similar to native T
4. This
indicates that none of the substitutions made in T
4 abolished
binding to actin. 1, actin +
Cys
T
4; 2, actin +
Cys
T
4; 3, actin +
Cys
T
4; 4, actin +
Cys
T
4; 5, actin +
Cys
T
4; 6, actin; 7, actin
+ T
4
Figure 6:
Reaction mixtures of different actin
derivatives cross-linked with CysT
4 were
analyzed on SDS-PAGE after 60 min. Yield of cross-linking was
determined by densitometric evaluation of the gel bands and was in good
agreement with the corresponding spectrophotometric values representing
the amount of ArS
released due to the cross-linking
reactions. For lanes 1-4, calculations of the yields of
cross-linking took into account that the actin derivatives were present
in excess (2:1) over T
4. 1,
actin
SS-(CH
)
-SSAr +
Cys
T
4 mixed at a ratio of 2:1 (yield of
cross-linking, 60%); 2,
actin
SS-(CH
)
-SSAr +
Cys
T
4 mixed at a ratio of 2:1 (yield of
cross-linking, 55%); 3,
actin
SS-(CH
)
-SSAr +
Cys
T
4 mixed at a ratio of 2:1 (yield of
cross-linking, 54%); 4, actin
SSAr +
Cys
T
4 mixed at a ratio of 2:1 (yield of
cross-linking, 29%); 5, actin; 6,
NEM-actin
SS-(CH
)
-SSAr
+ Cys
T
4 mixed at a ratio of 1:1 (yield of
cross-linking, 11%); 7,
NEM-actin
SS-(CH
)
-SSAr
+ Cys
T
4 mixed at a ratio of 1:1 (yield of
cross-linking, 9%); 8,
NEM-actin
SS-(CH
)
-SSAr
+ Cys
T
4 mixed at a ratio of 1:1 (yield of
cross-linking, 6%).
Based on extent and kinetics of the
ArS release, three types of reactions could be
distinguished. In the first type, the reaction proceeded rapidly
reaching its end point (>50%) within less than 10 min (Fig. 7, a-c). For one of these reactions, a
complete kinetic analysis was performed showing that the half-maximal
value was actually reached after about 1 min (data not shown). Reaction
kinetics of this type were taken as indicating the close proximity of
the two thiols in the protein complex. Based on this type of kinetics
it was possible to identify three sites of very close contact (
9.2
Å) between the two proteins. One of these contacts is between
Cys
actin and Cys
T
4.
Cross-linking at this site was almost independent of the length of the
cross-linker as yields and kinetics of
actin
SS-(CH
)
-SSAr were similar
when n was 3, 6, or 9. The proximity of
Cys
actin and Cys
T
4 was even
close enough to allow for zero-length cross-linking as shown for
actin
SSAr when allowed to complex with
Cys
T
4. However, zero-length cross-linking was
distinctly slower than the cross-linking reactions described first, and
thus belongs to the second type of kinetics described below. The two
other sites of close contact were identified from the rapid reactions
of the cross-linkers attached to the actin-bound ATP
S with
Cys
T
4 and, to a lower extent, with
Cys
T
4.
Figure 7:
Typical reaction kinetics of
CysT
4 with actin derivatives carrying
cross-linking reagents of different length at Cys
or
Cys
, as monitored by the release of ArS
.
The two proteins were mixed as described above, and the amount of
ArS
was determined after 10, 30 and 90 min. Curves a, b, and c represent reaction kinetics
defined as type 1 (see ``Results''); curve d represents the kinetics of a type 2 reaction; curve e represents the kinetics of a type 3 reaction. a,
actin
SS-(CH
)
-SSAr +
Cys
T
4; b,
actin
SS-(CH
)
-SSAr +
Cys
T
4; c,
actin
SS-(CH
)
-SSAr +
Cys
T
4; d, actin
SSAr
+ Cys
T
4; e,
NEM-actin
SS-(CH
)
-SSAr
+ Cys
T
4
In the second type of kinetics, yield
of cross-linking was low at the beginning (10% after 10 min) but
became extensive with time (Fig. 7d). It appears that
in this type of cross-linking reaction, the two thiols are not in close
proximity but can come close to each other due to the mobility of one,
or both, of the partners. Examples of this second type of kinetics are,
besides the reaction already mentioned, the cross-links between
actin
SS-(CH
)
-SSAr (n = 3, 6, 9) and the cysteines located in the central part of
thymosin
4. Particularly Cys
T
4, and to a
much lower extent also Cys
T
4 showed
considerable extents of cross-linking with cysteine 374 of actin,
although with low reaction rates. Cross-linking reactions of this type
were not regarded as identifying sites of strong contact.
