©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Contact Sites in the Actin-Thymosin 4 Complex by Distance-dependent Thiol Cross-linking (*)

(Received for publication, May 25, 1995; and in revised form, September 7, 1995)

Andreas Reichert (1) Daniela Heintz (1) Hartmut Echner (2) Wolfgang Voelter (2) Heinz Faulstich (1)(§)

From the  (1)Max-Planck-Institut für medizinische Forschung, 69120 Heidelberg, Germany, and the (2)Abteilung für Physikalische Biochemie des Physiologisch-chemischen Instituts der Universität Tübingen, 72076 Tübingen, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Binding sites of actin and thymosin beta4 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) (ATPS), the actin derivatives were reacted with synthetic thymosin beta4 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^6 of thymosin beta4, indicating that the N terminus of thymosin beta4 is in close proximity (leq9.2 Å) to the C terminus of actin. In contrast, only insignificant reactivity was measured for all thymosin beta4 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 beta4 with the ATPS derivative bound to actin, indicating that the hexamotif of thymosin beta4 (positions 17-22) is in close proximity (leq9.2 Å) to the nucleotide. The importance of the amino acids 17 and 28 in thymosin beta4 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.


INTRODUCTION

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 beta4 (Tbeta4), (^1)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 actinbulletTbeta4 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 Tbeta4, the exchange of the bound nucleotide in actin is retarded (8) . In a detailed study, Vancompernolle et al.(9) have shown that binding of Tbeta4 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 Tbeta4, whereas alterations in the C-terminal part (31-43) seem to be of minor importance (9) . As shown by ^1H NMR spectroscopy(10, 11) Tbeta4 does not contain an ordered conformation in aqueous solution but tends to form an alpha-helical conformation between residues 5 and 16 (11) . It has been proposed that Tbeta4 is likely to adopt a unique conformation upon binding actin(12) .

One of the binding sites of Tbeta4 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 actinbulletTbeta4 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 Tbeta4 does not contain any cysteine, Tbeta4 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 Tbeta4 analogs to the actin derivatives, the kinetics and extents of cross-linking were followed by spectrophotometric analysis of the 2-nitro-5-thiobenzoate released.


MATERIALS AND METHODS

Protein Purification

Actin was prepared from rabbit muscle as described by Spudich and Watt (17) and further purified by a gel filtration step on a Fractogel TSK HW 55 column (3 times 120 cm) (E. Merck, Darmstadt) in buffer G (2 mM Tris, 0.2 mM ATP, 0.1 mM CaCl(2), 0.02% NaN(3), pH 7.8). Thymosin beta4 was isolated from bovine lungs according to Spangelo et al.(18) or obtained by synthesis.

Preparation of Actin Derivatives

ActinSS-(CH(2))(n)-SSAr was prepared by reacting G-actin (3.8 times 10M) in buffer G with a 3 molar excess of the reagent ArSS-(CH(2))(n)-SSAr (n = 3, 6 or 9) (see Fig. 1). The cross-linkers were prepared according to (15) and (16) , and their corresponding lengths were 9.2, 13.8, or 18.4 Å, respectively. The mixture was kept at 4 °C until one equivalent of 2-nitro-5-thiobenzoate (ArS) was released ( = 14,150 M cm). By exhaustive dialysis in buffer G, the major part of excess reagent was removed together with ArS before the protein was purified on a Bio-Rad P2 column (2 times 45 cm) equilibrated with buffer G. Labeling of the actin derivative was 80-90% as determined from protein concentration of the purified derivative, and the amount of ArS detected at 412 nm after cleavage with excess of dithiothreitol (DTT). For zero-length cross-linking, we prepared actinSSAr by reacting G-actin with 3-fold excess of Ellman's reagent under the same conditions.


Figure 1: Reaction of the cross-linking reagent alkylene-bis-[5-dithio-(2-nitrobenzoic acid)], (ArSS-(CH(2))-SSAr), with cysteine 374 of actin. Yield of the actin derivative (actinSS-(CH(2))-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(2), 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 times g, the pellets were allowed to soften on ice in ADP buffer (2 mM Tris, 1 mM ADP, 0.02% NaN(3), 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-actinSS-(CH(2))(n)-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 times 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 ATPS, 2 mM Tris, 0.1 mM CaCl(2), 0.02% NaN(3), pH 8) as described above.

