Eur. J. Biochem. 209,829-835 (1992) Q FEBS 1992
Effects of nucleotide binding on thermal transitions and domain structure of myosin subfragment 1 Dmitrii I. LEVITSKY', Valery L. SHNYROV', Nikolai V KHVOROV'. Anna E. BUKATINA', Natalia S. VEDENKINA', Eugene A . PERMYAKOV2, Olga P. NIKOLAEVA' and Boris F POGLAZOV '
'
*
A. N. Bach Institute of Biochemistry of Russian Academy of Scicnces, Moscow, Russia lnstilute of Theoretical and Experimental Biophysics of Russian Academy of Sciences, Pushchino, Russia
(Received March 1 2/June 16, 1992) - EJB 92 0342
The thermal unfolding and domain structure of myosin subfragment 1 (SI) from rabbit skeletal muscles and their changes induced by nucleotide binding were studied by differential scanning calorimetry. The binding of ADP to S1 practically does not influence the position of the thermal transition (maximum at 47.2 "C), while the binding of the nun-hydrolysable analogue of ATP, adenosine 5'-[/Y~-imido]triphosphate (AdoPP[NH]P) to S1, or trapping of ADP in S1 by orthovanadate (V,), shift the maximum of the heat adsorption curve for S1 up to 53.2 and 56.loC, respectively. Such an increase of S1 thermostability in the complexes S1-AdoPP[NH]P and S1-ADPVi is confirmed by results of turbidity and tryptophan fluorescence measurements. The total heat adsorption curves for S1 and its complexes with nucleotides were decomposed into elementary peaks corresponding to the melting of structural domains in the S1 molecule. Quantitative analysis of the data shows that the domain structure of S1 in the complexes Sl-AdoPP[NH]P and S1-ADP-V, is similar and differs radically from that of nucleotide-free S1 and S1 in the S1-ADP complex. These data are the first direct evidence that the S1 molecule can be in two main conformations which may correspond to different states during the ATP hydrolysis : one of them corresponds to nucleotide-free S1 and to the complex S1-ADP, and the other corresponds to the intermediate complexes S1-ATP and S1-ADP-P,. Surprisingly it turned out that the domain structure of S1 with ADP trapped by p-phenylene-N, N'-dimaleimide @PDM) thiol cross-linking almost does not differ from that of the nucleotide-free S1. This means that pPDM-cross-linked S1 in contrast to S1-AdoPP[NH]P and S1ADP-V, can not be considered a structural analogue of the intermediate complexes S1-ATP and S1ADP-P,.
Muscle contraction and many other manifestations of biological motility are based on the cyclic interaction of myosin heads with actin, which is accompanied by ATP hydrolysis. During steady-state ATP hydrolysis the myosin head is subjected to conformational changes that can be detected by changcs in ultraviolet absorption [l]and intrinsic fluorescence 121. Such changes result from the fact that, in the ATPase reaction, the myosin head (M) passes through several intermediates: M . ATP, M . ADP . Pi, arid M . ADP differing from each other by intrinsic fluorescence parameters [3, 41. Isolated myosin heads, or myosin subfragment 1 (Sl); obtained by myosin proteolysis, and also stable analogues of S1 complexes with nucleotides, are often used for studies of thc structure and properties of the myosin ATPase interniediates. A complex of S1 with the non-hydrolysable analogue of ATP. adenosine S'-[fl,Y-irnido)triphosphate (AdoPP[NH]P), Covrespondencr to D. I. Levitsky, A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prospect 33, Moscow
117071, Russia Abbreviations. M, myosin; S1, myosin subfragment 1; AdoPP(NHIP, adenosine 5'-[fi,y-imido]triphosphate,Vi, orthovanadate; pPDM, IJ-plienylene-NN'-dimaleimide; DSC, diffcrential scanning calorimctry. Enzyme. Chymotrypsin (EC 3.4.21 .I).
is used as a stable analogue of the S1-ATP complex. Complexes of S1 with ADP and orthovanadate (Vi) [S, 61 and S1 cross-linked by p-phenylene-N,N-dimaleimide (pPDM) in the presence of ADP [7] are often used as stable analogues of the S1-ADP-Pi complex. The use of stable analogues allows the structure and properties of S1 in the intermediates of the ATPase reaction to be studied. There are numerous data suggesting that the stable analogues of the S1-ATP and S1-ADP-Pi intermediates are similar in structure, but differ from nucleotide-free S1 and S1-ADP [8-201, It should be noted that all these data are in fact indirect since they are based on either studies of the actinbinding properties of S1 [8 - 151 or studies of local conformational changes in S1 [16-201. In order to obtain direct proof of the assumption of two main structural states of S1, one must know the effects of nucleotide binding on the structure of the whole S1 molecule. In recent years the problem of elucidating the domain structure of the S1 molecule has attracted much attention. It is a very important problem since interdomain interactions inthe myosin head may play an essential role in force generation in muscles. At present there are data suggesting the existence of separate structural domains in S1 [21-241. How-
830 ever, the most distinct and general feature of a domain in a globular protein is that the structure of the domain folds and unfolds coopcrativcly in an all-or-none way with significant changes in enthalpy and entropy [25]. In accordance with this definition, domains can be revealed by studying the protein unfolding. The most effective method for studying thermal unfolding of proteins is differential scanning microcalorimetry. Earlier, one of us [26] introduced the ‘successive annealing’ method, which revealcd separate structural domains in proteins from appropriate calorimetric measurements. Recently we applied this method to S1 structure and revealed three domains in the S1 molecule [27 - 301. In the present work differcnt complexes of S1 with nucleotides were studied by microcalorimetry. The data obtained show that the domain structure of S1 in the S1AdoPP[NH]P and S1-ADP-Vi complexes is similar, and different from that or nucleotide-free S1 and S1 in the S1-ADP complex. So, these data are the first direct evidence that the S1 molecule can exist in two main conformational states, one of which occurs in the absence of nucleotides or in the complex of S1 with ADP, while the other occurs in the complexes S1ATP and S1-ADP-Pi. Unexpectedly it turned out that the domain structure of the pPDM-cross-linked S1 is practically the same as that of the nucleotide-free S1. This means that the p-PDM-cross-linked S1 can not be considered as a structural analogue of the intermediate complexes S1-ATP and S1-ADPPi. A preliminary report of this work has been published [31].
MATERIALS AND METHODS Reagents a-chymotrypsin was from Serva. Phenylmethylsulfonyl fluoride was from Merck. ATP, ADP, AdoPP[NH]P, pPDM and sodium vanadate (Na,VO,) were from Sigma Chemical Co. All other reagents were of analytical grade. 10 mM vanadate stock solution was prepared as described by Goodno [6].
