Bioconjugate Chem. 1992, 3, 328-336
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Photoaffinity Labeling of Scallop Myosin with 2 4 (4-Azido-2-nitrophenyl)amino]ethylDiphosphate: Identification of an Active Site Arginine Analogous to Tryptophan-130 in Skeletal Muscle Myosin+ Bruce A. Kerwin and Ralph G. Yount' Department of Biochemistry and Biophysics and Department of Chemistry, Washington State University, Pullman, Washington 99164-4660.Received April 6,1992
The ADP photoaffinity analogue 2-[(4-azido-2-nitrophenyl)aminolethyl diphosphate (NANDP) was used to photolabel the ATP binding site of scallop myosin. Approximately 1 mol of NANDP per mol of myosin was trapped at the active site by complexation with vanadate and manganese. ADP, but not AMP, inhibited trapping of NANDP. The trapped NANDP photolabeled up to 37% of the myosin upon UV irradiatioin. Papain subfragment-1 prepared from the photolabeled myosin was digested with trypsin, and the major photolabeled tryptic peptides were isolated by reversed-phase HPLC. The amino acid sequence of the major labeled peptide was X-Leu-Pro-Ile-Tyr-Thr-Asp-Ser-Val-Ile-Ala-Lys, where X represents the photolabeled amino acid ArglZ8. Previously, Trp130 of rabbit skeletal muscle myosin has been shown to be photolabeled by NANDP [Okamoto, Y., and Yount, R. G. (1985)Proc. Natl. Acad. Sci. U.S.A.82,1575-15801.Scallop and rabbit skeletal muscle myosin display a high degree of sequence similarity in this region with ArglZ8in an equivalent position as TrpI3O. These results suggest that the composition of the purine binding site is analogous in both myosins and that Arg and Trp play a similar role in binding ATP, despite the marked differences of their side chains.
INTRODUCTION
Myosin I1 (or conventional myosin) from skeletal muscle is the most extensively studied of the many actin-based molecular motors. It is a multidomain protein composed of six subunits: two heavy chains and four light chains of two types. The C-terminal halves of two heavy chains form an a-helical coiled-coil rod which make up the backbone of the thick filament while the N-terminus of each heavy chain folds into a globular head responsible for binding actin and hydrolyzing ATP. Each head contains a pair of light chains (one regulatory and one essential) which appear to modify or regulate contraction (for a review see Adelstein & Eisenberg, 1980). In smooth muscle this occurs via specific phosphorylation of the regulatory light chains (Sobieszek & Small, 1977;Sherry et al., 1978;for a review see Trybus, 1991). A similar myosin-based regulation occurs in the striated muscle of scallops (Kenrick-Jones et al., 1970; Szent-Gyorgyi & Szentkiralyi, 1973). Here, Ca2+ binding to a regulatory complex composed of the two light chains and a portion of the heavy chain (Kwon et al., 1990)regulates ATPase activity and tension development. In both of these systems, although the basic components of the regulatory mechanisms have been identified, little is known concerning the regionsof the active site which may be involved with regulation. In the absence of X-ray crystallographic data, amino acids within the active site of myosin can be identified using photoaffinity labeling with analogues of ATP or ADP (Yount et al., 1992). This technique has been used to map the amino acids near the ribose ring binding site of skeletal (Mahmood et al., 1989;Kennedy et al., 1991)and smooth muscle myosin (Cole & Yount, 1990)and portions of the ~~
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* Author to whom correspondence should be addressed. + Supported by grants from NIH (DK05195)and MDA. B.A.K. is an MDA postdoctoral fellow.