Incorporation of ATPS-S-(CH(2))(n)-SSAr was performed only with actin blocked at Cys and Cys in order to exclude any unspecific reaction. For this, ADPbulletG-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)(2)-actin was polymerized, and ATP was removed by the hexokinase reaction. The (NEM)(2)-actin pellet was allowed to soften on ice in the preformed softening buffer in order to incorporate the labeled ATPS. Directly before use, excess of ATPS-S-(CH(2))(n)-SSAr was removed on a Bio-Rad P2 column (1 times 18 cm) equilibrated with ADP buffer 2 (2 mM Tris, 0.2 mM ADP, 0.1 mM CaCl(2), 0.02% NaN(3), pH 7.8) yielding a fraction of actin that contained nearly one equivalent (95%) of the labeled nucleotide.

Synthesis of Thymosin beta4 Analogs

The thymosin beta4 analogs, (S-isopropylthio)-L-Cys^6bulletTbeta4, (S-isopropylthio)-L-CysbulletTbeta4, (S-isopropylthio)-L-CysbulletTbeta4, (S-isopropylthio)-L-CysbulletTbeta4, and (S-isopropylthio)-L-CysbulletTbeta4, were prepared with an Ecosyn P peptide synthesizer (Eppendorf-Biotronik, Maintal, Germany) using the Fmoc technique (21) and a 4-alkoxybenzyl alcohol resin(22) . The side chains of the Fmoc amino acids were protected as tert-butyl esters (glutamic and aspartic acid), tritylamides (glutamine and asparagine), tert-butyl ethers (threonine and serine), tert-butyloxycarbonyl derivatives (lysine), and S-isopropylthio derivatives (cysteines)(23) . After cleavage from the resin and deprotection with trifluoracetic acid/thioanisole/anisole/water (14.5/0.6/0.4/0.25 (v/v)), the peptides were precipitated with anhydrous diethyl ether and dried. Desalting of the crude peptides was performed by gel chromatography on a TSK-HW 40 S (Merck, Darmstadt, Germany) column (1.6 times 100 cm) with 5% acetic acid as eluent. The lyophilized products were further purified by HPLC on a Nucleosil 100 C18 (7 µm) (Macherey & Nagel, Düren, Germany) 250 times 20 mm column at 214 nm. The thymosin beta4 analogs were assayed for purity by analytical HPLC, and their composition was verified by an ion spray API III/TAGA 6000 E mass spectrometer (Sciex, Toronto, Ontario).

Cross-linking Studies

In the thymosin beta4 analogs, cleavage of the S-protecting isopropylthiol residue was achieved by incubating 1 mg of the Tbeta4 analog in a 200-fold excess of 2-mercaptoethanol in 2 mM Tris, pH 7.5, for 2 h at 4 °C. Excess reagent was removed on a Bio-Rad P2 column (1 times 15 cm) equilibrated with the same buffer. The concentration of CysbulletTbeta4 was determined by titrating an aliquot with Ellman's reagent and measuring the released ArS at 412 nm. The actin derivatives were mixed with the Cysbulletbeta4 analogs at ratios of 2:1 or 1:1 (concentrations of both proteins were in the range of 1-3 times 10M) and allowed to react at room temperature. At time intervals of 10, 30, and 90 min, the amount of ArS released due to the cross-linking reaction was measured, and the extent of cross-linking was determined (100% values were defined by the CysbulletTbeta4 concentration added). To verify that the extinctions measured at 412 nm reflected the cross-linking events quantitatively, aliquots of the reaction mixtures were taken after 60 min and applied to SDS-PAGE. The amount of actinbulletTbeta4 complex formed in the cross-linking reaction was determined by integrating the 47 kDa band.

All steps were performed in an argon atmosphere in order to minimize oxidation of the unprotected cysteine residue in the thymosin analogs.

