Temperature-induced changes in the flexibility of the loop between SH1 (Cys-707) and SH3 (Cys-522) in myosin subfragment 1 detected by cross-linking

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 290, No. 1, October, pp. l-6, 1991

Temperature-Induced Changes in the Flexibility of the Loop between SHI (Cys-707) and SH3 (Cys-522) in Myosin Subfragment 1 Detected by Cross-linking’ Rajesh Agarwal’ Department

and Morris

of Biology, Case Institute

Received February

Burke3 of Technology, Case Western Reserve University,

13, 1991, and in revised form May 29,199l

The ability of dibromobimane to cross-link SHl (Cys707) in the 21-kDa C-terminal segment to SH3 (Cys-522) in the 50-kDa middle segment of the myosin Sl heavy chain has been examined as a function of nucleotide binding and temperature. The results obtained indicate that, while the reagent rapidly reacts with SHl at both 25 and 4”C, its ability to cross-link to SH3 is highly dependent on temperature. At 25”C, substantial crosslinking from monofunctionally labeled SHl to SH3 occurs, in agreement with recent work of Mornet, Ue, and Morales (1985, Proc. Natl. Acad. Sci. USA 82, 165% 1662) and of Ue (1987, Biochemistry 26, 1889-1894) and with their conclusion that a loop, allowing SHl and SH3 to reside at the cross-linking span of dibromobimane, preexists in the protein. At 4”C, however, negligible amounts of cross-linking are observed whether or not a nucleotide is present, despite indications that SHl is labeled rapidly by the reagent at this temperature. The inability to form this cross-link is not due to an alternate cross-link between monofunctionally labeled SHl and another thiol in the 21-kDa segment. These results indicate that this loop exists at 25°C and does not exist (or o 1991 exists only transiently) at the lower temperature. Academic

Press,

Cleveland, Ohio 44106

Inc.

The binding of Mg nucleotide to myosin subfragment 1 (S1)4 results in changes in the conformation of the pro’ This work was supported by United States Public Health Service Grant NS 15319. * Present address: Department of Dermatology, Case Western Reserve University, Cleveland, OH 44106. 3 To whom correspondence should be addressed. 4 Abbreviations used: Sl, subfragment 1 of myosin; SlA2, subfragment 1 isozyme containing the A2 light chain; TSl, trypsinized subfragment 1; TSlA2, trypsinized isozyme of subfragment 1 containing the A2 light chain; SHP-NEM-Sl, subfragment 1 modified at the SH2 thiol with N000%9861/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form

Inc. reserved.

tein which have been detected by a variety of spectral and chemical techniques (l-6). However, the structural consequences of these conformational transitions have been more difficult to assess, since there is little detailed information about the folded structure of the catalytic heavy chain subunit. A number of biophysical techniques, such as low angle X-ray diffraction (7), relaxation of fluorescence polarization (8), and neutron scattering (9), have failed to reveal any gross change in the shape of Sl on binding nucleotide or actin. Cross-linking studies, however, have revealed that the binding of MgADP to Sl results in a marked increase in the flexibility of the heavy chain in the nine-residue segment separating SHl (Cys707) from SH2 (Cys-697), since these two thiols can be readily covalently bridged by reagents of cross-linking spans from 0.3 to 1.7 nm (10-13). Furthermore, the formation of these cross-links between these two thiols, or the formation of a disulfide linkage from them, results in the noncovalent trapping of the nucleotide in S1(11,14). This flexibility has also been sensed by fluorescence energy transfer (15, 16). It has also been shown that two other interthiol crosslinks could be identified, one of which required Mg nucleotide binding (SH2 to Cys-540) (17,18), while the other (SHl to Cys-522) was either insensitive (19, 20) or sensitive (21) to the presence of bound MgADP, depending on the particular reagents employed. In these cases, only those cross-links requiring Mg nucleotide resulted in trapping of the nucleotide. In addition to these thiol-specific reagents, the heterofunctional reagent p-nitrophenyliodoacetate resulted in linkage from Lys-184 (or Lys-

ethylmaleimide; DBB, dibromobimane; pPDM,p-phenylenedimaleimide; SBTI, soybean trypsin inhibitor; DTT, dithiothreitol; NEM, N-ethylmaleimide; DNP, 2,4dinitrophenyl group; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; Hepes, 4-(2.hydroxyethyl). 1-piperazineethanesulfonic acid; TLCK, 1-l-tosylamido-2-phenylethyl chloromethyl ketone. 1

