a very slow structural transition as seen by the expo- sure of 2.0 f 0.2 thiol groups per actin molecule. Once exposed, the second thiol group reacts with 5,5'-di-.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 8 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 266, No. 9, Issue of March 25, pp. 5508-5513, 1991 Printed in U.S.A.
A Reversible Conformational Transition in Muscle Actin Is Caused by Nucleotide Exchange and Uncovers Cysteine in Position 10” (Received for publication, October 3, 1990)
Gerard Drewes and Heinz FaulstichS From the Max Planck Znstitut fur Medizinische Forschung, W-6900Heidelberg, Federal Republic of Germany
ATP-G-actin in the absence of excess ATP and di- enough to be studied for its polymerization properties (6-8). valent metal ions was treated with ADP in amounts High concentrations of ADP (1 mM) seem to guarantee full large enough to ensurecomplete formationof ADP-G- complexation andincrease the stabilityof the actin monomer actin. Under these conditions the monomer undergoes a very slow structural transition as seen by the expo- (9). Rabbit muscle actin contains 5 cysteine residues, all existsure of 2.0 f 0.2 thiol groupsper actin molecule. Once ing in the reduced form (10-12). Under standard conditions, exposed, the second thiol group reacts with 5,5’-dithiobis-(2-nitrobenzoic acid) at a rate approximately only one of these thiol groups, that of cysteine 374 near the carboxyl terminus of the polypeptide chain, is accessible for of cysteine374.Labeling 10-fold higher than that experiments with 2,4-dinitrophenyl [ l-’4C]cysteinyl DTNB titration or reaction with NEM (13, 14). We reported disulfide followed by digestion and peptide analysis previously that denaturation, as occurring rapidly after loss showed(besides reaction with cysteine 374) nearly of the bound nucleotide, is associated with the exposure of exclusive labeling of cysteine 10. Since this residue is additional thiol groups (15, 16). The number of actin thiols completely shieldedin ATP-G-actin, exchangeof ATP exposed to solvent and their reaction rates are dependent on for ADP must have caused a partial unfolding of the the presence and natureof the bound nucleotide (16, 17). We protein uncovering the side chainof this cysteine. The two thiol groups are transition is reversible, because addition of ATP orof previously reported that in ADP-G-actin exposed as demonstrated by reaction with typical thiol reexcess divalent metal ions restored the conformation with only cysteine 374 exposed. Reversibility of the agents (15). Since there was evidence that this actin, ADPG(2S)-actin,represented a distinct species of monomeric transition allowed usto directly determine the relative a mixture of actin monomers suffering from affinities of ATP and ADP to monomeric actin in the actin rather than absence of Me2+ ions. By determination of the 50% progressive denaturation, a more detailed study of this actin exposure value of cysteine 10 from either side of the was started. We found that preparations of ADP-G-actin exist equilibrium we found a value of K A T P / K A D P = 30. The as a mixture of two species undergoing a slow and reversible rate of uncovering of the thiol of cysteine 10 at 0 “C conformational transition. Prolonged incubation on ice in the was distinctly slower (tllz= 9 h) than its reshielding absence of excess Me2+ions converts nearlyall ADP-G(-1s)by the addition of ATP (tllz = 3 h). The structural change was accompanied by a decrease in polymeri- actin to theform with two thiol groups exposed, ADP-G(2S)zation rate. Relative polymerization rates were deter- actin. The properties of the new actin species are described mined as ATP-G(1S)-actin:ADP-G(-1s)-actin:ADP- in the present study. G(2S)-actin = 1.0:0.35:0.1. From the data presented MATERIALSANDMETHODS here we conclude that preparations of ADP-G-actin remain undefined unless the number of thiol groups Reagents-All reagents were analytical grade and all solutionswere exposed has been determined. preparedwith doublydistilledwater. ATPand ADP were from Pharma-Waldhof (Dusseldorf, FRG), EGTA, NEM, N-(3-pyrenyl)maleimide, phalloidin, hexokinase (C-302), and DNase I were Under standard conditions the monomer of rabbit muscle from Sigma, DTNB and CNBr were from Serva (Heidelberg, FRG), actin (G-actin) contains one molecule of tightly bound ATP *‘CaCl, (specific activity 0.8 Ci/mmol) was from Amersham Corp., and one divalentmetal ion (in vivo Mg2+, i n vitro mostly Sephadex G-25 and G-50 Superfine, QAE-SephadexC-25, and Sephacryl S-200 HR were from Pharmacia LKB Biotechnology Inc. The Ca’+) bound to a single high affinity binding site(for reviews, ATP-bioluminescence CLS kitwas from Boehringer Mannheim.