The third
type of cross-linking reactions comprises those with very low amounts
(<10%) of ArS released during the first 10 min
followed by an only slight increase within 90 min (Fig. 7e). This reaction pattern was the most frequent
one and, in contrast to the two other types of kinetics, was taken as
an indication that the two thiol groups were remote from each other.
This type of kinetics was found e.g. in all experiments
involving Cys
T
4, suggesting that this position
in thymosin
4 must be located distant from both cysteine residues
in subdomain 1 of actin as well as from the actin-bound nucleotide.
This type of kinetics was likewise found in all cross-linking
experiments involving
NEM-actin
SS-(CH
)
-SSAr (n = 3, 6, 9).
Figure 8:
Polymerization kinetics of actin (10
µM) as followed by capillary viscometry in the absence or
presence of T4 or the T
4 analogs. The
-thymosin analogs
(1.5 eq) were mixed with native actin (1 eq) 30 min before the
polymerization conditions were established by the addition of KCl to a
final concentration of 100 mM. Values represent the average of
four measurements. Pure actin,
; actin + T
4,
;
actin + Cys
T
4,
; actin +
Cys
T
4,
; actin +
Cys
T
4,
; actin +
Cys
T
4,
; actin +
Cys
T
4,
.
Encouraged
by the good correlation found between polymerization inhibiting
capacities and cross-linking data, we assayed the retardation of the
nucleotide exchange rate of actin as another functional parameter of
T4. The influence of Cys
T
4 on the
nucleotide exchange was examined in comparison with native T
4 and
Cys
T
4, the latter as an example of a T
4
analog, which is ineffective in the thiol-specific cross-linking
reactions as well as in the polymerization-inhibiting assay. The
retardation effect of Cys
T
4 was found to be
indeed partly abolished. While the k value of
Cys
T
4 (k = 2.8
10
± 0.2
10
s
) was almost indistinguishable from that of native
T
4 (k = 2.9
10
±
0.2
10
s
), the nucleotide
exchange rate of Cys
T
4 was found to be
accelerated to a value of k = 4.7
10
± 0.2
10
s
, a value that approaches the k value of
pure actin (k = 6.2
10
± 0.3
10
s
) under
these conditions (Fig. 9).
Figure 9:
Time course of the exchange of actin-bound
ATP (0.3 µM) in the absence of T4 (+, upper
curve), in the presence of Cys
T
4 (
, middle curve), or in the presence of
Cys
T
4 (
, lower curve), showing
the effects of the two thymosin
4 analogs on the nucleotide
exchange rate.
In order to identify contact sites between actin and T4
we successfully used a method of selective cross-linking between thiols
that is able to measure the closest approach of two cysteines in the
protein complex. By using a set of cross-linking reagents of different
lengths, or the procedure of direct activation of one of the thiols
with Ellman's reagent, we were able to assay distances between
two thiols in the range from 0 to
18 Å.