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AGARWAL

189) to SH2 only when the ATP site of Sl was not occupied (22, 23). Other cross-linkers have established the proximity of residues residing in the three different heavy chain “domains” but the actual residues participating have not as yet been characterized (24-26). The availability of heterofunctional, photoactivatable cross-linkers has enabled this approach to be extended. In the case of myosin these were initially used to examine the topography of the regulatory light chains to the remainder of the scallop myosin molecule (27), and subsequently applied by Lu and her colleagues to examine the spatial relationships about the SHl thiol (28, 29). These studies showed that SHl was close to Glu-88 in the 27kDa5 “domain” (30) and that this relationship was not perturbed by the addition of nucleotide nor by mild thermal denaturation (31) where part of the 50-kDa “domain” does appear to unfold (32,33). Addition of MgADP, while not abolishing the SHl to Glu-88 link, was found to promote the formation of another cross-link from SHl to the 50-kDa domain (28) at about 55 kDa from the amino terminus. However, Muno and Sekine (34) found that photolysis of Sl, labeled at SHl with 1,2,4-trinitrobenzene, resulted in cross-linking to the 50-kDa segment only in a nucleotide-independent manner. The above observations are summarized in the schematics shown in Fig 1. We have recently shown that linkage of SHl to Cys522 can occur readily at 4°C with pPDM in SH2-NEMSl only in the presence of bound Mg nucleotide (21). This observation appears to be at odds with the recent work with DBB, where it was found that this cross-link occurred readily at 25°C in a nucleotide-independent manner (19). To obtain additional information about this apparent discrepancy, we have now examined the reaction of Sl and TSl with DBB at 4°C. The results suggest that, while SHl is reacted rapidly at both 4 and 25”C, the crosslinking to Cys-522 with DBB occurs at a significant rate only at the latter temperature. MATERIALS

AND

METHODS

Distilled water, further purified through a Millipore &TM system, was used throughout. DBB was purchased from Molecular Probe, and pPDM from Aldrich. TLCK-treated trypsin, SBTI, ATP, and ADP were obtained from Sigma and [‘%]ADP (sp act 2.4 X lOI cpm/mol) was from New England Nuclear. All other reagents were of reagent grade. The preparation of myosin and Sl and the separation of the Sl isozymes were done as described by Godfrey and Harrington (35) and Weeds and Taylor (36), respectively. TSl was prepared by digesting Sl (5.0 mg/ml) with trypsin at. a Sl/trypsin ratio of 1OO:l in 0.05 M Hepes, pH 7.8, for 35 min at 25°C followed by the addition of SBTI in double the amount of the trypsin used. The protein was then placed on ice. Protein concentrations were determined by absorption at 280 nm employing E’“” of 5.5 and 7.5 for myosin and Sl, respectively, or by the method of Bradford (37) using myosin or Sl as standards.

‘The heavy chain “domains” refer to the three sequential tryptic fragments of the heavy chain of molecular masses of 27,50, and 21 kDa based on their mobilities on SDSPAGE.

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1

.

.

m-493 ,z

. . .

5

0

100

200

300

400

Residue

500

600

700705

800

Number -

20

;

B cys-707

.

cys-540

.

.

Arg-239 0

. .

LYS-194 0

. . .

00 0

/” 100

200

300

400

Residue

500

600

700705

“”

” 800

Number

FIG. 1.

Schematic showing the locations and lengths of cross-links made in subfragment 1 from (A) SHl (Cys-707) and (B) SH2 (Cys-697) referred to in the text. The open symbols indicate the cross-links that are made in the absence of nucleotide or which are independent of nucleotide. The closed symbols represent those which require binding of nucleotide.