[cyssee Refs. 1and 2). On removalof the bound ATP denaturationteine-l-’4C]2,4-Dinitrophenylcysteinyldisulfide ([‘4C]DNPSSC) was of the protein proceeds rapidly (3, 4). A slow denaturation prepared as described recently (18). occurs when ADP is bound to the nucleotide-binding site (3, ADP was purified from traces of contaminating ATP by ion ex5 ) . ADP-G-actin’ remains native for a period of time long change Chromatography on QAE-Sephadex C-25 using a gradient of triethylammonium bicarbonate (pH 7.5,0.1-0.3 M). The final product * The costs of publication of this article were defrayed in part by contained approximately 0.1% ATP impurities as detectedusing the the payment of page charges. This article must therefore be hereby bioluminescence assay kit. Preparation of Proteins-Actin from rabbit skeletal muscle was marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 prepared from acetone powder essentially as described by Pardee and solely to indicate this fact. Spudich (19) with an additional Sephacryl S-200 gel filtration step $ T o whom correspondence should he addressed Max Planck Inand was pure as checked by sodium dodecyl sulfate-polyacrylamide stitut fur Medizinische Forschung, Jahnstrasse 29, W-6900 Heidelgel electrophoresis. G-actin was kept on ice in buffer 1 (2 mM Trisberg, FRG. Tel.: (0)6221-486224; Fax: 486351. ’ The abbreviations used are: ADP-G-actin, monomeric actin with HCI, pH 7.8, containing 0.2 mM ATP, 0.1 mM CaCl?, and 1 mM bound ADP; ATP-G-actin, monomeric actin with bound ATP; DNaseNaN:J.For some experimentsG-actin was labeled with “ T a by I, desoxyribonuclease I from calf thymus, EC 3.121.1; DNPSSC, 2,4- overnightincubationwith *‘CaCI2 on ice in buffer 1. Blocking of dinitrophenylcysteinyldisulfide; DTNB, 5,5’-dithiobis-(2-nitroben- cysteine 374 with NEM was achieved by incubation of G-actin with zoic acid); EGTA, [ethylenehis(oxyethylenenitrilo)]tetraacetic acid; a 10-fold excess of NEM in buffer 1 a t room temperature, quenching HPLC, high performance liquid chromatography; NEM, N-ethylmal- of the reaction after 30min with excessdithiothreitol, and separation of the protein on a small Sephadex G-25 column eluted with buffer eimide.
5508
Conformational Transition in Monomeric Actin
5509
1.F-actin was prepared by addition of EGTA/MgCl, (to final concentrations of 0.2 and 1mM, respectively) or EGTA/KCl (to 0.2 and 100 mM, respectively). Removal of the ATP was achieved by incubation with hexokinase (to a final concentration of 5 units/ml actin) and glucose (to 0.4 mM) for 1-2 h (7). Removal of ATP (i.e. less than 1 N M residual ATP) was ensured with the help of the ATP-bioluminescence assay according to Wendel and Dancker (20). From this actin pellets were prepared, allowed to soften on ice for a few hours in buffer 2 (2 mM Tris-HC1, p H 7.8, containing 1 mM ADP and 1 mM NaN:,), and then homogenized gently in buffer 2 in a Teflon/glass potter. The resulting solutions of monomeric ADP-actinwere clarified by ultracentrifugation at 300,000 X g for 1 h at 4 "C before use. 0.0 I Protein concentrations were determined on G-actin solutions spec0 2 4 6 E 10 = 26,460 M" cm" and a molecular mass trophotometrically using Time [min] of 42,300 daltons. Determination of Exposed Thiols-Thiol content of actin was deFIG. 