S1 preparations Myosin subfragment 1 (Sl) was obtained by digestion of myosin filainents from rabbit skeletal muscles by chymotrypsin [32]. S1 concentration was determined by absorbance, using an A of 7.5. The purity of S1 was checked by SDSjPAGE [33]. The preparation of S1 contained the Y5kDa heavy chain and the light chains 1 and 3 but light chain 2 was absent. pPDM-cross-linked S1 was obtained by modification of S1 with pPDM at 0°C in the presence of ADP, following Wells and Yount [7], and was purified as described by Chalovich et al. [Y]. In order to obtain the S1-ADP-V, complex, 17-50 pM S1 was incubated with 2 mM MgC12, 10 - 250 pM Vi and 10 - 250 pM ADP [6] for 30 min at 25 “C in a medium containing 10 mM Hepes pH 7.3. The excess ADP and Vi were removed by intensive dialysis of S1 against 10 mM Hepes, pH 7.3, contaiiiing 1 mM MgC12. The extent of inodification of S1 by pPDM or V, was controlled by measuring the K+-EDTA-ATPase activity of S1 by relcase of Pi into the medium containing 0.04mg/ml S1, 0.5 M KCl, 5 mM EDTA, 1 mM ATP and 50 mM Tris/HCl, pH 7.5. ATPase activity ofpPDM-cross-linked S1 and S1 modified by vanadate in the presence of ADY did not exceed 3% of the unmodified S1 preparation activity. Decomposition of the S1ADP-V, complex, i.e. dissociation of ADP and V, from S1,
was achieved by irradiation (> 320 nm) as described by Grammer et al. [34]. Differential scanning calorimetry (DSC) Calorimetric measurements were carried out on a differential adiabatic scanning microcalorimeter DASM-4 (Russia) whose construction and principlcs have been described elsewhere [35].In order to measure the base line, both identical measuring cells were filled with solvent. For the measurement of the excess partial heat sorption of a protein, one of the cells was filled with the protein solution. High reproducibility of the base line allowed measurement of the partial heat sorption of a 1 - 3 mg/ml protein solution with an error not exceeding 3%. Capillary construction of thc 0.47-ml platinum measuring cell prevents to a great extent the artifacts caused by thermally induced protein aggregation. The heating rate was 1 Klmin. At this rate the protein aggregation may only reduce the enthalpy of denaturation without causing any changes in other parameters such as shape of the heat sorption curve and the denaturation tcmperature [36, 371. The exothermic peaks sometimes observed in the DSC scan [38] are caused by the artifacts of measurement. Such artifacts caused by protcin precipitation do not take place in the capillary helical measuring cell. We have convinced ourselves of this by parallel experiments using DASM-1M and DASM-4 microcalorimeters with different constructions of the measuring cells [35]. Dccomposition of the complex heat sorption curves into elementary components was carried out by the method of ‘successive annealing’ as described earlier [26, 271. This method is applied to fully or partially irreversiblc thermal transitions. The essence of the method is as follows. After measurement of the total heat sorption curve for protein solution and determination of the mean values of the thermal transition temperatures (locally visible or experimentally determined maxima on the heat sorption curve), another portion of the protein solution is heated in the calorimeter up to the temperature of the first maximum. Then the protein solution is cooled to the initial temperature (the temperature at the beginning of the scanning), and is heated again to a temperature exceeding the second maximum by 0.5-1.0 K, and is then recooled to the initial tempcrature. The heating/cooling cycles are repeated until complete denaturation of the protein has been achieved. The result of these experimcnts is a set of curves; subtracting each curve from the previous one, we obtain the initial portion of the heat sorption peak for each transition. The portion of the peak after the maximum was constructed for each transition by assuming symmetry relative to the temperature of the maximum. The denaturation change in heat capacity, AC;, was obtained using a linear extrapolation of the heat capacity curves for the native and denatured protein states, from left and right, to the transition tempcrature Td (see Fig. 1, curve A). The denaturation enthalphy, AHTd,and Gibbs free energy of stabilization, AG1,were calculated as described by Privalov et al. [3Y, 401. Measurements of thermally induced protein aggregation Thermally induced aggregation of S1 and its complexes with nucleotides was detected by changes in apparent absorbance at 350nm measured on a Cary-219 spectrophotometer (Varian) equipped with a thermostatted cellholder. The temperature in the cell was measured by a thermo-
831 B ;\
..: ... .:. ...
_ . :. ..
. . .-.. . .... .. ..
50 TEMPERATURE
40
60 (OC)
Fig. 1. Temperature dependences of excess heat capacity (AC,) for (A) nucleotide-free S1 and for (B, C) the complexes of S 1 with ADP and Vi. Sl concentration was 17.5 pM; (Bj 0.25 mM ADP and 0.25 mM Vi; ( C ) 10 pM ADP and 10 pM Vi. 10 mM Hepes, plI 7.3; 1 m M MgCI2. Tdis denaturation temperature, 4 C : is the denaturational change of heat capacity for nucleotidc-free S1. Vertical bar corresponds to 500 J . K - ' kg-'.
couple. Heating rate was 1 K/min. The measurements were carried out on a 1.5-2.0-mg/ml protein solution containing 10 mM Hepes pH 7.3 and 1 mM MgCI2. Intrinsic fluorescence measurements
Tryptophan fluorescence of S1 with or without nucleotide w-as measured on a laboratory-built spectrofluorimeter with monochromatic excitation at 280.4 nm [41]. Fluorescence was collectcd from the front surface of the thermostatted cell. The mean heating rate was about 30 K/h; the S1 concentration was 0.5 mg/ml. The measurements were carried out in solutions containing 10 mM Hepes pH 7.3 and 1 mM MgC12. RESULTS The first part of the work was devoted to studying the thermal denaturation of S1 and its complexes with nucleotides. Fig. 1 shows data on the thermally induced unfolding of S1 and of the S1-ADP-Vi complex. The heat sorption curve for S1 has a single peak with the maximum at 47.2"C. The formation of thc complex essentially changes the character of the thermal denaturation of S1: the maximum of the thermogram shifts t o 56.1 "C and the transition becomes more distinct. Due to the high affinity of Sl to ADP and Vi, such a change occurs even at equimolar concentrations of SZ , 4 D P , and Vi. Fig. 1 also shows results of an experiment in which the concentrations of ADP and Vi added are insufficient for complete saturation of the S1 binding sites. In this case, when only 50 - 60% of the S1 molecules are able to complex with ADP and Vi, two heat sorption peaks are found, one of which is characteristic of nucleotide-free S1 and the other of the S1-ADP-Vi complex. Note the coinplete absence of any intermediate transitions. This suggests that all the S1 molecules are in one of two possible conformational states. Irradiation above 320 nm of the stable S1-ADP-Vi complex is known to result in covalent modification of the S1 and in a rapid release of trapped MgADP and Vi [34]. We have
40
50
60
T E M P E R A T U R E ('C) Fig. 2. Temperature dependences of excess heat capacity for the S1ADP- Vi complex. The figure demonstrates reversibility of changes cduscd by ADP and Vi binding to S1. The complex S1-ADP-Vi (S1 concentration 48 pM) was dialyzed for 4 h against 10 mM Hepes pH 7.3, 1 mM MgCl,, changing the buffer every hour. Then the preparation was irradiated with ultraviolet light, four times diluted by the same buffer (final S1 concentration about 12 pMj and used for calorimetric mcasurcments. (A) S1-ADP-V, complcx bcfore irradiation; (B, Cj the same preparation irradiated for 1 min and 3 min, respectively; (D) after 3-min irradiation before the dilution 0.25 mM ADP and 0.5 mM Vi were added to the preparation. Vertical bar corresponds to 500 J K . kg-
'.
used this phenomenon to study the reversibility of the effects of the MgADP and Vi binding on the S1 thermal transition. For this purpose the S1-ADP-Vi complex was dialysed to remove an excess of ADP and Vi and then irradiated by ultraviolet light, followed at once by calorimetric measurements. It is clearly seen in Fig. 2 that the irradiation causes appearance of the heat sorption peak characteristic of the nucleotide-free S1. An increase of the time of irradiation results in a decrease of the peak for the S1-ADP-Vi complex and an increase of the S1 peak, which is in good agreement with data on the kinetics of release of the trapped ADP and V i from S1 [34]. The ultraviolet irradiation of the S1-ADP-Vi complex causes an oxidation of Serl80 in the S1 heavy chain [42, 431, but such a modification by itself does not cause any significant changes of the S1 thermal transition (Fig. 2B, C). The photo-modified S1 is able to retrap MgADP and Vi [34]. The addition of MgADP and Vi after the 3-min irradiation results in an increase of the S1-ADP-Vi heat sorption peak and in a decrease of the S1 peak. So: the transition of the S1 molecule to a completely different conformational state, which is characteristic for the S 1-ADP-V, complex, is fully reversible and depends only on the presence of the trapped MgADP and Vi in the active site of S1. Thus, the trapping of ADP and Vi in the active site of S1 causes an essential change of S1 conformation which is reflected in the pronounced shift of the thermal transition and in an increase of its cooperativity (the peak becomes much narrower). Fig. 3 shows calorimetric data for other complexes of S1 with nucleotides. The binding of MgADP to S l by itself has practically no influence on the temperature of the thermal
832
B
40 50 TEMPERATURE
60
TEMPERATURE
(
C
(OC
Fig. 3. Tcmpcrature dependences of excess heat capacity for S1 in the presence of 0.5 mM MgADP (A), 0.1 mM Mg . AdoPPlNHlP (B) and pPDM-cross-linkcd S1 containing trapped MgADP (C). Conditions was as in Fig. 1. Vertical bar corresponds to 500 J . K-' . kg-'.
Fig. 4. Temperature dependence of turbidity of solutions of S1 and S1 in the complexes with nucleotides. The turbidity measurements wcre carried out at heating rate 1 K/min dctccting apparent absorption at 350 nm; 10 mM Hepes pH 7.3, 1 mM MgC12. Curve a. 17.5 pM nucleotide-free S1; curve b, 14.5 pM S1, 0.27 mM AdoPP[NH]P; curve c, 14.5 pM S1,0.5 mM ADP, 0.25 mM V,.
Table 1. Thermodynamic parameters obtained from the DSC data for S1 and S1-nucleotide complexes.