The proteins at 2.0 mg/ml in 0.05 M Hepes, pH 7.8, at 4 and 25°C were reacted with DBB using a 4.0-molar excess of the reagent in the presence and absence of 1.0 mM MgCl, and ADP. At desired incubation times 100 ~1 of the reaction mixture was removed and made 2.0 mM with respect to 2-mercaptoethanol to terminate the reaction. Aliquots were removed and diluted to 0.2 mg/ml for Ca” and K+/EDTA ATPase activity measurements by the method of Kielley and Bradley (38). The remainder of the protein was then subjected to SDS-PAGE by the procedure of Laemmli (39). The amount of Sl cross-linked by DBB at 4 and 25°C in the presence and absence of MgADP was obtained from densitometric analyses at 550 nm of SDS-PAGE of these samples using a Shimadzu CS-930 TLC scanner with a DR.2 data recorder. It was assumed that the color yields for the cross-linked species and the 95. kDa heavy chain were the same. This enabled the fraction of crosslinked Sl present in the reacted sample to be evaluated. The preparation of SHL-NEM-Sl involved reversible modification of Sl at SHl with 2,4-dinitrofluorobenzene as described by Bailin and Barany (40) and subsequent modification of SH2 by reaction at pH 7.8 with NEM (1.3 molar excess) in the presence of 1.0 mM MgC1, and ADP as described previously (41), followed by thiolysis of the DNP group from SHl by an overnight incubation at 4’C in 0.01 M DTT (18). Modification of SHL-NEM-Sl at 2.0 mg/ml by DBB was done in 0.05 M Hepes, pH 7.8, at 4 and 25°C using a 4.0-molar excess of DBB in the presence and absence of 1.0 mM MgCl, and ADP for 40 min and the

MYOSIN

SUBFRAGMENT

reaction was stopped by making the solution 2.0 mM in P-mercaptoethanol. The samples were then analyzed by SDS-PAGE using the method of Laemmli (39). Nucleotide trapping was determined by using [i4C]ADP during the reaction of Sl or SH2-NEM-Sl with DBB at 4 and 25°C. The reacted protein was precipitated with saturated ammonium sulfate to 66% saturation and centrifuged. The pellet was dissolved in 0.05 M Hepes, pH 7.8, at 4°C and residual untrapped nucleotide was removed by centrifugation through Sephadex G-50, equilibrated in the same buffer, as described by Penefsky (42). Aliquots of these samples were used for radioactivity and for protein concentration, to estimate the amount of nucleotide trapped in the total Sl including uncross-linked (samples at 4’C) and cross-linked (samples at 25’C) Sl and SHB-NEM-Sl. From densitometric analyses of SDS-PAGE electrophoretograms of the modified Sl species, the amount of cross-linked species was evaluated allowing for the stoichiometry of trapping to be determined. The interaction of F-actin with Sl premodified with DBB at 4 and 25°C was determined by adding F-actin (1.0 mg in actin buffer) to Sl and Sl premodified with DBB at 4 and 25’C (1.0 mg/ml in each case) in 0.05 M Hepes, pH 7.8. The samples were then centrifuged at 100,OOOg for 60 min at 4°C and the protein concentration in supernatants was determined. The pellets from various samples were dissolved in 0.5 ml of the same buffer, and both supernatants and pellet solutions were subjected to SDS-PAGE analysis. For comparison, samples of F-actin, Sl, and Sl premodified with DBB at 4 and 25°C were separately centrifuged and analyzed for protein concentration in supernatants, and SDS-PAGE in supernatants and pellets under similar conditions. Gel electrophoresis was performed by the procedure of Laemmli (39) using 12.5% acrylamide gels in the presence of 0.1% SDS and 0.1% 2mercaptoethanol. The gels were stained with Coomassie brilliant blue.

A

i: 20 7

OL 0

I 10

20 Reaction

30

40

50

60

Time (min)

100n

1

60

0

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20 Reaction

30

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60

Time (min)

FIG. 2. Effect of modification time with DBB on the ATPase properties of Sl. (A) Changes in the Ca*’ ATPase and (B) K+/EDTA ATPase activities of Sl at 4’C (0) and 25°C (0). The 100% values correspond to 7.7 and 19 s-i, respectively.