1. Titration of actin thiols with DNPSSC. G-actin (2 X termined spectrophotometrically by reaction with DNPSSC (or, alM) in 2 mM Tris-HC1, pH 7.8, containing 0.1 mM CaC12, 1 mM ternatively, DTNB) using the extinction coefficient of the dinitrowith a10-fold excess of NaN3, and 1 mM nucleotidewasreacted = 12,700 M-' cm"). The reaction was started thiopenolateions DNPSSC and the reaction monitored spectrophotometricallya t 408 by adding a 10-fold excess of reagent (as a 10 mM solution in 1% nm and 4 "C: trace I, G-actin in the presence of ADP; trace 2, GNaHCO:,) to sample andreference cuvette andrecording the increase actin preblocked with NEM at cysteine 374 in the presence of ADP; in absorbance a t 408 or 412 nm. The spectrometer (Aminco DW-2) trace 3, G-actin in the presenceof ATP. was equipped with a thermostated cell compartment cooled to 4 "C. Labeling of Thiols and Determinution of the Labeled ResidueLabeling of actin thiols with [14C]DNPSSC and degradation of the nonpolymerizable material which was typically lessthan 10%. labeled protein with CNBr after blocking of the remaining thiols in Previously reported methods for the preparation of ADP-G8 M urea with NEM was performed as described recently (18).The actin, which used an about 10-fold molar excess of ADP and CNBr digest was applied to a Sephadex G-50 Superfine column(250 a shorter depolymerization time, resulted in up to 30% of the X 1.5 cm) eluted with 25% acetic acid essentially as described by total actin remainingunpolymerizable (8). Elzinga (21). Radioactive fractions were further purified by removing The ADP-G(2S)-actinspecies could also be prepared from insoluble peptides on Sephadex G-10 (22). Final purification of the monomeric ATP actin. Solutions of G-actin containing 0.2 labeled peptides was achieved by reversedphase HPLC ona HD-Gel RP-7s-300 column(Orpegen, Heidelberg,FRG) using an acetonitrile/ mM ATP and 0.1 mM CaC12 were treated twice with Dowex water gradient containing 0.05% trifluoroacetic acid (18). Labeled 1-X2 to remove excess ATP. When ADP, purified of traces peptides were identified by amino acid analysis. of ATP, was added to this solution atofinal concentrationof PolymerizationMeasurements-Polymerization was followed a t 1 mM and allowed to stand onice overnight, thesecond thiol 25 "C by the fluorescence enhancement of pyrene-labeled actin inan SLM 4000 spectrofluorimeter equipped with a thermostated cell group became uncovered. The ADP-G(2S)-actin prepared in compartment. The excitation andemission wavelengths were366 and this way was indistinguishable from that prepared from F405 nm, respectively. Labeling of actin with N-(3-pyrenyl)maleimide actin in the way described above. Both preparations of the was performed according to Cooper et al. (23). Actin samples were monomeric actin species were polymerizable on the addition mixed with 5 7 % pyrene-labeled actin before the preparation of the of KC1 with high yield, about 90% as determined photometrF-actin fromwhich ADP-actin was preparedas described above. ically on the supernatant of an aliqout after ultracentrifugaPolymerization was induced by addition of KC1 up toa final concentration of 100 mM. In some experiments an aliquotof the ADP-actin tion. When the filamentsof both preparations were collected step in ATP wasused for thepreparation of a10 WM solution ofKC1 seeds and submitted to aseconddepolymerization according to Pollard (24) and added up0.2% to of total actin in order containing buffer, the resulting G-actin was indistinguishable to eventually circumvent nucleation effects. Serial dilutions of actin from control actin. solutions for critical concentration measurements were performed in For determining the number of thiol groups exposed we Elkay polystyrene disposable cuvettes. Computer fitting of fluoresor our own reagent, DNPSSC, used Ellman's reagent (DTNB) cence data was performed using the "GraFit" program (Erithacus an activated unsymmetric alkylaryl disulfide withequal effect Software Ltd.). Other Procedures-Metal ion content of G-actin samples was de- (18). Both reagents indicated the presence of two solventtermined using isotope labeling for calcium and atomic absorption exposed thiol groups in ADP-G-actin prepared as described spectroscopy for determination of magnesium using a Perkin Elmer above. Remarkably, independent of the reagentused, the two HGA4000 AtomicAbsorption Spectrophotometeras describedby (Fig. 1). While thefirst Selden etal. (25). Inhibition of DNase I activity by monomeric actin thiolsreacted at differentrates substitution was finished within approximately 1 min, the was measured as described previously (26, 27).