Other Cross-linking Experiments

G-actin (3 times 10M) in 2 mM MOPS, pH 6.0, containing 0.2 mM ATP, and 0.1 mM CaCl(2) was reacted with 1-ethyl-3(3-dimethyl-aminopropyl)carbodiimide(2 mM)/N-hydroxysuccinimide (5 mM) (EDC/NHS) for 30 min at 4 °C. After adjusting the pH to 7.8 by adding 2 mM MOPS buffer, pH 9.5, an equimolar amount of the beta-thymosins was added. Reaction was stopped after 30 min by the addition of glycine and the mixture analyzed on 10% SDS-PAGE.

Viscosimetric Measurements

Actin polymerization was monitored in a Cannon capillary viscosimeter, using a 10 µM concentration of G-actin in buffer G. Polymerization conditions were established by the addition of KCl to a final concentration of 100 mM. To investigate the inhibitory capacities of the different thymosin beta4 analogs, the peptides were added to the actin to a final concentration of 15 µM and allowed to incubate for 30 min at room temperature before polymerization was started.

Nucleotide Exchange

Nucleotide exchange was measured in a Spex fluorolog (Spex Industries Inc.) at 410 nm (excitation: 350 nm) using 0.2 mM 1-N^6-ethenoadenosine 5`-triphosphate (24) in a buffer containing 2 mM Tris, 0.1 mM CaCl(2), 0.02% NaN(3), pH 7.5. The reaction was started by the addition of actin, whose concentration was 0.3 µM in all cases.

For measuring the effects of the different thymosins on the nucleotide exchange rate, the Tbeta4 analogs (8 µM) were added to the -ATP buffer. For comparing the nucleotide exchange rates of (NEM)(2)-actin containing either the ATPS derivative or ATP as the bound nucleotide, both actin derivatives were purified on a Bio-Rad P2 column (1 times 18 cm) equilibrated with ADP buffer 2, assayed for concentration, and applied to the nucleotide exchange measurements just after elution from the column.


RESULTS

A method was developed that allowed the investigation of contact sites between actin and thymosin beta4 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.

Anchoring of the Cross-linkers to Actin

The actin derivatives of the type actinSS-(CH(2))(n)-SSAr (n = 3, 6, 9) were obtained in a nearly quantitive reaction of G-actin with a 3-fold excess of the cross-linkers (Fig. 1). The stoichiometry of the actin derivatives was proved by the release of approximately 1 equivalent of ArS during the cross-linking reaction, as well as by analysis of the purified actin derivative, which released 0.8-0.9 equivalents of ArS on the addition of DTT. It was shown that actin derivatives of this type polymerized similar to normal actin apart from a slightly increased critical concentration. In SDS-PAGE, these actin derivatives were indistinguishable from G-actin (Fig. 2a). Likewise we found that binding to native Tbeta4 was not altered by the modification, as concluded from cross-linking experiments with EDC/NHS yielding the typical 47 kDa band (Fig. 2b). Densitometric evaluation yielded an extent of 25 ± 1% cross-linking for all actin derivatives and G-actin.


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(2))(3)-SSAr; 3, (NEM)(2)-actin bullet ATPSS-(CH(2))(9)-SSAr; 4, NEM-actinSS-(CH(2))(9)-SSAr. b, SDS-PAGE of native actin and several actin derivatives cross-linked with native Tbeta4 using EDC/NHS. The presence of similar amounts of the 47 kDa band representing the covalently linked actinbulletTbeta4 complex indicates that none of the cross-linking reagents attached to actin inhibited binding of Tbeta4. 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 Tbeta4; 3, actinSS-(CH(2))(3)-SSAr cross-linked with native Tbeta4; 4, NEM-actinSS-(CH(2))(9)-SSAr cross-linked with native Tbeta4.