Preparation
Td
s1 S1-ADP pPDM-S1 Sl-AdoPP(NH)P S 1-ADP-V,
"C 47.2 47.8 45.4 53.2 56.1
k 0.1 k 0.1 &- 0.1 k 0.1 i 0.1
AC;
AH T ~
kJ . K - ' . mol-' 22.6 k 0.2
kJimol 1130 f 30 1200 k 30 1120 k 30 1460 f 40 1330 f 40
0.2 12.3 0.3 10.3 f 0.2 12.3 & 0.2 16.0
334
t
0 0
20
transition but increases its sharpness, while the binding of Mg . AdoPP[NH]P to S1 essentially increases the temperature of the thermal transition, i. e. causes changes similar to those induced by the effect of the trapping of MgADP by V,. On the other hand, even the tight trapping of MgADP in S1 by means of the pPDM-cross-linking does not cause any significant change of S1 thermostability (Fig. 3). Thermodynamic parameters obtained from the calorimetric measurements on various complcxes of S1 with nucleotides are summarized in Table 1. An analysis of the main parameters of the melting (denaturation temperature, Td, and denaturation enthalphy, AHT,) shows that all the preparations studied can be separated into two groups. One of the groups comprises nucleotide-free S1, S1-ADP, and pPDM-crosslinkcd S1, while the other contains Sl-AdoPP[NH]P and S1ADP-V,. Some differences in the thermodynamic parameters of the preparations exist within each group, but they are small compared with those of the preparations in different groups. So, data from DSC studies show that the S1-AdoPPWHlP and S1-ADP-Vicomplexes differ in their main thermodynamic parameters (Td, AHTd) from all the other s 1 preparations studied. We decided to confirm the differences by other methods. The experiments were carried out in the same medium and at the same heating rate as in the calorimetric experiments. Fig. 4 shows temperature dependenccs of thc thermally induced aggregation of S1, SI-AdoPP[NH]P, and S1-ADPV,. For these preparations the temperatures of the half-maxima1 effect are 40.5"C, 47.0' C, and 49.0"C, respectively. These results show that the binding of Mg . AdoPP[NH]P or
40 T E M P ERR TU RE ( OC
60
Fig. 5. Temperature dependences of tryptophan fluorescence spectrum position (A) for S1 ( O ) ,S1-ADP (A),pPDM-cross-linked Sl (fl), S1AdoPPlNHlP (0)and S1-ADP-Vi ( A ) . Conditions were as in Figs 1 and 3.
trapping of MgADP by Vi essentially increase the stability of S1 against the thermally induced aggregation of S1 and the effect of ADP and Vi is more pronounced compared to that of AdoPP[NH]P which correlates well with the DSC data. At the same time, the binding of MgADP or the trapping of MgADP by the pPDM cross-linking do not increase and sometimes even slightly decrcase (by 1-2°C) the S1 stability against the thermally induced aggregation (data are not shown). Fig. 5 shows temperature dependences of the tryptophan fluorescence spectrum position (wavelcngth at the peak) for S1 and its complexes with nucleotides. This parameter (2) is the least sensitive to possible artifacts caused by the protein aggregation. Such artifacts are caused mainly by protein precipitation induced by heating. It should be noted that usually the thermal denaturation of a protein is accompanied by an unfolding of the protein structure and a transfer of some fluorescent tryptophan residues to the water environment which is reflected by a red spectral shift. In the case of the S1 preparations, the thermal denaturation causes the opposite effect [28] which seems to be duc to the fact that the thermal transitions in S1 inducc an increase of intermolecular interactions, i. e. of protein aggregation. In the course of this process
833 40 20 0
-
-20
A
E"
*CP
\
2 40 d.
v
Q 2o 0
- 20 -4 0
40 50 60 TEMPERATURE ( "C)
Fig. 6. Decomposition by the 'successive annealing' method of the total heat sorption curves (-) for nucleotide-free SI (A), pPDM-crosslinked S1 (B), S1-AdoPPINHIP (C) and S1-ADP-Vi (D) into elementary peaks (- - - -) corresponding to the melting of separate domains. Conditions were as In Figs 1 and 3 Vertical bar corresponds to 500 J.K kg-'.
some of tryptophan residues, which initially were at the protein surface, are transferred into a purely protein environment in the regions of intermolecular contacts. It is clearly seen from Fig. 5 that an increase of temperature induces a blue shift of the tryptophan fluorescence spectrum of S1, S1-ADP and pPDM-cross-linked S1. The shift occurs within the temperature region 40- 50'C. For the S1-AdoPP[NH]P a weak blue shill takes place at higher temperatures but is completcly absent in the temperature region studied in the case of S1ADP-Vi. These data corroborate the fact that the complexes S1-AdoPP[NH]P and S1-ADP-V, essentially differ in thermostability from S1-ADP, pPDM-cross-linked S1 containing trapped ADP, and nucleotide-free S1. So, all the data presented above show that, according to the thermal sensitivity and to the thermal transition paramcters, all the preparations studied are separated into two groups, one of which contains S1-AdoPP[NH]P and S1-ADP-Vi and the other Sl -ADP, pPDM-crosslinked S1 and nucleotide-free S1. It would be reasonable to assume that the preparations of different groups essentially differ in conformational state of S1 molecule. In order to chcck this assumption we have applied the 'successive annealing' method to reveal the domain structure of S1 in some of the complexes with nucleotides and to compare it with the domain structure of the nucleotide-free s1. Fig. 6 A shows results of a decomposition of thc total heat sorption curve of S1 into elementary peaks. Each peak corresponds to the melting or a separate cooperative region (domain) in the S1 molecule. It is clearly seen that S1 contains three such domains which melt with maxima at 39 "C, 47.2 "C and 50.8 "C. pPDM-cross-linked S1 containing tightly trapped MgADP has a similar three-domain structure though with other contributions and temperatures of the peaks (41.2,45.5 and 48.8") (Fig. 6B). S1-ADP also has a similar three-domain structure (the data are not shown). On the other hand, the S1 molecule has an absolutely different domain structure in the Sl-AdoPP[NH]P and S1-ADP-Vi complexes (Fig. 6C,
- 60
0
20
40
60
0
20
4060
TEMPERATURE ('C) Fig. 7. Temperature dependences of Gibbs energy of stabilization for the S1 domains (see Fig. 6). Lettering of the domains is in the order of increased maximum temperature. (A-C) Nucleotide-free S1 ( 0j and pPDM-cross-linked3 (0); (A) the first domain(39Tand41.2'C for S1 and pPDM-cross-linked S1, respectively); (B) the sccond domain (47.2'C and 45.SnC, respcctivcly); (C) third doinain ( 5 0 3 ° C and 48.8 "C, respectively). (D - F) S1 -AdoPP[NH]P ( A j and S1-ADP-Vi (0); (D) the first domain (42.0"C and 52.4"C for Sl-AdoPP[NH]P and S1-ADP-Vi, respectively); (Ej the second domain (47.8"C for SI-AdoPP[NH]P, the peak is absent for S1-ADP-Vi); (Fj the third domain (53.2"C and 562°C for Sl-AdoPP[NFT]P and S1-ADP-Vi, respectivelyj.
