Proc. Nati Acad. Sci. USAVol. 79, pp. 958r-962, February 1982Biochemistry

Tryptic digestion as a probe of myosin S-1 conformation(nucleotide sensitivity/limited proteolysis/protein structure/actin-myosin interaction)

ANDRAS MUHLRAD* AND TETSU HOZUMIt*Department of Oral Biology, Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel; tDepartment of Physiology, Nagoya City UniversityMecal School, Nagoya, Japan; and Cardiovascular Research Institute 841-HSW, University of California, San Francisco, California 94143

Communicated by Manuel F. Morales, September 28, 1981

ABSTRACT One of the products of the limited tryptic hydrol-ysis of chymotryptic myosin subfragment 1 is the 27,000-daltonNH2-terminal fragment. This fragment is generated by two par-allel routes from either the 75,000- or 95,000-dalton peptide of theheavy chain: (i) through a 29,500-dalton precursor or (ii) directlywithout participation of a precursor. Lowering of pH and tem-perature and increasing of ionic strength inhibited route i diges-tion in comparison to route ii. MgATP and its derivatives in mil-limolar concentration substantially suppressed route i digestion.Suppression of route i digestion depended on the concentrationofMgATP. Itoccurred after a lag phase when the ratio ofMgATPto subfragment 1 concentrations was >0.5. In contrast, theMgATP-induced increase in tryptophan fluorescence of myosinsubfragment 1 appeared without a lag phase. The generation ofthe 27,000-dalton fragment by either route was not affected by F-actin; however, the suppression of route i digestion induced byMgADP was abolished when myosin subfragment 1 was in ternarycomplex with actin and MgADP. We conclude that the 27,000/50,000-dalton hinge region is a flexible domain of the myosin headand that conformation of this region is sensitive to the presenceof nucleotides and actin and to variations in ambient factors.

Myosin subfragment 1 (S-1) is the segment of the myosin mol-ecule containing the active site of ATPase and also the site atwhich actin interacts. Because the interactions ofnucleotide andactin occur on S-1, studies of changes in S-1 conformation ac-companying these interactions are highly significant. We havestudied these interactions by using a method based on prote-olysis. The reasoning is that access of the proteolytic enzymeto the S-l bond that will be cut is modulated by the conformationof the vulnerable S-1 region. Balint et al. first showed that, inlimited tryptic proteolysis of S-1, mainly three large fragments[27,000, 50,000, and 20,000 daltons (Dal)] were produced (1)and that the 27,000-Dal fragment contained the NH2 terminusof the myosin heavy chain (2). Mornet et al. (3) and Yamamotoand Sekine (4) showed that the 50,000/20,000-Dal cut was re-lated to the actin interaction because it was abolished in theacto-(S-1) complex and because the cut prevented the charac-teristic activation of myosin MgATPase by actin (but not theintrinsic MgATPase of S-1 alone).

Recently, we studied the proteolytic generation of the NH2-terminal 27,000-Dal fragment-i.e., the 27,000/50,000-Dal cut(5). We found that it was generated by two routes proceedingin parallel: in one case, a 29,500-Dal fragment was first pro-duced and then degraded to 27,000 Dal; in the other, a 27,000-Dal fragment was directly produced without any precursor.These studies suggested the existence of a flexible 2500-Dalhinge region between the stable 27,000- and 50,000-Dal frag-ments. This flexible region must be between the 27,000- and'50,000-Dal fragments because the 27,000-Dal fragment con-

tains the original NH2-terminus of the myosin heavy chain (2)and the 29,500-Dal fragment, which is the precursor of the27,000-Dal fragment, must contain the same region by definition.

Because the binding ofactin, ATP, and other polyphosphatesto the myosin head is known to produce conformational changesat identifiable sites (6-10) and because conformational changesinduced elsewhere by putting bulky substitutes at SH1 (11) orby organic solvents (12, 13) produce changes at the ATPase site,we decided to study whether these conformational changes ex-tend to the newly described 2500-Dal hinge region. In this pa-per we present evidence that ATP and its derivatives and var-ious reaction conditions affect the conformation ofthe 2500-Dalhinge region.