3

1 FLEXIBILITY

kDa

A

95-

abcde

a

b

c

d

e

FIG. 3. SDSPAGE electrophoretograms of Sl(A2) and TSl(A2) on modification with DBB at 4 and 25°C. (A) Lanes: (a) unmodified SlA2; (b) and (c) are modified at 4°C for 60 min in the absence and presence of 1 mM MgADP, respectively; (d) and (e) same as for (b) and (c) respectively, except that the modifications were done at 25°C. (B) similar to (A) except done with TSl(A2). Xl and X2 represent the major crosslinked products observed with Sl(A2) and TSl(AB), respectively.

RESULTS

Previous studies have shown that when Sl was modified at SHl with DBB at 25°C the Ca2+ and K+/EDTA ATPase activities were inhibited by about 40 and 60%, respectively and MgATP had only a slight effect on the inhibitory property of the modification on the ATPase (19). The results of the present study, as shown in Fig. 2, indicate that when Sl is modified with DBB in the presence or absence of MgADP, there is sharp inhibition of about 40 and 60% in the Ca2+ and K+/EDTA ATPase activities, respectively, in the first 5 min of reaction, independent of temperature. This finding at 25°C is in agreement with the work reported by Mornet et al. (19). These observations suggest that a very rapid modification at SHl by DBB occurs at the lower temperature. Similar results were obtained when TSl was used instead of native Sl (data not shown). To examine whether cross-links were formed from SHl when Sl or TSl were reacted with DBB at 4 and 25”C, the peptide composition of the reaction mixture as a function of time was examined by SDS-PAGE. These results are shown in Fig. 3 for cross-linking studies done at the two temperatures in the presence and absence of MgADP. At 4”C, negligible amounts of cross-linking were observed independent of the presence of Mg nucleotide (Fig. 3A, lanes b and c) as evidenced by two very faint bands shown as Xl of lower mobility than the unchanged heavy chain. The reaction at 25’C resulted in a significant amount of cross-linking as evidenced by the large increase in the band designated as Xl, again independent of MgADP (Fig. 3A, lanes d and e). Densitometric analyses of the amounts of Xl and heavy chain bands indicated that Xl amounted to less than 2% of the total at 4’C, while at 25°C it was about 42%. The results of reacting TSl with DBB were essentially the same, since little if any cross-linking was observed at 4°C (Fig. 3B, lanes b and c), whereas at 25°C significant cross-linking occurred

4

AGARWAL kDa

kDa

95 502720A2-

A2-

a

b

c

d

FIG. 4. SDS-PAGE electrophoretograms. Lanes: (a) SlA2 reacted for 5 min at 4°C; (b) SlA2 reacted for 5 min at 4”C, then centrifuged through Sephadex G-50 and allowed to incubate at 25°C for 40 min; (c) and (d) correspond to (a) and (b), respectively, except that the reactions were done with TSlAZ. Xl and X2 are the cross-linked species formed in (b) and (d), respectively.

as denoted by the band designated as X2 in Fig. 3B (lanes d and e), independent of MgADP. When these electrophoretograms of reacted Sl were examined under uv illumination, significant fluorescence appeared in the 95-kDa heavy chain band in the case of samples modified at 4°C after 5 min of reaction (data not shown), consistent with the ATPase changes that at 4°C DBB reacted at SHl in native Sl. This observation was further substantiated with TSl reacted at 4”C, since in this case the fluorescence was confined to the 21-kDa segment (data not shown). However, when electrophoretograms of Sl or TSl reacted with DBB at 25°C were examined under uv illumination, we observed the same pattern of fluorescence associated with the new peptides formed by cross-linking as reported by Mornet et al. (19) and Ue (20). To determine whether the failure to detect significant levels of cross-linking to Cys-522 at 4°C could be attributed to cross-linking to a thiol in the 21-kDa segment the following reactions were examined. Sl and TSl were modified with DBB at 4°C for 5 min and then subjected to centrifugation through Sephadex G-50 (42) to remove residual reagent. Aliquots of these samples were then incubated for an additional 40 min at 25°C (Fig. 4). It is evident that, after this incubation, significant cross-linking has occurred in these samples, as seen in lanes b and d of Fig. 4. These data, together with the ATPase results shown in Fig. 2, demonstrate that only monofunctional modification at SW1 has occurred at 4°C. Furthermore, similar results were also observed when SHB-NEM-Sl was reacted with DBB at 4 and 25”C, in the presence or absence of MgADP, indicating that prior blocking of SH2 does not affect these reactions (data not shown). The possibility that the nucleotide was trapped during the cross-linking reaction from SHl to the 50-kDa segment using DBB was also investigated. The amount of [14C]ADP present in Sl or SHS-NEM-Sl, after modification with DBB at 4 and 25”C, was obtained from the radioactivity present in a known concentration of the pu-