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-actinSS-(CH(2))(n)-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 beta4 (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 ATPS was identified by its ^1H NMR spectrum (^2)as well as by UV spectrometry (Fig. 3a). The presence of a 1:1 adduct of ATPS 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 times 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 ATPS part and the cross-linking part. The modified nucleotide was exchanged for ADP in (NEM)(2)-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)(2)-actinbulletATPSS-(CH(2))(n)-SSAr (n = 3, 9). Since the affinity of the modified ATPS for actin is lower than that of ATP (see below), loading with the labeled nucleotide was optimized by separating the excess of unbound, labeled ATPS 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(2))(9)-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 ATPS bound to (NEM)(2)-actin for -ATP. This exchange rate was found to be accelerated 5-fold in comparison with normal ATP bound to (NEM)(2)-actin (k = 2.8 times 10 ± 0.2 times 10 s in comparison with k = 5.8 times 10 ± 0.3 times 10 s).

Preparation of the Thymosin beta4 Analogs

Since thymosin beta4 does not contain any cysteine residue, five different Tbeta4 analogs were synthesized, each containing one cysteine in a defined position (Fig. 4). The distribution of the cysteines in the Tbeta4 sequence was such as to replace hydrophobic residues only. To ensure that these substitutions did not influence binding of Tbeta4 to actin, cross-linking studies using EDC/NHS were performed with all analogs. These studies showed that all analogs were still able to bind actin similar to normal Tbeta4 as indicated by the occurrence of the 47 kDa band in SDS-PAGE (Fig. 5). To confirm this result, and to ensure that the substitution even in the hexamotif of Tbeta4 had no significant effect on the affinity to actin, the K(D) value of CysbulletTbeta4 was determined according to (13) . It was shown to be 0.8 µM ± 0.1 µM and thus in the same range as the K(D) value of native Tbeta4, which was reported to be 0.7-2.0 µM(4, 7) .


Figure 4: Thymosin beta4 does not contain any cysteine (a). Therefore five different Tbeta4 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 Tbeta4 analogs using EDC/NHS. Comparable amounts of the 47 kDa band representing the covalently linked actinbulletTbeta4 complex were formed from all thiol-protected analogs similar to native Tbeta4. This indicates that none of the substitutions made in Tbeta4 abolished binding to actin. 1, actin + Cys^6bulletTbeta4; 2, actin + CysbulletTbeta4; 3, actin + CysbulletTbeta4; 4, actin + CysbulletTbeta4; 5, actin + CysbulletTbeta4; 6, actin; 7, actin + Tbeta4



Cross-linking Studies

Cross-linking reactions were detected by measuring changes in absorbance at 412 nm that occur when actin derivatives and thymosin beta4 analogs were allowed to form a complex. In order to prove that the DeltaOD really reflected cross-link formation between the two proteins, the reaction mixtures were investigated in parallel by SDS-PAGE (Fig. 6). The amounts of the 47 kDa bands, representing the covalently linked actin-thymosin beta4 complex, were measured by integration of the gel bands and compared with the absorbance values detected at the same time. From the good agreement of the two sets of data, it was concluded that the release of ArS measured by UV spectroscopy at 412 nm indeed reflected the formation of cross-links. Proof of the disulfide nature of the linkage between the two proteins was obtained by SDS-PAGE where the 47 kDa band disappeared in the presence of DTT. Since each of the two proteins in the complex was exposing only one thiol group, a positive cross-linking reaction could be taken as evidence that the two thiols had approached to a distance that could be bridged by the length of the cross-linker. In total, more than 40 kinds of cross-linking experiments were performed, the results of which are compiled in Table 1. In control experiments, it was shown that native Tbeta4 when added to the actin derivatives did not induce the release of ArS .