In obtaining
reliable results from this kind of study it was essential that the
cross-linking reaction was absolutely thiol-specific and that each of
the two proteins exposed only one thiol group. The first condition was
assured by the fact that the disulfide-exchange reaction runs with
thiols only(28) . The second requirement was met for T4 in
that the synthetic T
4 analogs used contained only one cysteine
each. As for actin, we made use of the fact that actin in buffer G
exposes only cysteine 374(29) , which could either be reacted
with the cross-linking reagents or be blocked with NEM. By exchanging
ATP for ADP in
NEM-actin, cysteine 10 could be
selectively uncovered(20) , thus providing another distinct
thiol group to be reacted with the cross-linking reagents. In order to
obtain a third well defined anchoring point in actin, the cross-linkers
were attached to the ATP analog ATP
S. The modified nucleotide was
characterized by UV and
H NMR spectroscopy and shown to
contain adenosine and the cross-linking reagent at a ratio of 1:1.
Although the exchange rate of the modified actin-bound nucleotide was
increased by a factor of five over that of ATP, binding of the labeled
ATP
S was regarded as tight enough to provide the third point of
attachment for the cross-linkers. For all actin derivatives, it was
shown that they behaved similarly, or even identically, to G-actin with
respect to polymerization, appearance in SDS-PAGE, and binding to
native T
4. For all T
4 analogs, it was proven that the
substitutions did not impair complex formation with actin.
As in
titrations using Ellman's reagent, formation of a cross-link
between an actin derivative and a T4 analog was accompanied by the
release of ArS
detectable at 412 nm, which allowed
easy determination of the extent and kinetics of cross-link formation
by UV spectrometry. Since these data were in very good agreement with
those obtained by integrating the corresponding gel bands in SDS-PAGE,
it was concluded that the release of ArS
reflected
the cross-link formation quantitatively. The extent of cross-linking as
determined by spectrophotometry was independent on whether one of the
components was used in excess (2:1) and never exceeded 75%. The
incompleteness of the reaction may be explained by the K
value of the actin
T
4 complex (
1 µM)
limiting complex formation. In addition, the extent of cross-linking
may be lowered by the fact that all actin derivatives were labeled only
up to 80-90%. Finally, it cannot be excluded that the unprotected
cysteine in the T
4 analogs was partially oxidized during the
cross-linking reaction. On the other hand, in all reactions classified
as negative, the release of ArS
was never zero. We
suppose that the small amounts of ArS
(<10%)
detected in these experiments were released by unspecific reactions in
which the small T
4 reacted to some extent in a way similar to a
low molecular weight thiol.
Three major reaction types could be
distinguished on the basis of kinetics and the extent of cross-linking.
Fast reactions with high extents of cross-linking (50-75%) within
a few minutes were taken as indicating close proximity of the two
cysteines in the protein complex. According to this classification, one
major contact was identified between the C terminus of actin and the N
terminus of T4. In particular, there is evidence that the thiols
of Cys
in actin and Cys
in T
4 approach
to within 9.2 Å. This finding confirms previous data that
identified Cys
as a part of a short distance cross-link
with T
4(13) . Contact in this region must indeed be very
close since it was even possible to form a zero-length cross-link
between Cys
of actin and Cys
T
4,
although at a low rate. As a second major contact site the hexamotif of
T
4 (position 17-22) was identified as located near the
actin-bound nucleotide, since the distance of
Cys
T
4 and the sulfur atom of ATP
S could be
bridged by a cross-linker of 9.2 Å in length. Lower, but still
significant yields of cross-linking were found also between
Cys
T
4 and the modified ATP
S, suggesting
that the whole central part of T
4 is in proximity to the
-phosphate of the nucleotide. Considering the different yields of
these two cross-linking reactions, Cys
may be located
closer to the nucleotide than Cys
, provided sterical
influences can be excluded. Fig. 10illustrates the position of
the two major contact sites within a space-filling model of G-actin
according to Kabsch et al.(30) . Due to the mobility
of the cross-linkers, only spheres of contact can be defined with
dimensions determined by the length of the cross-linkers.