v
other took about 10 min for complete reaction, corresponding to the reaction rate of cysteine 374 in ATP-G-actin. When the new actin monomer species was prepared from F-actin in Conditions for Uncovering of the Additional Thiol GroupDuring our studies on theinfluence of the nucleotide on the which cysteine 374 was previously blocked by reaction with exposure of the cysteine residues in muscle actin we detected NEM, only the fast reactingthiol group was found. It is the uncovering of a second thiol group when ATPwas replaced therefore concluded that thesecond thiol group uncovered in at by ADP (15). This was achieved by depolymerizing an F-actin ADP-G-actin reacts with Ellman-type thiol reagentsa rate pellet in a buffer containing a 50-100-fold excess of ADP approximately 10-fold higher than that of cysteine 374. Identification of the Uncovered Cysteine Residue-The thiol (typically 1 mM) over the protein. Before depolymerization, the pellet was freed from ATP eitherby a washing stepusing group uncovered in ADP-G-actin was identified by reaction nucleotide-free buffer (2 mM Tris-HC1, pH 7.8, 100 mM KC1) with ['4C]DNPSSC, a recently developed thiol reagent that or, moreconveniently and with minimal denaturation, by allows radioactive labeling of thiol groups to be followed by incubation of the polymerized actin with glucose and hexo- photometry (18).The gel filtration elution profile (Sephadex kinase. Depolymerization was allowed to proceed for 1-2 days G-50) of the cyanogen bromide digest of the labeled ADPon ice. ADP-G-actin prepared this way showed 1.8-2.2 S H G(2S)-actin shows a typical and highly reproducible pattern groups when titrated with DTNB or DNPSSC (Fig. 1).Na- (10, 22) containing two major radioactive peaks (Fig. 2). The tiveness of ADP-G(aS)-actin was checked by the amount of peak eluting in the second position disappeared when actin RESULTS
5510
Conformational Transitionin Monomeric Actin TABLEI Amino acid composition (residues per mole)of the actin peptide labeled during reaction with p4C]DNPSSC
0
.o, o
. 40
,
60
50
' b,O."~O.,,d "
9 , '
-
9
.0I
.
70 80 90 100 Fraction Number
\,A
'
110
120
FIG.2. Separation of cyanogen bromide peptides obtained from G-actin exposing 2 thiols labeled with ['4C]DNPSSC. A Sephadex G-50 Superfine column (250 X 1.5 cm) eluted with 20% acetic acid was used. Fractions indicated by the bar were pooled and lyophilized. Inset, the lyophilized material was solved in 70% formic acid and applied to a Sephadex G-10 column (100 X 1.5 cm) eluted with 50 mM pyridine/acetic acid, pH 6.8.
-I 1 v
Amino acid
Amino-terminal CB-13 peptide (10)
Labeled peptide
ASP Thr Ser HSer Glu Pro G~Y Ala
6 2 2 1 3 3 6
5.9 2.0 1.8 0.7 3.9 3.1
Val
5 1 2 0
6.7
5
Ile Leu TYr Phe LYS His Arg
4.9 4.8 1.0 2.1 0.1 2.1
2
1 1 3
1.0 1.3 2.7
I
I
0.2
10
2.0 -
0
2
l.E -
0
.*>
0.0' 0
'
'
5
1
L
'
10
15
20
25
Z
1.6
?
1.4 -
5
1.2 -
2
1.0'
~
10
30
Time [rnin]
FIG. 3. Reversed phase HPLC of the radioactive fraction pooled from the SephadexG-10 column (Fig. 2 , inset). Radioactivity content of the fractions is indicated by the bars. An acetonitrile/water gradient containing 0.05% trifluoroacetetic acid was applied to the column as indicated by the dashed line. The radioactive fractionelutedbetween 24 and 25 min at 34%acetonitrile was submitted toamino acid analysis (Table I).