Figure 6: Reaction mixtures of different actin derivatives cross-linked with Cys^6bulletTbeta4 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 Tbeta4. 1, actinSS-(CH(2))(9)-SSAr + Cys^6bulletTbeta4 mixed at a ratio of 2:1 (yield of cross-linking, 60%); 2, actinSS-(CH(2))(6)-SSAr + Cys^6bulletTbeta4 mixed at a ratio of 2:1 (yield of cross-linking, 55%); 3, actinSS-(CH(2))(3)-SSAr + Cys^6bulletTbeta4 mixed at a ratio of 2:1 (yield of cross-linking, 54%); 4, actinSSAr + Cys^6bulletTbeta4 mixed at a ratio of 2:1 (yield of cross-linking, 29%); 5, actin; 6, NEM-actinSS-(CH(2))(9)-SSAr + Cys^6bulletTbeta4 mixed at a ratio of 1:1 (yield of cross-linking, 11%); 7, NEM-actinSS-(CH(2))(6)-SSAr + Cys^6bulletTbeta4 mixed at a ratio of 1:1 (yield of cross-linking, 9%); 8, NEM-actinSS-(CH(2))(3)-SSAr + Cys^6bulletTbeta4 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 (leq9.2 Å) between the two proteins. One of these contacts is between Cysbulletactin and Cys^6bulletTbeta4. Cross-linking at this site was almost independent of the length of the cross-linker as yields and kinetics of actinSS-(CH(2))(n)-SSAr were similar when n was 3, 6, or 9. The proximity of Cysbulletactin and Cys^6bulletTbeta4 was even close enough to allow for zero-length cross-linking as shown for actinSSAr when allowed to complex with Cys^6bulletTbeta4. 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 ATPS with CysbulletTbeta4 and, to a lower extent, with CysbulletTbeta4.


Figure 7: Typical reaction kinetics of Cys^6bulletTbeta4 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, actinSS-(CH(2))(9)-SSAr + Cys^6bulletTbeta4; b, actinSS-(CH(2))(6)-SSAr + Cys^6bulletTbeta4; c, actinSS-(CH(2))(3)-SSAr + Cys^6bulletTbeta4; d, actinSSAr + Cys^6bulletTbeta4; e, NEM-actinSS-(CH(2))(9)-SSAr + Cys^6bulletTbeta4



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 actinSS-(CH(2))(n)-SSAr (n = 3, 6, 9) and the cysteines located in the central part of thymosin beta4. Particularly CysbulletTbeta4, and to a much lower extent also CysbulletTbeta4 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 CysbulletTbeta4, suggesting that this position in thymosin beta4 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-actinSS-(CH(2))(n)-SSAr (n = 3, 6, 9).

Functional Interactions between the Thymosin beta4 Analogs and Actin

All five thymosin beta4 analogs obtained by peptide synthesis were able to bind actin as shown from the cross-linking studies illustrated above (Fig. 5). In order to assay the influence of the substitutions in Tbeta4 on the polymerization-inhibiting capacity, polymerization of G-actin was monitored in the presence of each of the analogs. Generally, we found that all Tbeta4 analogs, which proved positive in one of the cross-linking reactions, also showed a reduced inhibitory capacity on actin polymerization (Fig. 8). Lowest inhibitory capacities were found for those two analogs that showed the highest yields in the cross-linking reactions with the actin-bound nucleotide (CysbulletTbeta4 and CysbulletTbeta4). In line with this, even CysbulletTbeta4, which reacted with the actin-bound nucleotide only to a small amount, had likewise lost part of its inhibitory capacity. The substitution in position 6 of Tbeta4 resulted in an only slight alteration in the polymerization-inhibiting capacity, whereas the substitution in position 40 of Tbeta4 had no influence at all. In accordance with this, the capacity of the latter to inhibit actin polymerization was virtually indistinguishable from that of native Tbeta4.


Figure 8: Polymerization kinetics of actin (10 µM) as followed by capillary viscometry in the absence or presence of Tbeta4 or the Tbeta4 analogs. The beta-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, circle; actin + Tbeta4, bullet; actin + CysbulletTbeta4, ; actin + CysbulletTbeta4, box; actin + CysbulletTbeta4, ; actin + Cys^6bulletTbeta4, down triangle; actin + CysbulletTbeta4, Delta.