D). In this case the total heat sorption curve is decomposed into the components with maxima at 42.0"C, 47.8"C, and 53.2OC for S1-AdoPP[NH]P (Fig. 6C) and at 52.4"C and 56.2"C for S1-ADP-V, (Fig. 6D). Moreover, for the S1-ADPVi preparation an additional very small heat sorption peak was found at 40°C (see, for example, Fig. 6D). Probably a similar peak is contained in the thermograms of other preparations but it is not revealed because in these cases it is located within the limits of the total heat sorption contour and practically coincides in position with the peak of the most thermolabile domain (Fig. 6). It seems most likely that this peak corresponds to the melting of the alkali light chains in the S1 preparation [30]. The most convenient measure for the quantitative description of the stability of a cooperative domain, or of a singledomain protein, is the difference in the Gibbs free energies of the denatured and native states of a protein. Fig. 7 shows the temperature dcpendences of the free energies of stabilization for the experimentally revealed domains. Taking into account the fact that the structural stability of a multi-domain protein is conditioned by the stability of its domains, we can assert that the stability of S1 and pPDM-cross-linked S1 at 25°C is the same. A small decrease of free energy for the second domain inpPDM-cross-linked S1 (Fig. 7 B) is completely compensated by an increase o r free energy of the first domain
834 Shriver and Kamath [38] could not show separate structural domains in the myosin head though they postulated their existence. Wc have succeeded in obtaining direct evidence for the existence of domain structure in S1 which essentially changes upon the binding of AdoPP[NH]P or ADP and Vi to S1 (Fig. 6). Thus, we can definitively subdivide all the S1 samples studied into two classes with different domain structures. Since SI-AdoPf"NH]P and S1-ADP-Vi are stable analogues of the intermediates of the ATPase reaction, S1-ATP and S1-ADPPi, we can conclude that the domain structure of S1 in these intermediates is different from that of nucleotide-free S1 or S1 in the complex with ADP. Probably, the formation of these intermediates is accompanied by the domain -domain interactions in the S1 molecule resulting in the change of S1 DISCUSSION domain structure. Thus, the data obtained are direct evidence The data obtained lead to the conclusion that the binding for the existence of S1 in two main conformational states, one of Mg . AdoPP[NH]P to S1 or the trapping of MgADP in S1 of which is realized in the absence of nucleotides or in the S1by Vi result in a pronounced conformational change of the ADP complex and the other one corresponds to the S1-ATP whole S1 molecule, while the binding of MgADP and the and S1-ADP-Picomplexes. At the same time, S1-ADP slightly trapping of MgADP by pPDM-cross-linking cause only local differs from S1, and S1-ATP - from S1-ADP-Pi, but such conformational changes in S1. The global conformational relatively small differences are negligible compared to the changes are reflected in a pronounced increase of S1 global differences between the main confornutional states. thermostability and in a significant change of S1 domain It is surprising that the domain structure ofpPDM-crossstructure. The decrease of the number of the structural do- linked S1 differs from the structure of nucleotide-free S1 much mains in S1-ADP-Vi in comparison with other S1 preparations less than from the domain structure of SZ in the S1(see Fig. 6) seems to be due to interdomain interactions caus- AdoPP[NH]P and S1-ADP-Vi complexes (Fig. 6). The point ing a tightening of the S l structure which makes it more here is that such S1 cross-linked by pPDM in the presence of compact. This assumption is corroborated by the data of ADP displays, like S1-ATP and S1-ADP-Vi, 'weak' binding Aquirre et al. [44] demonstrating that the formation of the to actin [9, 46,471 and is supposed to be a structural analogue S1-ADP-Vi complex causes spatial translocations in the S1 of S1-ATP or S1-ADP-Pi [19,20]. We can propose the followmolecule that dccreasc distances between some parts of the ing explanation of this seeming contradiction. One of the heavy chain and the nucleotide binding site in the active site actin-binding sites in the S1 heavy chain is located in the of S1. Moreover, it is confirmed by the electron microscopic region of the SH1 and SH2 groups (Cys707 and Cys697, redata of Katayama [13] who showed that the addition of spectively) [48] and possibly even between these SH-groups MgADP and Vi significantly changes the shape of the actin- since synthetic peptides with similar amino acid sequences bound S1 molecules making them shorter and rounder in interact with F-actin [49, 501. Morita et al. [46, 471 suggested comparison with the more elongated S1 molecules bound to that this site is responsible for 'strong' binding of S1 to actin actin in the presence of MgADP or in the absence of which is completely abolished by the pPDM-cross-linking of nucleotides. Recently published data on the ADP- and Vi- the SH1 and SH2 groups. Probably, the 'weak' binding of S1 induced change in rotational correlation coefficient of S1 to F-actin caused by the modification of the 'strong' binding obtained by transient electrical birefringence techniques [45] site can be achieved in two ways: either by the change of the also confirm the assumption that S1 molecule becomes more structure of the whole S1 molecule in S1-ATP or S1-ADPcompact when Vi binds. Pi, or as a result of the pPDM-cross-linking inducing local Very recently Shriver and Kamdth [38] also studied the conformational changes just in the region of this site. thermally induced unfolding of S1 and heavy meromyosin by Literature data on the structural similarity of pPDMDSC and showed that the binding of AdoPP[NH]P and ADPcross-linked S1 with S1-ADP-Pi were obtained by indirect Vi cause a pronounced shift of the S1 calorimetric peak methods such as circular dichroic spectroscopy in the neartowards higher temperatures by 7-K and 10-K, respectively, ultraviolet region [ 191 or ultraviolet difference spectroscopy while the binding of ADP induces only a small (2-K) shift. [20] which seem to show a similarity in local conformation. Despite the fact that these data were mostly obtained with The data of Cremo ct al. [51], who studied the trapping in S1 heavy meromyosin and in quite different conditions (at higher of ADP analogues with fluorescent groups attached at the pH values, higher ionic strengths and at higher nucleotide concentrations), they correlate well with our data (see Figs 1 hydroxyls of the ribose ring by pPDM cross-linking or by Vi, and 3). This means that the effects caused by nucleotide bind- indicate that in both cases the trapping causes similar changes ing arc revealed in a rather wide range of solvent conditions of fluorescence. These authors concluded that the trapping by and do not depend on the type of the preparation used (S1 or either pPDM or Vi induces similar conformational changes in heavy meromyosin). It is note worthy that, because of artifacts the rcgion ncar the ribose binding site in S1. Thus, the 'weak' caused by the protein aggregation, these authors did not deter- binding of S1 to actin by itself is not a criterion for global mine thermodynamic parameters for the unfolding of S1 and conformational changes in the S1 molecule. its complexes with nucleotides. Due to the special construction of the measuring cell in our calorimeter [35] we avoided these Wc are very grateful to Prof. Manuel E . Morales for correcting the difficulties (see Materials and Methods) and wcre able to English of the manuscript. This work was supported by Internahnal measure thermodynamic parameters of the thermal unfolding Science Cooperation Grant DMB 9003692 from thc US National of S1 and its complexes with nucleotides (Table 1). Moreover, Scicncc Foundation and Russian Academy of Sciences.
(Fig. 7A), the free energy of the third domain being constant (Fig. 7 C ) . As a result, the Gibbs free energy of stabilization at 25 "C is about 60 kJ/mol for both preparations. In contrast to this, the Gibbs free energy of stabilization for thc S1AdoPP[NH]P and S1-ADP-Vi complexes (about 100 kJ/mol) is almost twice that for S1 andpPDM-cross-linked Sl. At the same time, SI-AdoPP[NH]P and S1-ADP-Vi practically do not differ in Gibbs free energy of stabilization at 25°C. A small decrease of free energy for two domains in SI-AdoPP[NH]Pin comparison with S1-ADP-Vi (Fig. 7D, F) is compensated by the presence of an additional domain at 47.8'C (Fig. 7E) which is absent in S1-ADP-Vi.