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rifled protein, and from densitometric analysis of SDSPAGE electrophoretograms which yielded the amount of cross-linked sample present in the modified protein. These results showed a negligible amount of ADP (0.053 k 0.006 mol) trapped per mole of cross-linked Sl or SHP-NEMSl, in the cases of reactions at both 4 and 25°C. To examine whether actin binding was altered by monofunctional modification of Sl at SHl or by crosslinking from SHl to Cys-522, F-actin was added to Sl, reacted earlier for 60 min with DBB at 4 and 25”C, respectively, and after centrifugation at 100,OOOgthe supernatant and pellets were examined by SDS-PAGE. These results are presented in Fig. 5. It is evident that both forms of reacted Sl bind to actin since the corresponding pellets showed significant levels of these proteins cosedimenting with actin (Fig. 5, lanes c and d) and very low amounts were found in the supernatants (Fig. 5, lanes e and f). In the absence of actin few if any of these Sl species were pelleted under otherwise identical conditions. From the amounts of unmodified and modified Sl species present in the supernatants of the centrifuged actoS1 samples (Fig. 5, lanes e, f, and h), it appears that the modifications at SHl with or without cross-linking show similar or only slightly weakened binding to actin, consistent with the findings of Mornet et aE. (19). DISCUSSION

Cross-linking approaches have provided significant information about the proximity of certain residues in Sl in different conformational states (e.g., in the absence and presence of Mg nucleotide) through the ability to form cross-links between them. In the case of conventional cross-linking, using homofunctional and heterofunctional reactive groups, formation of a cross-link depends on a number of factors among which are: (i) the range of separation of the functional groups that the crosslinker can adopt due to bond rotational freedom and (ii)

kDa

-Xl 95Acth-

A2-

a

FIG. 6.

b

c

d

e

f

9

h

SDS-PAGE electrophoretograms examining actin binding of Sl modified with DBB. Lanes: (a) actin (1.0 mg/ml) and Sl (1.0 mg/ ml) premodified with DBB at 4’C; (b) same as (a) except modification of Sl was done at 25’C; pellets (c) and (d) and supernatants (e) and (f) obtained by centrifugation of (a) and (b), respectively; (g) pellet and (h) supernatant after centrifugation of unmodified Sl and actin.

MYOSIN

SUBFRAGMENT

the availability of residues of the appropriate chemical specificity at the appropriate separation and orientation in the folded native structure of Sl to combine with the reagent. In addition, the influence of molecular dynamics (flexibility) of the folded structure on the time frame in which the two residues reside in the correct spatial geometry for cross-linking, as well as the reactivities of the residues, will also play a significant role in determining whether a cross-link will be formed. Assuming that the functional groups of the cross-linker are not hydrolyzable at a significant rate and that condition (ii) is also met, then the extent of cross-linking will depend on the additional factors just mentioned. In general, the cross-link will involve two intermediate steps, the first being a bimolecular reaction involving monofunctional substitution at one residue, followed by a second intramolecular reaction which represents the coupling to the second residue. In the case where there is unusually high specificity (reactivity) at both of the two residues involved, the use of close to stoichiometric amounts of cross-linker assures that there should be little likelihood for monofunctional substitution at both residues to compete with the cross-linking reaction. From these considerations, it may be concluded that a high level of cross-linking is an indication that the coupled residues do reside for a significant time at separations conducive to cross-linking. This appears to be the case for cross-linking of SHl to Cys-522 in Sl and TSl with DBB at 25°C (19,20), but the present work suggests that this is not occurring at 4”C, since insignificant levels of cross-linking are observed at the latter temperature. The failure to observe cross-linking at 4°C cannot be attributed to a marked reduction in the first step of the reaction, since the changes in ATPase activities accompanying the initial stages of incubation at 4°C (Fig. 2) suggest that the same residue, presumably SHl, is modified at about the same rate at the lower temperature as observed at 25°C. The assignment that SHl is the thiol modified by DBB is based primarily on the previous work by Mornet et al. (19) and by Ue (20), who showed that SHl was linked to Cys-522 under these reaction conditions. We have also found that Sl, premodified at SH2 with NEM, can form the cross-link between the 21- and 50-kDa domains with DBB at 25’C (unpublished results) consistent with SHl being the thiol in the Sl-kDa segment, which is modified in the first step of the reaction. The failure to form significant levels of cross-linking from SHl to Cys-522 at 4°C cannot be attributed to reaction at other thiols in the 21-kDa segment, since the cross-link to Cys-522 does subsequently occur if the protein, premodified at SHl at 4”C, is warmed to 25°C (Fig. 4). This would indicate that the residual bromoacetyl group of DBB, monofunctionally attached to SH1 at 4°C is still free to react at 25°C. Thus, the present data indicate that, while the bimolecular reaction between DBB and SHl is not markedly sensitive to temperature in the