MATERIALS AND METHODSChemicals. TPCK-trypsin, soybean trypsin inhibitor, and a-

chymotrypsin were from Worthington. Pyruvate kinase andphosphoenolpyruvate (PEP) were from P-L Biochemicals. ATP,ADP, and adenosine 5'-[f3, y-imido]triphosphate were fromBoehringer Mannheim or Sigma. All other chemicals were ofreagent grade.

Proteins. Myosin and actin from back and leg muscles of rab-bits were prepared by the methods of Tonomura et al. (14) andSpudich and Watt (15), respectively. S-1 was prepared by diges-tion of myosin filaments with a-chymotrypsin (16) and purifiedby filtration through Sephacryl S-200 in 20 mM Tris HCl/0. 1mM dithiothreitol/0.1 mM NaN3, pH 7.3. S-1 and actin con-centrations were calculated from their absorbance by assumingabsorption coefficients A"~m 280 nm = 7.5 and Alm" 290 nm =6.3, respectively; in each case, a light-scattering correction wasapplied. We assumed that S-1 and actin monomer had molec-ular weights of 110,000 and 43,000, respectively.

Tryptic Digestion of S-1. S-1 was digested with 1% (wt/wt)of its concentration of TPCK-trypsin at 250C, if not stated oth-erwise. The digestion was started by addition of the trypsin. Atprescribed time intervals, aliquots were withdrawn and soybeantrypsin inhibitor was added to twice the enzyme concentration(wt/wt) to terminate digestion. Finally, an equal volume of 0.1M Tris-HCl, pH 6.8/2% NaDodSOJ2% 2-mercaptoethanoV21% (vol/vol) glycerol/0.002% bromphenol blue was added;the samples were then incubated in a boiling water bath for 3min. Mixtures were subjected to NaDodSOjpolyacrylamidegel electrophoresis on either 15% slab gels (17) or 7-18% poly-acrylamide gradient slab gels (18). After electrophoretic sepa-ration, a gel slice containing a Coomassie blue-stained band ofthe fragment of interest was extracted with 20% (vol/vol) pyr-idine in water for 5 hr at 370C, and the fragment concentrationwas taken to be proportional to the absorbance ofthe eluted dyeat 605 nm. Myosin fragments ofthis size give absorbances thatobey Beer's law (19). Alternatively the relative band intensities

Abbreviations: 51, myosin subfragment 1; Dal, daltons; PEP, phos-phoenolpyruvate; RLR, reactive lysine residue.

958

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Proc. NatL Acad. Sci. USA 79 (1982) 959

were assessed by scanning the gels in a Helena Quick Scan den-sitometer. Molecular weights of fragments were estimated bycomparing electrophoretic mobilities to those of authenticmarker proteins and of myosin light chains ofknown molecularweight.

Fluorescence Measurement of S-1. A Hitachi spectrofluo-rometer was used to.measure the ATP-induced fluorescenceemission increase of S-1 at room temperature. To S-1 at 1 mg/ml in a solution containing pyruvate kinase at 1 mg/ml, 5 mMPEP, 2 mM MgCl2, and 100 mM KHCO3 (pH 8.0), ATP wasadded. The solution was excited at 295 nm and.its 345 nmemission was recorded before and after addition of ATP.

Assay of Pyruvate Kinase Activity. This activity was mea-sured in a solution containing S-1 at 0.5 mg/ml, 0.5 mM ATP,5 mM PEP, and 100 mM KHCO3 (pH 8.0) by measuring thepyruvate concentration (20) after a 20-min incubation at 250C.The activity was measured also after incubation ofthe above testsolution with TPCK-trypsin (5 ug/ml) for 15 min at 25°C andaddition ofsoybean trypsin inhibitor (10 ,g/ml) to stop the tryp-tic digestion. No difference was observed in activity ofpyruvatekinase before and after digestion with trypsin.