835 REFERENCES 1. Morita, F. (1967) J . Biol. Chem. 242, 4501 -4506. 2. Werber, M., Szent-Gyorgyi, A. G. & Fasman, G. (1972) Biochemistry 11, 2872 - 2882. 3. Bagshow, C. R. & Trentham, D. R. (1974) Biochem. J . 141,331 349. 4. Johnson, K. A. & Taylor, E. W. (1978) Biochemistry 17, 34323442. 5. Goodno, C. C. (1979) Proc. Nut1 Acad. Sci. USA 76, 2620-2624. 6. Goodno. C. C. (1982) Methods Enzymol. 85, 116-123. 7. Wells, J. A. & Yount, R. G. (1982) Methods Enzymol. 115, 93116. 8. Goodno, C. C. & Taylor, E. W. (1982) Proc. Nut1 Acad. Sci. U S A 79, 21 -25. 9. Chalovich, J. M., Greene, L. E. & Eisenherg, E. (1983) Proc. Nut1 Acad. Sci. U S A 80,4909 -491 3. 10. Chalovich, J. M., Stein, L. A,, Greene, L. E. & Eisenherg, E. (1984) Biochemistry 23,4885-4889. 11. Craig, R., Grccnc, L. E. & Eisenberg, E. (1985) Proc. Nut/ Acud. Sci. USA 82. 3247-3251. 12. King, R. T. & Greene, L. E. (1987) J . Biol. Chem. 262, 61286134. 13. Katayama, E. (1989) J. Biochem. (Tokyo) 106,751 -770. 14. Arata, T. (1984) J . Biochem. (Tokyo) 96, 337-347. 15. Arata, T. (1986) J. Mol. Biol. 191, 107-116. 16. Shrivcr, J. W. & Sykes, B. D. (1981) Biochemistry 20,2004-2012. 17. Kamath, U. & Shriver, J. W. (1989) J . Biol. Chem. 262, 55865592. 18. Barnett, V. A. & Thomas, D. D. (1987) Biocltemisfry 26, 314323. 19. Burkc. M., Reisler, E. & Harrington, W. F. (1976) Biochernis/r,v 15, 1923- 1927. 20. Cunningham, K. & Shriver, J. W. (1984) Biophys. J. 45,227a. 21. Walker, M. L. & Trinick, J. A. (1988) J . Muscle Res. Cell Motil. 9. 359 366. 22. Winkclmann, D. A,, Mekeel, H. & Rayment, I. (1985) J . Mol. Biol. 18J, 487-501. 23. Winkclmann, D. A., Baker, T. S. & Rayment, I. (1991) J . Cell Biol. 114, 701 - 71 3. 24. Highsmith, S. & Eden; D. (1986) Biochemistry 25, 2237-2242. 25. Privalov, P. L. (1982) Adv. Protein Chem. 35, 1-104. 26. Shnyrov, V. L., Zhadan, G. G. & Akoev, I. G. (1984) Bioelectroma~netics5, 41 1 -418. 27. Shnyrov, V. L., Levitsky, D. I., Vedenkina, N. S., Nikolaeva, 0. P., Khvorov, N. V., Permyakov, E. A. & Poglazov, B. F. (1989) Dokl. h a d . Nauk lJSSR 304, 1497- 3499 (in Russian). 28. Levitsky, D. I., Khvorov, N. V., Shnyrov, V. L., Vedenkina, N. S., Permyakov, E. A . & Poglazov, B. F. (1990) FEBSLett. 264, 176-178. ~~
29. Levilsky, D. I., Nikolaeva, 0. P., Vedenkina, N. S.. Shnyrov, V. L., Golitsina, N . L., Khvorov, N. V., Permyakov, E. A. & Pogla7ov, B. F. (1993) Biomed. Sci. 2, 140-147. 30. Golitsina, N. L., Shnyrov. V. L. & Levitsky, D. I. (1992) FEBS L.at 303, 255-257. 31. Levitsky, D. I., Khvorov, N. V., Shnyrov, V. L., Bukatina, A. E., Vedenkina, N. S . , Permyakov, E. A. & Poglazov, B. F. (1991) J . Muscle Res. Cell Motil. 12, 107. 32. Weeds, A. G. & Taylor, R. S. (1975) Nature [London) 257, 5456. 33. Lacmmli, U . K. (1970) Nature (London) 227, 681 -685. 34. Grammer, J. C., Cremo, C. R. & Yount, R. G. (1988) Biochemislry 27, 8408 - 8415. 35. Privalov, P. L. & Potekhin, S. A. (1986) Merhods Enzymol. 131, 4-51. 36. Maleev, V. Ya. & Gasan, A. G. (1974) Biopolymers 13, 24092422. 37. Sturtevant, J. M. (1974) Annu. Rev. Biophys. Bioeng. 3, 35-51. 38. Shriver, J. W. & Kamath, U. (1990) Biochemistry 29,2556- 2564. 39. Privalov, P. L. & Khechinashvili, N . N. (1974) J . M o l . Bid. 86, 665 - 684. 40. Privalov, P. L., Griko, Yu. V., Venyaminov, S. Yu. & Kutyshenko, V . P. (1986) J . Mol. B i d . 190,487-498. 41. Permyakov, ti. A., Burstein, E. A,, Sawada, Y . & Yamazaki, I. (1977) Biochim. Biophys. Acta 491, 149-154. 42. Crcmo, C. R . , Grammer, J. C. & Yount, R. G. (1988) Biochemistry 27, 8415 - 8420. 43. Cremo, C. R., Grammer, J. C . & Yount, R. G. (1989) .I. Bid. Chem. 264,6608 -661 1. 44. Aquirre, R., Lin, S.-H., Gonsoulin, F., Wang, C.-K. & Cheung, H. C. (3989) Biochemistry 28, 799 -807. 45. Highsmith, S. & Eden, D. (1990) Biochenzistry 29,4087-4093. 46. Katoh, T., Imae, S. & Morita, F. (1984) J . Biochem. (T0k.w) 95, 447-454. 47. Katoh, T. & Morita, F. (1984) J . Biochem. (Tokyo) 96, 12231230. 48. Katoh, T., Katoh, H. & Morita, F. (1985) J . Riol. Chem. 260, 6723 - 6727. 49. Suzuki, R., Nishi, N., Tokura, S. & Morita, F. (1987) J . Biol. Chem. 262, 11410-11412. 50. Keane, A. M., Trayer, I. P., Levine, B. A,, Zeugner, C. & Ruegg, J. C. (1990) Nature (London) 344,265-268. 51. Cremo, C. R., Neuron, J. M. & Yount, R. G. (1990) Bi0chemistr.l) 29,3309-3319.
Effects of nucleotide binding on thermal transitions and domain structure of myosin subfragment 1 Dmitrii I. LEVITSKY', Valery L. SHNYROV', Nikolai V KHVOROV'. Anna E. BUKATINA', Natalia S. VEDENKINA', Eugene A . PERMYAKOV2, Olga P. NIKOLAEVA' and Boris F POGLAZOV '
'
*
A. N. Bach Institute of Biochemistry of Russian Academy of Scicnces, Moscow, Russia lnstilute of Theoretical and Experimental Biophysics of Russian Academy of Sciences, Pushchino, Russia
(Received March 1 2/June 16, 1992) - EJB 92 0342
The thermal unfolding and domain structure of myosin subfragment 1 (SI) from rabbit skeletal muscles and their changes induced by nucleotide binding were studied by differential scanning calorimetry. The binding of ADP to S1 practically does not influence the position of the thermal transition (maximum at 47.2 "C), while the binding of the nun-hydrolysable analogue of ATP, adenosine 5'-[/Y~-imido]triphosphate (AdoPP[NH]P) to S1, or trapping of ADP in S1 by orthovanadate (V,), shift the maximum of the heat adsorption curve for S1 up to 53.2 and 56.loC, respectively. Such an increase of S1 thermostability in the complexes S1-AdoPP[NH]P and S1-ADPVi is confirmed by results of turbidity and tryptophan fluorescence measurements. The total heat adsorption curves for S1 and its complexes with nucleotides were decomposed into elementary peaks corresponding to the melting of structural domains in the S1 molecule. Quantitative analysis of the data shows that the domain structure of S1 in the complexes Sl-AdoPP[NH]P and S1-ADP-V, is similar and differs radically from that of nucleotide-free S1 and S1 in the S1-ADP complex. These data are the first direct evidence that the S1 molecule can be in two main conformations which may correspond to different states during the ATP hydrolysis : one of them corresponds to nucleotide-free S1 and to the complex S1-ADP, and the other corresponds to the intermediate complexes S1-ATP and S1-ADP-P,. Surprisingly it turned out that the domain structure of S1 with ADP trapped by p-phenylene-N, N'-dimaleimide @PDM) thiol cross-linking almost does not differ from that of the nucleotide-free S1. This means that pPDM-cross-linked S1 in contrast to S1-AdoPP[NH]P and S1ADP-V, can not be considered a structural analogue of the intermediate complexes S1-ATP and S1ADP-P,.
Muscle contraction and many other manifestations of biological motility are based on the cyclic interaction of myosin heads with actin, which is accompanied by ATP hydrolysis. During steady-state ATP hydrolysis the myosin head is subjected to conformational changes that can be detected by changcs in ultraviolet absorption [l]and intrinsic fluorescence 121. Such changes result from the fact that, in the ATPase reaction, the myosin head (M) passes through several intermediates: M . ATP, M . ADP . Pi, arid M . ADP differing from each other by intrinsic fluorescence parameters [3, 41. Isolated myosin heads, or myosin subfragment 1 (Sl); obtained by myosin proteolysis, and also stable analogues of S1 complexes with nucleotides, are often used for studies of thc structure and properties of the myosin ATPase interniediates. A complex of S1 with the non-hydrolysable analogue of ATP. adenosine S'-[fl,Y-irnido)triphosphate (AdoPP[NH]P), Covrespondencr to D. I. Levitsky, A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prospect 33, Moscow
117071, Russia Abbreviations. M, myosin; S1, myosin subfragment 1; AdoPP(NHIP, adenosine 5'-[fi,y-imido]triphosphate,Vi, orthovanadate; pPDM, IJ-plienylene-NN'-dimaleimide; DSC, diffcrential scanning calorimctry. Enzyme. Chymotrypsin (EC 3.4.21 .I).
is used as a stable analogue of the S1-ATP complex. Complexes of S1 with ADP and orthovanadate (Vi) [S, 61 and S1 cross-linked by p-phenylene-N,N-dimaleimide (pPDM) in the presence of ADP [7] are often used as stable analogues of the S1-ADP-Pi complex. The use of stable analogues allows the structure and properties of S1 in the intermediates of the ATPase reaction to be studied. There are numerous data suggesting that the stable analogues of the S1-ATP and S1-ADP-Pi intermediates are similar in structure, but differ from nucleotide-free S1 and S1-ADP [8-201, It should be noted that all these data are in fact indirect since they are based on either studies of the actinbinding properties of S1 [8 - 151 or studies of local conformational changes in S1 [16-201. In order to obtain direct proof of the assumption of two main structural states of S1, one must know the effects of nucleotide binding on the structure of the whole S1 molecule. In recent years the problem of elucidating the domain structure of the S1 molecule has attracted much attention. It is a very important problem since interdomain interactions inthe myosin head may play an essential role in force generation in muscles. At present there are data suggesting the existence of separate structural domains in S1 [21-241. How-
830 ever, the most distinct and general feature of a domain in a globular protein is that the structure of the domain folds and unfolds coopcrativcly in an all-or-none way with significant changes in enthalpy and entropy [25]. In accordance with this definition, domains can be revealed by studying the protein unfolding. The most effective method for studying thermal unfolding of proteins is differential scanning microcalorimetry. Earlier, one of us [26] introduced the ‘successive annealing’ method, which revealcd separate structural domains in proteins from appropriate calorimetric measurements. Recently we applied this method to S1 structure and revealed three domains in the S1 molecule [27 - 301. In the present work differcnt complexes of S1 with nucleotides were studied by microcalorimetry. The data obtained show that the domain structure of S1 in the S1AdoPP[NH]P and S1-ADP-Vi complexes is similar, and different from that or nucleotide-free S1 and S1 in the S1-ADP complex. So, these data are the first direct evidence that the S1 molecule can exist in two main conformational states, one of which occurs in the absence of nucleotides or in the complex of S1 with ADP, while the other occurs in the complexes S1ATP and S1-ADP-Pi. Unexpectedly it turned out that the domain structure of the pPDM-cross-linked S1 is practically the same as that of the nucleotide-free S1. This means that the p-PDM-cross-linked S1 can not be considered as a structural analogue of the intermediate complexes S1-ATP and S1-ADPPi. A preliminary report of this work has been published [31].