1 FLEXIBILITY

5

range of 4 to 25°C the subsequent intramolecular coupling to Cys-522 is extremely temperature dependent. A number of factors may be contributing to this effect. It is possible that at 4°C Cys-522 is in an environment that lowers its intrinsic reactivity, or alternatively, at the lower temperature Cys-522 falls only infrequently into the cross-linking range of DBB attached to SHl. The effect of nucleotide binding was previously shown to have no effect on the ability of DBB to cross-link SHl to Cys-522 at 25°C (19), a result which was confirmed in the present work. The present data, however, also indicate that the presence of Mg nucleotide is ineffective in promoting cross-linking between these thiols at 4°C with this reagent. It is unlikely that the failure to observe crosslinking with DBB at 4°C in the presence of nucleotide can be attributed to a decrease in the reactivity of Cys522. On the contrary, evidence does exist that Cys-522 is more reactive in the presence of MgADP since at 4°C cross-linking of SHl, monofunctionally labeled with pPDM, to Cys-522 requires nucleotide (21). We have also observed the same nucleotide requirement to produce this cross-link with 4,4’-difluoro-3,3’-dinitrodiphenylsulfone at 4°C (unpublished results). Therefore, the differences in the capacity to form cross-links between SHl and Cys522 with these reagents appear to reflect changes in the flexibility of the intervening segment of Sl, and consequently, in the spatial and temporal relationships between these two thiols induced by either temperature or nucleotide binding. The high levels of cross-linking observed with DBB at 25°C suggest that the coupling rate is markedly enhanced at the higher temperature. This could conceivably arise from either a change in the intrinsic reactivity of Cys522 or, alternatively, from temperature-induced changes in the conformation of Sl which allow SHl and Cys-522 to reside for longer durations at the cross-linking range of DBB. It should be noted that a temperature-dependent equilibrium between two states for nucleotide-bound Sl has been reported based on NMR (43) and fluorescence data (44). It is conceivable that the extents of cross-linking of Sl with DBB in the absence of nucleotide, observed at 4 and 25”C, reflect a change in the equilibrium distribution of two corresponding states. It is also of interest to comment on the inability of the DBB cross-link between SHl and Cys-522 to trap nucleotide, while the same cross-link produced with wider separation by pPDM does lead to a very large increase in the off-rate for MgADP (21). It is possible that the longer linkage produced by pPDM indirectly causes a steric barrier to retard the dissociation of nucleotide from the active site; studies are underway to delineate the factors which contribute to nucleotide trapping by cross-linking. REFERENCES 1. Morita, F. (1967) J. Bid. Chem. 242, 4501-4506. 2. Murphy, A. d. (1974) Arch. Biochem. Biophys. 163, 290-296.

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13,2014-2022. 42. Penefsky, H. S. (1977) J. Biol. Chem. 252,2891-2899. 43. Shriver, J. W., and Sykes, B. D. (1981) Biochemistry 20,6357-6362. 44. Aguirre, R., Lin, S-H., Gonsoulin, F., Wang, C-K., and Cheung, H. C. (1989) Biochemistry 28,799-807.