RESULTSIn. order to assess the effect of ambient factors and nucleotideson the tryptic digestion of S-1, we developed a quantitativekinetic model. This model was based on our former observationsof the two routes of tryptic hydrolysis of S-1 (5) except that nowwe also took into account generating 29,500- or 27,000-Dal frag-ments directly from the 95,000-Dal intact heavy chain of S-1.We took into account only those kinetically significant frag-ments that were seen in the NaDodSO4 gels. All rate constantswere assumed to be pseudo first-order. The behavior of themodel was computer simulated and a number of theoreticalcurves were generated with different sets ofassumed rate con-stants. The experimental curves were compared to .the theo-retical ones, and the set of values giving the best fit was chosenby a least squares method.

In the absence of F-actin, k, is an order of magnitude greaterthan either k3 or k2, and flow over the lower branches is sup-pressed in both routes. Under these circumstances, k2 and k2are hard to measure accurately; however, k2 and k3 refer to thecleavage of the same bond, and so do k' and k , so we assumedthat k2 = k3 and k' = kC. According to this argument, k4 wouldalso be equal to k' or k' because it also is referred to the cuttingof the same bond, but in certain circumstances (see below) we

can show that it is different. The reason for this difference maybe that, although the bond cut in the k2, k', and k4 reactionsis the same, only the k4 reaction separates a short (2500 Dal)fragment. In the presence of F-actin, k, is almost zero (3, 4),flow along the upper branches is suppressed in both routes, andk3 and k' are not measurable.

Route i 95,000

Route ii 95,000

k3 50000*75,000 3

29,500 4 27,000-20,000

- 705000k4

29,500 27,000

k' 50,00075,000

k, / 27,00020,000

70,000k'2

27,000

Increasing NaCl from 30mM to 500 mM in the tryptic diges-tion medium decreased k3 without affecting kC. This effect in-creases the flow over route ii (production ofthe 27,000-Dal frag-ment without involvement of precursor) and could be char-acterized quantitatively by a decrease in kJk, (Fig. 1; Table 1).The k4 value also decreased at 500 mM NaCl, which sloweddown the disappearance of the 29,500-Dal precursor. Furtherincrease in NaCl up to 1 M did not affect the rate constants.Lowering the pH of the tryptic digestion from 7.2 to 6.2 de-creased kj, k3, k', and k4; however, k' was the least affectedamong the calculated rate constants. These changes also favoredroute ii over route i and also were characterized by a decreasein kJk' ratio (Table 1). Increase in pH from 7.2 to 8.2 essentiallydid not affect the rate constants. The flow in the two routes alsodepended on temperature. All rate constants decreased whenthe temperature ofdigestion was lowered from 25°C to 0°C, butk' was less affected than the others. The kJk' ratio decreasedand the digestion shifted in favor of route ii. The k3k' ratio wasvirtually unchanged when the temperature of the digestion in-

A

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FIG. 1. Effect of NaCl concentration on time course of tryptic digestion of 28 pM S-i1 in 20 mM Tris-HCl (pH 8.0) and NaCl as indicated. (A)Electrophoresis on 7-18% gradient acrylamide slab gel: Left, 0.03 M NaCI; Center, 0.5 M NaCl; Right, 1.0 M NaCl. (B) Distribution of 29,500-(e, o) and 27,000-Dal (A, A) fragments during the digestion in 30 mM NaCl (o, A) at 500 mM NaCl (o, A).

Biochemistry: Muhlrad and Hozumi

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960 Biochemistry: Muhlrad and Hozumi

Table 1. Effect of experimental conditions on the rate constantsof tryptic digestion of S-1

Rate constants, x 103 sec-'Variable ki k3 k' k4* k3/k3 k2 k2 k2/k'

30 mM NaCl 10.0 1.09 1.21 1.14 0.90 - - -

300 mM NaCl 11.3 0.69 1.41 0.30 0.49 - - -1000 mM NaCl 8.9 0.51 1.16 0.38 0.44 - - -

pH 6.2 7.3 0.52 0.76 0.52 0.68 - - -pH 7.2 10.1 0.89 0.97 0.83 0.92 - - -pH 8.2 10.6 0.95 1.00 0.82 0.95 - - -00C 1.9 0.09 0.22 0.21 0.41 - - -250C 11.2 1.07 1.30 1.02 0.82 - - -370C 19.2 2.32 2.63 3.27 0.88 - - -