MATERIALS AND METHODS Reagents a-chymotrypsin was from Serva. Phenylmethylsulfonyl fluoride was from Merck. ATP, ADP, AdoPP[NH]P, pPDM and sodium vanadate (Na,VO,) were from Sigma Chemical Co. All other reagents were of analytical grade. 10 mM vanadate stock solution was prepared as described by Goodno [6].
S1 preparations Myosin subfragment 1 (Sl) was obtained by digestion of myosin filainents from rabbit skeletal muscles by chymotrypsin [32]. S1 concentration was determined by absorbance, using an A of 7.5. The purity of S1 was checked by SDSjPAGE [33]. The preparation of S1 contained the Y5kDa heavy chain and the light chains 1 and 3 but light chain 2 was absent. pPDM-cross-linked S1 was obtained by modification of S1 with pPDM at 0°C in the presence of ADP, following Wells and Yount [7], and was purified as described by Chalovich et al. [Y]. In order to obtain the S1-ADP-V, complex, 17-50 pM S1 was incubated with 2 mM MgC12, 10 - 250 pM Vi and 10 - 250 pM ADP [6] for 30 min at 25 “C in a medium containing 10 mM Hepes pH 7.3. The excess ADP and Vi were removed by intensive dialysis of S1 against 10 mM Hepes, pH 7.3, contaiiiing 1 mM MgC12. The extent of inodification of S1 by pPDM or V, was controlled by measuring the K+-EDTA-ATPase activity of S1 by relcase of Pi into the medium containing 0.04mg/ml S1, 0.5 M KCl, 5 mM EDTA, 1 mM ATP and 50 mM Tris/HCl, pH 7.5. ATPase activity ofpPDM-cross-linked S1 and S1 modified by vanadate in the presence of ADY did not exceed 3% of the unmodified S1 preparation activity. Decomposition of the S1ADP-V, complex, i.e. dissociation of ADP and V, from S1,
was achieved by irradiation (> 320 nm) as described by Grammer et al. [34]. Differential scanning calorimetry (DSC) Calorimetric measurements were carried out on a differential adiabatic scanning microcalorimeter DASM-4 (Russia) whose construction and principlcs have been described elsewhere [35].In order to measure the base line, both identical measuring cells were filled with solvent. For the measurement of the excess partial heat sorption of a protein, one of the cells was filled with the protein solution. High reproducibility of the base line allowed measurement of the partial heat sorption of a 1 - 3 mg/ml protein solution with an error not exceeding 3%. Capillary construction of thc 0.47-ml platinum measuring cell prevents to a great extent the artifacts caused by thermally induced protein aggregation. The heating rate was 1 Klmin. At this rate the protein aggregation may only reduce the enthalpy of denaturation without causing any changes in other parameters such as shape of the heat sorption curve and the denaturation tcmperature [36, 371. The exothermic peaks sometimes observed in the DSC scan [38] are caused by the artifacts of measurement. Such artifacts caused by protcin precipitation do not take place in the capillary helical measuring cell. We have convinced ourselves of this by parallel experiments using DASM-1M and DASM-4 microcalorimeters with different constructions of the measuring cells [35]. Dccomposition of the complex heat sorption curves into elementary components was carried out by the method of ‘successive annealing’ as described earlier [26, 271. This method is applied to fully or partially irreversiblc thermal transitions. The essence of the method is as follows. After measurement of the total heat sorption curve for protein solution and determination of the mean values of the thermal transition temperatures (locally visible or experimentally determined maxima on the heat sorption curve), another portion of the protein solution is heated in the calorimeter up to the temperature of the first maximum. Then the protein solution is cooled to the initial temperature (the temperature at the beginning of the scanning), and is heated again to a temperature exceeding the second maximum by 0.5-1.0 K, and is then recooled to the initial tempcrature. The heating/cooling cycles are repeated until complete denaturation of the protein has been achieved. The result of these experimcnts is a set of curves; subtracting each curve from the previous one, we obtain the initial portion of the heat sorption peak for each transition. The portion of the peak after the maximum was constructed for each transition by assuming symmetry relative to the temperature of the maximum. The denaturation change in heat capacity, AC;, was obtained using a linear extrapolation of the heat capacity curves for the native and denatured protein states, from left and right, to the transition tempcrature Td (see Fig. 1, curve A). The denaturation enthalphy, AHTd,and Gibbs free energy of stabilization, AG1,were calculated as described by Privalov et al. [3Y, 401. Measurements of thermally induced protein aggregation Thermally induced aggregation of S1 and its complexes with nucleotides was detected by changes in apparent absorbance at 350nm measured on a Cary-219 spectrophotometer (Varian) equipped with a thermostatted cellholder. The temperature in the cell was measured by a thermo-
831 B ;\
..: ... .:. ...
_ . :. ..
. . .-.. . .... .. ..
50 TEMPERATURE
40
60 (OC)
Fig. 1. Temperature dependences of excess heat capacity (AC,) for (A) nucleotide-free S1 and for (B, C) the complexes of S 1 with ADP and Vi. Sl concentration was 17.5 pM; (Bj 0.25 mM ADP and 0.25 mM Vi; ( C ) 10 pM ADP and 10 pM Vi. 10 mM Hepes, plI 7.3; 1 m M MgCI2. Tdis denaturation temperature, 4 C : is the denaturational change of heat capacity for nucleotidc-free S1. Vertical bar corresponds to 500 J . K - ' kg-'.
couple. Heating rate was 1 K/min. The measurements were carried out on a 1.5-2.0-mg/ml protein solution containing 10 mM Hepes pH 7.3 and 1 mM MgCI2. Intrinsic fluorescence measurements
Tryptophan fluorescence of S1 with or without nucleotide w-as measured on a laboratory-built spectrofluorimeter with monochromatic excitation at 280.4 nm [41]. Fluorescence was collectcd from the front surface of the thermostatted cell. The mean heating rate was about 30 K/h; the S1 concentration was 0.5 mg/ml. The measurements were carried out in solutions containing 10 mM Hepes pH 7.3 and 1 mM MgC12. RESULTS The first part of the work was devoted to studying the thermal denaturation of S1 and its complexes with nucleotides. Fig. 1 shows data on the thermally induced unfolding of S1 and of the S1-ADP-Vi complex. The heat sorption curve for S1 has a single peak with the maximum at 47.2"C. The formation of thc complex essentially changes the character of the thermal denaturation of S1: the maximum of the thermogram shifts t o 56.1 "C and the transition becomes more distinct. Due to the high affinity of Sl to ADP and Vi, such a change occurs even at equimolar concentrations of SZ , 4 D P , and Vi. Fig. 1 also shows results of an experiment in which the concentrations of ADP and Vi added are insufficient for complete saturation of the S1 binding sites. In this case, when only 50 - 60% of the S1 molecules are able to complex with ADP and Vi, two heat sorption peaks are found, one of which is characteristic of nucleotide-free S1 and the other of the S1-ADP-Vi complex. Note the coinplete absence of any intermediate transitions. This suggests that all the S1 molecules are in one of two possible conformational states. Irradiation above 320 nm of the stable S1-ADP-Vi complex is known to result in covalent modification of the S1 and in a rapid release of trapped MgADP and Vi [34]. We have
40
50
60
T E M P E R A T U R E ('C) Fig. 2. Temperature dependences of excess heat capacity for the S1ADP- Vi complex. The figure demonstrates reversibility of changes cduscd by ADP and Vi binding to S1. The complex S1-ADP-Vi (S1 concentration 48 pM) was dialyzed for 4 h against 10 mM Hepes pH 7.3, 1 mM MgCl,, changing the buffer every hour. Then the preparation was irradiated with ultraviolet light, four times diluted by the same buffer (final S1 concentration about 12 pMj and used for calorimetric mcasurcments. (A) S1-ADP-V, complcx bcfore irradiation; (B, Cj the same preparation irradiated for 1 min and 3 min, respectively; (D) after 3-min irradiation before the dilution 0.25 mM ADP and 0.5 mM Vi were added to the preparation. Vertical bar corresponds to 500 J K . kg-
'.