0.E
1
10
100
1000
[ADPI/[ATPl
FIG. 4. Thiol exposure as dependent on the concentration ratio ADP/ATP. Opensymbols, different amounts of ATPwere M ) , prepared from EGTA/MgCI,added toG-ADP-actin (2 X polymerized actin freed from ATP and depolymerized in the presence of 1mM ADP. Closed symbols, increasing amountsof ADP were added to ATP-G-actin containing 2 equivalents of ATP. Thiolswere titrated after 40-h incubation on ice. To ensure that the end point of the transition had been reached, the titrations were repeated 8 h later. Other conditions asdescribed in the legend of Fig. 1.
previously blocked with NEM was used. Consequently, this peakcontainedthepeptide with the labeled cysteine 374 residue, which could be used as a standard for the incorpora78-85 tion of 1 equivalent of label into the protein. Fractions containing 50-60% of the total radioactivity were combined equivalent of MeZ+ present) the binding of ADP to actin is and subjected to further gel filtration on a Sephadex G-10 about 30-fold weaker than that of ATP. Influence of Divalent Metal Ions-Uncovering of the cyscolumn (Fig. 2, inset), where the labeled peptide eluted with the void volume. The pooled fraction was submitted to further teine 10 residue did not strongly depend on the presence of the metal ion bound to the high affinity binding site of Gpurification by reversed phase HPLC resulting in a single radioactive peak (Fig. 3). Table I gives the amino acid com- actin. The transitionwas observed when either Mg2+or CaZ+ positionfor this peptide, which indicates that the labeled ions were bound to the high affinity site, but also in prepapeptide is the amino-terminal cyanogen bromide peptide CB rations containing virtually no MeZ+ ions bound to this site 13 (residues 1-44) containing only the cysteine residue in (Table 11). For ADP-G-actin containing Ca2+, however, we values, position 10. About 10% of the radioactivity incorporated into found considerable scattering in the thiol titration ion, when bound to thehigh affinity the proteinwas detected in fraction 85-90 (Fig. 2) represented suggesting that the Ca2+ by the peptide containing the cysteine residue in position 257. site, may have some influence on the transition, possibly on Reversibility of the Effect-Exposure of cysteine 10 in ADP- the rate of the conformationalchange. Standardization of metal ion content of the ADP-G-actin G-actin can be reversed by the addition of ATP. The number under investigation was achieved by assaying the metal ion of exposed thiols in relation to the ADP/ATP ratio in the medium is illustrated in Fig. 4. One set of data was obtained content of the polymerfrom which it was prepared.For from a [45Ca]-doted, EGTA/ by incubating G-actin in a mediumcontaining about50 equiv- example, ADP-G-actin prepared alents of ADP with various amounts of ATP as indicated, the KC1-polymerized F-actin was found to contain less than 0.1 other by the addition of excess ADP to G-actinexposing one equivalent of ["Ca] per mol actin andwas regarded as metalwere depolymerizedinbuffer thiol group in a medium containing 2 equivalents of ATP. free. Whenthesefilaments Fromboth sides of the equilibrium the half-value of the containing 1 mM ADP the resulting G-actindeveloped the 2transition, corresponding to the exposure of 1.5 thiols, was thiol state asreadily as ADP-G-actin prepared from EGTA/ reached at the same ratioof ADP/ATP (Fig. 4). The conclu- MgC12 polymerized actin, in which about 1 equivalent Mg2+ sion can be drawn that under the given conditions (about 1 per mole actin was bound as determinedby atomic absorption
5511
Conformational Transition i n Monomeric Actin TABLEI1 Thiol groups titratableby DNPSSC or DTNB in G-actin prepared from pellet actin as described under "Materials and Methods" as dependent on the kind of nucleotide in the depolymerization buffer(2 mM Tris-HC1,pH 7.8, containing either1 mM A T P or ADP) and the conditions under which theF-actin had been prepared The reaction was followed spectrophotometricallyat 4 "C until a constant value was reached. The results are average values (fstandard deviation) for the number of determinations given in parentheses. The amount of unpolymerizable actinwas determined from the supernatants of the solutions after polymerization with KC1 and phalloidin and ultracentrifugation. The amount of 45Cainlabeledsampleswasmeasured as describedunder "Materials and Methods." Polymerization buffer 2 mM Tris-HCI, pH 7.8, containing
Equivalents of "Ca bound
0.2 mM EGTA/100 mM KC1