No addition 9.5 0.95 1.12 0.37 0.84 - - -ADP 10.0 0.42 1.38. 0.27 0.31 - - -MgADP 10.1 0.21 1.50 0.30 0.14 - - -

No addition 7.8 0.79 0.91 0.33 0.87 - - -F-actin - - - 1.21 1.33 0.91

F-actin + MgADP 0.92 1.46 0.63

S-1/trypsin = 100:1 in 50mM NaCl/20 mMTris HCl, pH 8.0, at 250Cif not stated otherwise. S-1 was 28 ,M for studies of NaCl, pH, andtemperature effects and 10 ,uM for studies of nucleotide and actin ef-fects. Results shown are mean values from three consecutive experi-ments in each case. The variation in the values of individual rate con-stants was 5-15%.* The value of k4 was found to depend on the concentration of S-1 andtrypsin. This might be caused by the fact that the reactions werepseudo first order; when the concentration of the reactants waschanged, the observed rate constants changed as well.

creased from 250C to 370C (Table 1).Addition of 5 mM ATP to the medium of tryptic digestion

drastically decreased the generation of the 29,500-Dal precur-sor (Fig. 2). ATP analogs such as. ADP (Fig. 3; Table 1), aden-osine 5'-[3,y-imido]triphosphate, and pyrophosphate had asimilar effect. The suppression of the precursor production wasobserved both in the presence and absence of Mg2+; however,the presence ofMg2' enhanced the inhibition (Fig. 3 and Table1). MgADP greatly decreased kJk3 by decreasing k3 and in-creasing k3 and almost eliminated route i digestion but did notaffect k, (Table 1). Divalent cations (Mg2+ or Ca2+) or EDTAdid not affect the tryptic digestion of S-1 in the absence ofnucleotides.Low concentration of ATP ([ATP]/[S-1] = 0.5) only slightly

inhibited the production of the 29,500-Dal precursor (Fig. 4).When these experiments were carried out both in the presenceand in the absence of an ATP-regenerating system consisting

a b a b a b a b a bkDal

75 _, _m~ _.0m50 - Im _-~

29.5%27--.20-

3 5 10Time, min

15 30

FIG. 2. Effect of MgATP on the time course of tryptic digestion ofS-1. S-1 was 18 ,uM in 100 mM KHCO3/10 mM MgCl2, pH. 8.0. Elec-trophoresis was on a 7-18% gradient acrylamide slab gel. Lanes: a, noATP; b, with 5 mM ATP.

of PEP and pyruvate kinase, the result was the same. The de-pendence of suppression of route i digestion on ATP concen-tration was measured and compared to the ATP concentrationdependence of the nucleotide-induced fluorescence enhance-ment of S-1 (Fig. 5). The suppression of route i digestion (de-crease in the production of the 29,500-Dal fragment and in-crease in the direct generation of the 27,000-Dal fragment)became evident only when the [ATP]/[S-1] ratio was >0.5 andplateaued at [ATP]/[S-1] = 2. In contrast to suppression ofroute i, the fluorescence emission increase had no lag phase,even at the lowest ATP concentration, and it plateaued at alower [ATP]/[S-1] ratio than the change oftryptic susceptibilityof the 2500-Dal hinge region at the 27,000/50,000 site.

Addition of F-actin to the tryptic digest (Fig. 6) completelyinhibited k1 and slightly increased k2 and k' in comparison tok3 and k3 (the kinetically significant rate constants in the absenceof F-actin). However, k2/k2, which defines the ratio of the tworoutes in the presence of actin, was not affected (Table 1). Thepresence ofF-actin accelerated the digestion ofthe "alkali" lightchain LC1 to a 23,000-Dal fragment. The suppressing effect ofMgADP on the route i digestion (or on the kJk' ratio) wasgreatly decreased in the presence of F-actin (Table 1; Fig. 6).According to our calculations, which were based on the asso-ciation constants of Greene and Eisenberg (21), S-1 was dis-tributed among the species actin-(S-1)-ADP, (S-1)-ADP, ac-tin-(S-1), and (S-1) in the proportions 82%:12%:6%:0.04%,respectively.