used this phenomenon to study the reversibility of the effects of the MgADP and Vi binding on the S1 thermal transition. For this purpose the S1-ADP-Vi complex was dialysed to remove an excess of ADP and Vi and then irradiated by ultraviolet light, followed at once by calorimetric measurements. It is clearly seen in Fig. 2 that the irradiation causes appearance of the heat sorption peak characteristic of the nucleotide-free S1. An increase of the time of irradiation results in a decrease of the peak for the S1-ADP-Vi complex and an increase of the S1 peak, which is in good agreement with data on the kinetics of release of the trapped ADP and V i from S1 [34]. The ultraviolet irradiation of the S1-ADP-Vi complex causes an oxidation of Serl80 in the S1 heavy chain [42, 431, but such a modification by itself does not cause any significant changes of the S1 thermal transition (Fig. 2B, C). The photo-modified S1 is able to retrap MgADP and Vi [34]. The addition of MgADP and Vi after the 3-min irradiation results in an increase of the S1-ADP-Vi heat sorption peak and in a decrease of the S1 peak. So: the transition of the S1 molecule to a completely different conformational state, which is characteristic for the S 1-ADP-V, complex, is fully reversible and depends only on the presence of the trapped MgADP and Vi in the active site of S1. Thus, the trapping of ADP and Vi in the active site of S1 causes an essential change of S1 conformation which is reflected in the pronounced shift of the thermal transition and in an increase of its cooperativity (the peak becomes much narrower). Fig. 3 shows calorimetric data for other complexes of S1 with nucleotides. The binding of MgADP to S l by itself has practically no influence on the temperature of the thermal
832
B
40 50 TEMPERATURE
60
TEMPERATURE
(
C
(OC
Fig. 3. Tcmpcrature dependences of excess heat capacity for S1 in the presence of 0.5 mM MgADP (A), 0.1 mM Mg . AdoPPlNHlP (B) and pPDM-cross-linkcd S1 containing trapped MgADP (C). Conditions was as in Fig. 1. Vertical bar corresponds to 500 J . K-' . kg-'.
Fig. 4. Temperature dependence of turbidity of solutions of S1 and S1 in the complexes with nucleotides. The turbidity measurements wcre carried out at heating rate 1 K/min dctccting apparent absorption at 350 nm; 10 mM Hepes pH 7.3, 1 mM MgC12. Curve a. 17.5 pM nucleotide-free S1; curve b, 14.5 pM S1, 0.27 mM AdoPP[NH]P; curve c, 14.5 pM S1,0.5 mM ADP, 0.25 mM V,.
Table 1. Thermodynamic parameters obtained from the DSC data for S1 and S1-nucleotide complexes.
Preparation
Td
s1 S1-ADP pPDM-S1 Sl-AdoPP(NH)P S 1-ADP-V,
"C 47.2 47.8 45.4 53.2 56.1
k 0.1 k 0.1 &- 0.1 k 0.1 i 0.1
AC;
AH T ~
kJ . K - ' . mol-' 22.6 k 0.2
kJimol 1130 f 30 1200 k 30 1120 k 30 1460 f 40 1330 f 40
0.2 12.3 0.3 10.3 f 0.2 12.3 & 0.2 16.0
334
t
0 0
20
transition but increases its sharpness, while the binding of Mg . AdoPP[NH]P to S1 essentially increases the temperature of the thermal transition, i. e. causes changes similar to those induced by the effect of the trapping of MgADP by V,. On the other hand, even the tight trapping of MgADP in S1 by means of the pPDM-cross-linking does not cause any significant change of S1 thermostability (Fig. 3). Thermodynamic parameters obtained from the calorimetric measurements on various complcxes of S1 with nucleotides are summarized in Table 1. An analysis of the main parameters of the melting (denaturation temperature, Td, and denaturation enthalphy, AHT,) shows that all the preparations studied can be separated into two groups. One of the groups comprises nucleotide-free S1, S1-ADP, and pPDM-crosslinkcd S1, while the other contains Sl-AdoPP[NH]P and S1ADP-V,. Some differences in the thermodynamic parameters of the preparations exist within each group, but they are small compared with those of the preparations in different groups. So, data from DSC studies show that the S1-AdoPPWHlP and S1-ADP-Vicomplexes differ in their main thermodynamic parameters (Td, AHTd) from all the other s 1 preparations studied. We decided to confirm the differences by other methods. The experiments were carried out in the same medium and at the same heating rate as in the calorimetric experiments. Fig. 4 shows temperature dependenccs of thc thermally induced aggregation of S1, SI-AdoPP[NH]P, and S1-ADPV,. For these preparations the temperatures of the half-maxima1 effect are 40.5"C, 47.0' C, and 49.0"C, respectively. These results show that the binding of Mg . AdoPP[NH]P or
40 T E M P ERR TU RE ( OC
60
Fig. 5. Temperature dependences of tryptophan fluorescence spectrum position (A) for S1 ( O ) ,S1-ADP (A),pPDM-cross-linked Sl (fl), S1AdoPPlNHlP (0)and S1-ADP-Vi ( A ) . Conditions were as in Figs 1 and 3.
trapping of MgADP by Vi essentially increase the stability of S1 against the thermally induced aggregation of S1 and the effect of ADP and Vi is more pronounced compared to that of AdoPP[NH]P which correlates well with the DSC data. At the same time, the binding of MgADP or the trapping of MgADP by the pPDM cross-linking do not increase and sometimes even slightly decrcase (by 1-2°C) the S1 stability against the thermally induced aggregation (data are not shown). Fig. 5 shows temperature dependences of the tryptophan fluorescence spectrum position (wavelcngth at the peak) for S1 and its complexes with nucleotides. This parameter (2) is the least sensitive to possible artifacts caused by the protein aggregation. Such artifacts are caused mainly by protein precipitation induced by heating. It should be noted that usually the thermal denaturation of a protein is accompanied by an unfolding of the protein structure and a transfer of some fluorescent tryptophan residues to the water environment which is reflected by a red spectral shift. In the case of the S1 preparations, the thermal denaturation causes the opposite effect [28] which seems to be duc to the fact that the thermal transitions in S1 inducc an increase of intermolecular interactions, i. e. of protein aggregation. In the course of this process
833 40 20 0
-
-20
A
E"
*CP
\
2 40 d.
v
Q 2o 0
- 20 -4 0
40 50 60 TEMPERATURE ( "C)
Fig. 6. Decomposition by the 'successive annealing' method of the total heat sorption curves (-) for nucleotide-free SI (A), pPDM-crosslinked S1 (B), S1-AdoPPINHIP (C) and S1-ADP-Vi (D) into elementary peaks (- - - -) corresponding to the melting of separate domains. Conditions were as In Figs 1 and 3 Vertical bar corresponds to 500 J.K kg-'.
some of tryptophan residues, which initially were at the protein surface, are transferred into a purely protein environment in the regions of intermolecular contacts. It is clearly seen from Fig. 5 that an increase of temperature induces a blue shift of the tryptophan fluorescence spectrum of S1, S1-ADP and pPDM-cross-linked S1. The shift occurs within the temperature region 40- 50'C. For the S1-AdoPP[NH]P a weak blue shill takes place at higher temperatures but is completcly absent in the temperature region studied in the case of S1ADP-Vi. These data corroborate the fact that the complexes S1-AdoPP[NH]P and S1-ADP-V, essentially differ in thermostability from S1-ADP, pPDM-cross-linked S1 containing trapped ADP, and nucleotide-free S1. So, all the data presented above show that, according to the thermal sensitivity and to the thermal transition paramcters, all the preparations studied are separated into two groups, one of which contains S1-AdoPP[NH]P and S1-ADP-Vi and the other Sl -ADP, pPDM-crosslinked S1 and nucleotide-free S1. It would be reasonable to assume that the preparations of different groups essentially differ in conformational state of S1 molecule. In order to chcck this assumption we have applied the 'successive annealing' method to reveal the domain structure of S1 in some of the complexes with nucleotides and to compare it with the domain structure of the nucleotide-free s1. Fig. 6 A shows results of a decomposition of thc total heat sorption curve of S1 into elementary peaks. Each peak corresponds to the melting or a separate cooperative region (domain) in the S1 molecule. It is clearly seen that S1 contains three such domains which melt with maxima at 39 "C, 47.2 "C and 50.8 "C. pPDM-cross-linked S1 containing tightly trapped MgADP has a similar three-domain structure though with other contributions and temperatures of the peaks (41.2,45.5 and 48.8") (Fig. 6B). S1-ADP also has a similar three-domain structure (the data are not shown). On the other hand, the S1 molecule has an absolutely different domain structure in the Sl-AdoPP[NH]P and S1-ADP-Vi complexes (Fig. 6C,
- 60
0
20
40
60
0
20
4060
TEMPERATURE ('C) Fig. 7. Temperature dependences of Gibbs energy of stabilization for the S1 domains (see Fig. 6). Lettering of the domains is in the order of increased maximum temperature. (A-C) Nucleotide-free S1 ( 0j and pPDM-cross-linked3 (0); (A) the first domain(39Tand41.2'C for S1 and pPDM-cross-linked S1, respectively); (B) the sccond domain (47.2'C and 45.SnC, respcctivcly); (C) third doinain ( 5 0 3 ° C and 48.8 "C, respectively). (D - F) S1 -AdoPP[NH]P ( A j and S1-ADP-Vi (0); (D) the first domain (42.0"C and 52.4"C for Sl-AdoPP[NH]P and S1-ADP-Vi, respectively); (Ej the second domain (47.8"C for SI-AdoPP[NH]P, the peak is absent for S1-ADP-Vi); (Fj the third domain (53.2"C and 562°C for Sl-AdoPP[NFT]P and S1-ADP-Vi, respectivelyj.