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 Tbeta4. The influence of CysbulletTbeta4 on the nucleotide exchange was examined in comparison with native Tbeta4 and CysbulletTbeta4, the latter as an example of a Tbeta4 analog, which is ineffective in the thiol-specific cross-linking reactions as well as in the polymerization-inhibiting assay. The retardation effect of CysbulletTbeta4 was found to be indeed partly abolished. While the k value of CysbulletTbeta4 (k = 2.8 times 10 ± 0.2 times 10 s) was almost indistinguishable from that of native Tbeta4 (k = 2.9 times 10 ± 0.2 times 10 s), the nucleotide exchange rate of CysbulletTbeta4 was found to be accelerated to a value of k = 4.7 times 10 ± 0.2 times 10 s, a value that approaches the k value of pure actin (k = 6.2 times 10 ± 0.3 times 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 Tbeta4 (+, upper curve), in the presence of CysbulletTbeta4 (times, middle curve), or in the presence of CysbulletTbeta4 (circle, lower curve), showing the effects of the two thymosin beta4 analogs on the nucleotide exchange rate.




DISCUSSION

In order to identify contact sites between actin and Tbeta4 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 Tbeta4 in that the synthetic Tbeta4 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 ATPS. The modified nucleotide was characterized by UV and ^1H 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 ATPS 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 Tbeta4. For all Tbeta4 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 Tbeta4 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(D) value of the actinbulletTbeta4 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 Tbeta4 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 Tbeta4 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 Tbeta4. In particular, there is evidence that the thiols of Cys in actin and Cys^6 in Tbeta4 approach to within 9.2 Å. This finding confirms previous data that identified Cys as a part of a short distance cross-link with Tbeta4(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^6bulletTbeta4, although at a low rate. As a second major contact site the hexamotif of Tbeta4 (position 17-22) was identified as located near the actin-bound nucleotide, since the distance of CysbulletTbeta4 and the sulfur atom of ATPS 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 CysbulletTbeta4 and the modified ATPS, suggesting that the whole central part of Tbeta4 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 Tbeta4 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(2))(3)-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 alpha-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 CysbulletTbeta4. 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 Tbeta4 analogs may rely on a partial occlusion of the corresponding cysteine residue due to binding to actin.

The N terminus of Tbeta4 has been described as tending to form an alpha-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 Tbeta4 and the C terminus of actin on one hand, and the hexamotif of Tbeta4 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 Tbeta4 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 Tbeta4 analog approached the C terminus. This correlation indicates that the highly flexible C terminus of Tbeta4 is most probably directed away from the actin-bound nucleotide. Since no thiol-specific cross-link formation was found in any experiment involving CysbulletTbeta4, this part of Tbeta4 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 Tbeta4 analogs followed the third type of reaction kinetics. According to our classification, we believe that Tbeta4 is not in direct contact with that side of actin bearing Cys, the latter known to be part of a beta-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 Tbeta4 may be understood on the basis of the maximal possible reaction range of the cross-linking reagent (geq9.2Å) 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 Tbeta4(14, 31) .

Finally, we assayed whether the replacement of five hydrophobic amino acids in Tbeta4 had caused any functional deficits. For all Tbeta4 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 CysbulletTbeta4, 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 Tbeta4.


FOOTNOTES

*
This work was supported by a grant from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Max-Planck-Institut für medizinische Forschung, Jahnstraße 29, 69120 Heidelberg, Germany. Tel.: 49-6221-486-224; Fax: 49-6221-486-351.

(^1)
The abbreviations used are: Tbeta4, thymosin beta4; ArSS-(CH(2))-SSAr, alkylene-bis-[5-dithio-(2-nitrobenzoic acid)]; ArS, 2-nitro-5-thiobenzoate; NEM, N-ethylmaleimide; DTT, dithiothreitol; EDC/NHS, 1-ethyl-3(3-dimethlyaminopropyl)carbo-diimide/N-hydroxysuccinimide; MOPS, 3-[N-morpholino]propanesulfonic acid; HPLC, high performance liquid chromatography; ATPS, adenosine 5`-O-(thiotriphosphate); Fmoc, N-(9-fluorenyl)methoxycarbonyl.

(^2)
A. Reichert, D. Heintz, H. Echner, W. Voelter, and H. Faulstich, unpublished data.


ACKNOWLEDGEMENTS

We thank Wolfgang Kabsch for discussion of the data and for preparation of Fig. 10.


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