Control

95q175

.g- .

50

29.527

..._ .___ _~~~~~~~~~~~~~~~~~~~~....20 _~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

ADP MgADP

0 2 5 10 20 30 0 2510203002 5 10 20 30Time, min

FIG. 3. Effect of ADP and MgADP on the time course of tryptic digestion of S-1. S-1 was 10 ALM in 30 mM KC1/4 mM MgC12/20 mM Tris HCl,pH 8.0. Electrophoresis was on a 7-18% gradient acrylamide slab gel. When present, ADP was 2 mM.

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Proc. Natl. Acad. Sci. USA 79 (1982)

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Proc. Natl. Acad. Sci. USA 79 (1982) 961

S-1 a b a b a b a b

SiS S i b~.., PK..J- - ,,,50w~~~~~~~~~~~~~~~~~o *60

LC-$~ 27

LC3 "** AW .1

5 7.5 10Time, min

15

FIG. 4. Effect of substochiometric concentration of MgATP on thetime course of tryptic digestion of S-i. S-1 was 18 uM in 100 mMKHCO3/10mM MgCl2/pyruvate kinase (PK) at 2 mg/ml/10mM PEP,pH 8.0. Electrophoresis was on 15% acrylamide slab gel. Lanes: a, no

ATP; b, ATP at 9 tLM.

DISCUSSIONIn a previous study (5), extended here, we showed that the27,000-Dal tryptic fragment of S-1 is formed by two routes. Inone case, a 29,500-Dal precursor was generated first and thendegraded to a 27,000-Dal fragment; in the other case, the27,000-Dal fragment was formed directly from larger heavychain fragments (95,000 or 75,000 Dal). In this work, it was

possible to describe our observations quantitatively by formu-lating a kinetic model of the cleavages and assigning numericalvalues to the first-order velocity constants. The relative materialflux over the two routes then depended on a single value, kJk'.The simplest interpretation of our evidence for two routes

of forming the 27,000-Dal fragment is that in the typical S-ipreparation there are actually two populations of heavy chainsdiffering in their 50,000/27,000 junctional regions. We cannotspeculate at present as to whether these different chains are intwo populations of homodimeric myosin molecules or in a sin-gle population of heterodimeric molecules [Tonomura's hy-pothesis (22)]. Because S-1 was in large molar excess over tryp-sin in all our experiments, we cannot tell whether the differencebetween populations refers to the binding of trypsin at theseregions or to the hydrolytic susceptibility to bound trypsin.Nevertheless, our observations are the equivalent of having a

kDal Control

95

75

_ _um

5043

29.527020

F-Actin

[ATP]/[S-1I

FIG. 5. Dependence of the generation of 29,500- and 27,000-Dalfragments and tryptophan fluorescence enhancement of S-1 on ATPconcentration. S-1 was 18 ,uM in 100 mM KHCO3/10 mM MgCl2/py-ruvate kinase at 2 mg/ml/10 mM PEP, pH 8.0. ATP was as indicatedon the abscissa; tryptic digestion was for 10 min. Electrophoresis wason a 15% acrylamide slab gel. Amounts of 29,500- and 27,000-Dal frag-ments were determined by extraction of the Coomassie blue-stainedbands with pyridine. Ratio of 29,500-Dal fragment to 27,000-Dal frag-ment after 10-min digestion without ATP was 0.5. This value wastaken as 100% on the left-hand ordinate, and the relative changes inthe concentrations of 27,000- (v) and 29,500-Dal (e) fragments at dif-ferent ATP concentrations were presented.

probe at a new location in S-1, and the probe is characterizedby the magnitude of kJk'.