D). In this case the total heat sorption curve is decomposed into the components with maxima at 42.0"C, 47.8"C, and 53.2OC for S1-AdoPP[NH]P (Fig. 6C) and at 52.4"C and 56.2"C for S1-ADP-V, (Fig. 6D). Moreover, for the S1-ADPVi preparation an additional very small heat sorption peak was found at 40°C (see, for example, Fig. 6D). Probably a similar peak is contained in the thermograms of other preparations but it is not revealed because in these cases it is located within the limits of the total heat sorption contour and practically coincides in position with the peak of the most thermolabile domain (Fig. 6). It seems most likely that this peak corresponds to the melting of the alkali light chains in the S1 preparation [30]. The most convenient measure for the quantitative description of the stability of a cooperative domain, or of a singledomain protein, is the difference in the Gibbs free energies of the denatured and native states of a protein. Fig. 7 shows the temperature dcpendences of the free energies of stabilization for the experimentally revealed domains. Taking into account the fact that the structural stability of a multi-domain protein is conditioned by the stability of its domains, we can assert that the stability of S1 and pPDM-cross-linked S1 at 25°C is the same. A small decrease of free energy for the second domain inpPDM-cross-linked S1 (Fig. 7 B) is completely compensated by an increase o r free energy of the first domain
834 Shriver and Kamath [38] could not show separate structural domains in the myosin head though they postulated their existence. Wc have succeeded in obtaining direct evidence for the existence of domain structure in S1 which essentially changes upon the binding of AdoPP[NH]P or ADP and Vi to S1 (Fig. 6). Thus, we can definitively subdivide all the S1 samples studied into two classes with different domain structures. Since SI-AdoPf"NH]P and S1-ADP-Vi are stable analogues of the intermediates of the ATPase reaction, S1-ATP and S1-ADPPi, we can conclude that the domain structure of S1 in these intermediates is different from that of nucleotide-free S1 or S1 in the complex with ADP. Probably, the formation of these intermediates is accompanied by the domain -domain interactions in the S1 molecule resulting in the change of S1 DISCUSSION domain structure. Thus, the data obtained are direct evidence The data obtained lead to the conclusion that the binding for the existence of S1 in two main conformational states, one of Mg . AdoPP[NH]P to S1 or the trapping of MgADP in S1 of which is realized in the absence of nucleotides or in the S1by Vi result in a pronounced conformational change of the ADP complex and the other one corresponds to the S1-ATP whole S1 molecule, while the binding of MgADP and the and S1-ADP-Picomplexes. At the same time, S1-ADP slightly trapping of MgADP by pPDM-cross-linking cause only local differs from S1, and S1-ATP - from S1-ADP-Pi, but such conformational changes in S1. The global conformational relatively small differences are negligible compared to the changes are reflected in a pronounced increase of S1 global differences between the main confornutional states. thermostability and in a significant change of S1 domain It is surprising that the domain structure ofpPDM-crossstructure. The decrease of the number of the structural do- linked S1 differs from the structure of nucleotide-free S1 much mains in S1-ADP-Vi in comparison with other S1 preparations less than from the domain structure of SZ in the S1(see Fig. 6) seems to be due to interdomain interactions caus- AdoPP[NH]P and S1-ADP-Vi complexes (Fig. 6). The point ing a tightening of the S l structure which makes it more here is that such S1 cross-linked by pPDM in the presence of compact. This assumption is corroborated by the data of ADP displays, like S1-ATP and S1-ADP-Vi, 'weak' binding Aquirre et al. [44] demonstrating that the formation of the to actin [9, 46,471 and is supposed to be a structural analogue S1-ADP-Vi complex causes spatial translocations in the S1 of S1-ATP or S1-ADP-Pi [19,20]. We can propose the followmolecule that dccreasc distances between some parts of the ing explanation of this seeming contradiction. One of the heavy chain and the nucleotide binding site in the active site actin-binding sites in the S1 heavy chain is located in the of S1. Moreover, it is confirmed by the electron microscopic region of the SH1 and SH2 groups (Cys707 and Cys697, redata of Katayama [13] who showed that the addition of spectively) [48] and possibly even between these SH-groups MgADP and Vi significantly changes the shape of the actin- since synthetic peptides with similar amino acid sequences bound S1 molecules making them shorter and rounder in interact with F-actin [49, 501. Morita et al. [46, 471 suggested comparison with the more elongated S1 molecules bound to that this site is responsible for 'strong' binding of S1 to actin actin in the presence of MgADP or in the absence of which is completely abolished by the pPDM-cross-linking of nucleotides. Recently published data on the ADP- and Vi- the SH1 and SH2 groups. Probably, the 'weak' binding of S1 induced change in rotational correlation coefficient of S1 to F-actin caused by the modification of the 'strong' binding obtained by transient electrical birefringence techniques [45] site can be achieved in two ways: either by the change of the also confirm the assumption that S1 molecule becomes more structure of the whole S1 molecule in S1-ATP or S1-ADPcompact when Vi binds. Pi, or as a result of the pPDM-cross-linking inducing local Very recently Shriver and Kamdth [38] also studied the conformational changes just in the region of this site. thermally induced unfolding of S1 and heavy meromyosin by Literature data on the structural similarity of pPDMDSC and showed that the binding of AdoPP[NH]P and ADPcross-linked S1 with S1-ADP-Pi were obtained by indirect Vi cause a pronounced shift of the S1 calorimetric peak methods such as circular dichroic spectroscopy in the neartowards higher temperatures by 7-K and 10-K, respectively, ultraviolet region [ 191 or ultraviolet difference spectroscopy while the binding of ADP induces only a small (2-K) shift. [20] which seem to show a similarity in local conformation. Despite the fact that these data were mostly obtained with The data of Cremo ct al. [51], who studied the trapping in S1 heavy meromyosin and in quite different conditions (at higher of ADP analogues with fluorescent groups attached at the pH values, higher ionic strengths and at higher nucleotide concentrations), they correlate well with our data (see Figs 1 hydroxyls of the ribose ring by pPDM cross-linking or by Vi, and 3). This means that the effects caused by nucleotide bind- indicate that in both cases the trapping causes similar changes ing arc revealed in a rather wide range of solvent conditions of fluorescence. These authors concluded that the trapping by and do not depend on the type of the preparation used (S1 or either pPDM or Vi induces similar conformational changes in heavy meromyosin). It is note worthy that, because of artifacts the rcgion ncar the ribose binding site in S1. Thus, the 'weak' caused by the protein aggregation, these authors did not deter- binding of S1 to actin by itself is not a criterion for global mine thermodynamic parameters for the unfolding of S1 and conformational changes in the S1 molecule. its complexes with nucleotides. Due to the special construction of the measuring cell in our calorimeter [35] we avoided these Wc are very grateful to Prof. Manuel E . Morales for correcting the difficulties (see Materials and Methods) and wcre able to English of the manuscript. This work was supported by Internahnal measure thermodynamic parameters of the thermal unfolding Science Cooperation Grant DMB 9003692 from thc US National of S1 and its complexes with nucleotides (Table 1). Moreover, Scicncc Foundation and Russian Academy of Sciences.
(Fig. 7A), the free energy of the third domain being constant (Fig. 7 C ) . As a result, the Gibbs free energy of stabilization at 25 "C is about 60 kJ/mol for both preparations. In contrast to this, the Gibbs free energy of stabilization for thc S1AdoPP[NH]P and S1-ADP-Vi complexes (about 100 kJ/mol) is almost twice that for S1 andpPDM-cross-linked Sl. At the same time, SI-AdoPP[NH]P and S1-ADP-Vi practically do not differ in Gibbs free energy of stabilization at 25°C. A small decrease of free energy for two domains in SI-AdoPP[NH]Pin comparison with S1-ADP-Vi (Fig. 7D, F) is compensated by the presence of an additional domain at 47.8'C (Fig. 7E) which is absent in S1-ADP-Vi.
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