Figure 10:
Schematic representation of the two major
contact sites identified in actin for T4 as illustrated by two
spheres fitted into the structure of G-actin in a space-filling model
according to Kabsch et al.(30) . The spheres are
centered either at the
-phosphate of ATP (sphere A), or
at C
(sphere B), and both have a
radius of 10 Å representing the maximal possible reaction range
for the cross-linkers of the type
ArSS-(CH
)
-SSAr. Sphere B was centered
at C
as the last defined position in
G-actin as determined by x-ray analysis. Cys
, to which
the cross-linker was attached, can be assumed to be ca. 3Å apart
from C
, given an
-helical
conformation at the C terminus of actin. The numbers in the
illustration denote the four subdomains of the actin molecule. This
figure was prepared by the PLUTO program written by Sam Motherwell at
the Chemical Laboratory, Cambridge, UK.
Different
from the first type of reaction, a second one was distinguished from
its distinctly slower kinetics. In this type, rather high yields of
cross-linking were obtained but only after a prolonged reaction time
(40% yield after 90 min). Examples were the formation of the
zero-length cross-link mentioned above as well as the reaction of the
Cys
of actin and Cys
T
4. It
appears that cross-linking in these cases depends on processes that
proceed at a low rate, as for example mobility of the C terminus of
actin(30) . For this reason, cross-linking reactions proceeding
slowly were not used in identifying sites of closest contact. Finally,
more than 50% of all cross-linking experiments proceeded not only at
very slow kinetics but on the average reached yields of only 9% of
cross-linking (highest yield, 18%) even after prolonged incubation.
This third type of reaction kinetics was taken as excluding a proximity
of the two thiols concerned. However, the possibility still exists that
a limited reactivity in some of the positions in the T
4 analogs
may rely on a partial occlusion of the corresponding cysteine residue
due to binding to actin.
The N terminus of T4 has been
described as tending to form an
-helix between the residues 5 and
16(10) . Given that this structure is stabilized in contact
with actin as hypothesized by Sun et al.(3) , the
helix might stretch along subdomain 1 with contacts between the N
terminus of T
4 and the C terminus of actin on one hand, and the
hexamotif of T
4 and the actin-bound nucleotide on the other.
According to Kabsch et al.(30) the distance between
Cys
and the ribose unit is around 28 Å, a distance
that can easily be spanned by the length of the two cross-linkers plus
the 11 amino acids between the residues 6 and 17 of the T
4
molecule, even when they are in helical conformation. The yield of
cross-linking with the actin-bound nucleotide decreased when the
position of the cysteine residue in the T
4 analog approached the C
terminus. This correlation indicates that the highly flexible C
terminus of T
4 is most probably directed away from the actin-bound
nucleotide. Since no thiol-specific cross-link formation was found in
any experiment involving Cys
T
4, this part of
T
4 appears to be located in a domain of actin that is different
from subdomain 1 and remote from the nucleotide region.
Reactions of
cysteine 10 of actin with all T4 analogs followed the third type
of reaction kinetics. According to our classification, we believe that
T
4 is not in direct contact with that side of actin bearing
Cys
, the latter known to be part of a
-sheet(30) . Nevertheless, low yield cross-linking
reactions with Cys
were measured by spectrophotometry, and
confirmed by SDS-PAGE. The existence of these reactions between the
cross-linker attached to Cys
and T
4 may be understood
on the basis of the maximal possible reaction range of the
cross-linking reagent (
9.2Å) that reaches far beyond the
thiol of Cys
. The reaction range of the cross-linker may
be comparable with that of the first four amino acids of actin, which
are believed to form a mobile structure (30) and have been
reported to be involved in EDC cross-links with
T
4(14, 31) .
Finally, we assayed whether the
replacement of five hydrophobic amino acids in T4 had caused any
functional deficits. For all T
4 analogs, a clear correlation was
found between the extents of cross-linking with the actin-bound
nucleotide and the decrease in the inhibitory capacities on actin
polymerization. The strongest reduction of inhibitory capacities was
found for substitutions by cysteine in or near by the hexamotif. This
observation is in line with the results of Vancompernolle et al.(9) who showed the hexamotif to be most important for
binding and function. Interestingly, in the case of
Cys
T
4, the greatest extent of cross-linking was
paralleled not only by the most strongly reduced inhibitory capacity,
but also by a significant decrease of the retardation effect on the
nucleotide exchange rate in comparison to native T
4.