The binding of ATP, nucleotide derivatives, and polyphos-phates such as pyrophosphate to S-i can be detected by singleprobes located at various places [e.g., a tryptophan (7, 8), theSH, thiol (9), and the reactive lysine residue (RLR) (10)] or bychanging the separation between pairs of probes at differentplaces [e.g., a tryptophan and the binding site of 8-anilino-1-naphthalene sulfonate (ANS) (6) and RLR and SH, (23)]. Herewe have shown that this binding is also detected at a new lo-cation, the 50,000/27,000 junction. It seems unlikely that allthese effects of ATP binding result from direct contact of thesite and the nucleotide. Various solvents known to disrupt struc-ture through their general affinities for hydrophobic regions ofproteins (12, 13) and also bulky substitutes placed at the SH,thiol (11) dramatically affect ATPase, so reciprocally one mightexpect events at the ATP binding site to be propagated over a

F-Actin +MgADP

0 2 5 10 20 30 0 2 5 10 20 30 0 2 5

Time, min10 20 30

FIG. 6. Time course of tryptic digestion of S-1 in the presence of F-actin and MgADP. S-1 was 10 gM with 56 AM F-actin (monomer concentration)in 4 mM MgCl2/2 mM ADP/30 mM KCI/20 mM Tris-HCl, pH 8.0. Electrophoresis was on a 7-18% gradient acrylamide slab gel.

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Biochemistry: Muhlrad and Hozumi

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962 Biochemistry: Muhlrad and Hozumi

sizable structural region. Our data showing effects at points both27,000 and 29,500 Dal "distant" from the NH2 terminus alsosuggest that nucleotide binding changes are propagatedthroughout a region. Thus, our observations strengthen theview that substantial conformational change results from ATPbinding and the speculation that this change may be part of anintersite signaling system (24).An interesting feature emerged from our study of the de-

pendence of the behavior of our probe on ATP concentration:the lag at low ATP concentration (Fig. 5). One interpretationof this phenomenon is that advanced by Inoue and Tonomura(25)-that ATP binding to one of two heavy chains postulated(the more responsive in terms of kJk3) is weaker than to theother.

Ambient factors (NaCl concentration, pH, temperature)were observed to.affect the ratio oftwo routes in the generationofthe 27,000-Dal fragment. It is unlikely that the changes werecaused by the direct effect of these factors on trypsin activitybecause not only the absolute values of the rate constants buttheir ratio (kJk3) was changed by varying the factors, so it seemsprobable that the 50,000/27,000 hinge region was directly af-fected. Because Na' and Cl- both affect the structure ofmyosin(26), it is possible that the decrease in kJk' as NaCl concen-tration increases results from opening up of a segment of thepolypeptide chain. The effect of increasing pH in the region6.2-7.2 and not elsewhere suggests some histidine involve-ment. The increased flow over route ii when temperature waslowered from 25TC to 0C could be related to a change in themechanism ofATP hydrolysis by myosin observed in the sametemperature range (27).We observed that actin prevents the MgADP-induced di-

version from route i to route ii. The most obvious way in whichactin binding to S-1 can antagonize a nucleotide effect on S-1is by causing the dissociation of nucleotide (28). In our exper-iment, however, with great excess ofADP over S-1, no signif-icant dissociation occurred. Therefore, actin induces a confor-mational change in the 50,000/27,000 hinge region whichantagonizes the effect of nucleotide over the same site. It isprobable that the actin-induced conformational change propa-gates over a distance in this case because actin is thought to bebound not to this region but to the 50,000- and 20,000-Dal frag-ments (29).We conclude that the 2500-Dal hinge region between 27,000

and 29,500 Dal from the NH2 terminus of the myosin heavychain is a specifically sensitive domain of the myosin head. Ad-dition of ATP or its derivatives or actin or variation of ambientfactors induces conformational changes in this region. Becausethe nucleotide binding site is located near this sensitive region(30), it is conceivable that it may have a specific role in myosinfunction.

The authors are indebted to Professor M. F. Morales for his continuedinterest in the work, for several helpful discussions during preparation

of the manuscript, and for his kind hospitality. This research was sup-ported by a grant from the Muscular Dystrophy Association and alsoby Grants HL-16683 (U.S. Public Health Service) and CI-8 (AmericanHeart Association). During the preparation ofthe manuscript A. M. wasthe recipient of a Career Investigator Visiting Scientist Award of theAmerican Heart Association.

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Proc. Nad Acad. Sci. USA 79 (1982)

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