purine binding site of Acanthamoeba myosin I1 (Atkinson et al., 1986)and smooth muscle myosin (Garabedian & Yount, 1990,1991). The region of the active site of skeletal muscle myosin which binds near C-2 of the adenine ring of ATP has been mapped using 2-azido-ATP (Yount et al., 1987;H. Kuwayama, unpublished results) and the photoreactive ADP analogue NANDP (Okamoto and Yount, 1985). NANDPl (Figure 1) has proven to be exceptionally useful in studying skeletal myosin due to its high photoincorporation and the stability of its photoadducts (Nakamaye et al., 1985). Trp'30of skeletal MHC was identified as the major photolabeled amino acid when either 2-azido-ATP or NANDP was used. Analysis of the amino acid sequence of scallop MHC (Nyitray et al., 1991) shows that ArglZ8replaces Trpl30 (skeletal). The introduction of a positively charged residue at the adenine binding site is surprising and may reflect a structural change that is important in myosin-based regulation. As a first step toward identifying amino acids in the purine binding region which may be involved in myosinbased regulation, we have used NANDP to photolabel the active site of scallop myosin. Here we demonstrate that NANTP is an excellent substrate for scallop myosin and that NANDP is stably trapped at the active site with Mn2+ and Vi. This step allowed the unbound NANDP to be separated from the myosin complex before irradiation. Approximately 35 5% of the trapped NANDP photoincorporated into the heavy chain with less than 2 % photoincorporation into the light chains. Arg128was identified as the major photolabeled amino acid, suggesting that ArglZ8(scallop) and Trp130 (skeletal) are in analogous Abbreviations used: high-performance liquid chromatography, HPLC; myosin heavy chain, MHC; myosin regulatory light chain, RLC; myosin subfragment 1,S1; 2-[(4-azido-2-nitropheny1)aminolethyl diphosphate, NANDP; phenylthiohydantoin, PTH; polyacrylamide gel electrophoresis, PAGE; sodium dodecy1 sulfate, SDS; trifluoroacetic acid, TFA; vanadate, Vi. 0 1992 American Chemical Society
Bioconjbp~teChem,, Vol. 3, No. 4, 1992 929
Photoaffinity Labellng of Scallop Myosin 0
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Figure 1. Schematic diagram of 2 4(4-azide2-nitrophenyl)ami-
nolethyl diphosphate, NANDP.
positions within the purine binding site. Thus, it is highly likely that this region of the ATP binding site is similar for the two myosins even though they contain amino acids with quite different properties. EXPERIMENTAL PROCEDURES
Purification of Scallop Myosin. Bay scallops (Aequipecten irradiana), preserved in 50% polyethylene glycol, were a generous gift from Dr. Andrew G. SzentGyBrgyi. Myosin was purified from the striated adductor muscle according to the method of Chantler and SzentGy6rgyi (1978) as modified by Szent-GyBrgyi (personal communication). Briefly, 10-50 g of scallops was homogenized in ice cold buffer in a Waring blender by pulsing three times for 10 s and three times for 15 s. The homogenate was placed on ice for 2-3 min between each pulse. The myofibrils were spun down at 7000g and resuspended with a Dounce homogenizer (A. H. Thomas) in 40 mM NaC1,5 mM NaPi, pH 7.0,3 mM MgC12,O.l mM EGTA, and 0.1 mM DTT. The pH of the myofibril solution was adjusted to 7.5 with 0.5 M Na2HP04 and fresh DTT was added to 1.0 mM. MgC12 and ATP were then added to 5 mM and NaCl was added to 0.6 M while the pH was kept constant at 7.5 using 0.5 M NazHP04. The solution was centrifuged at 16000gfor 20 min a t 4 "C. MgC12 and ATP were then added to the supernatant to 10mM, and either solid or saturated solution of ammonium sulfate was added to 45% saturation while the pH was kept constant at 7.0 using0.5 M Na2HP04. The suspension was centrifuged at l6000g for 20 min a t 4 "C to remove precipitated actin and tropomyosin. The decanted supernatant was brought to 65 % saturation by addition of solid ammonium sulfate. The myosin precipitate was centrifuged a t l6000g for 20 min; the pellet was resuspended in a minimal volume of 20 mM NaC1,lO mM NaPi, pH 6.5,O.l mM EGTA, 3 mM MgC12,O.l mM DTT, and 0.1 mM NaN3 and dialyzed overnight against 6 L of the same buffer. The resulting myosin filaments were either washed extensively with 30 mM KC1 and 20 mM Tris, pH 7.5 after centrifugation or were dialyzed into 30 mM KC1, 20 mM Tris, pH 7.5, and 0.1 mM DTT, and then washed extensively as above. This is referred to as filamentous myosin. Protein concentrations were determined using = 5.3, after dissolving the myosin filaments in 0.5 M K81 and 20 mM Tris, pH 8.0, at 4 "C. The myosin was used immediately for trapping of [@-32P]NANDP. ATPase and NANTPase Assays. CaATPase assays were done as described by Wells et al. (1979) except that the release of Pi was measured after 2 and 8 min. Synthesis of NANTP and [8-32P]NANDP. NANTP and [/3-32PlNANDPwere prepared as described by Nakamaye et al. (1985) and Okamoto and Yount (19851, respectively. Carrier-free 32P (10 mCi in 1 mL) waa purchased from New England Nuclear (Du Pont). Purity of each compound was determined by chromatography on silica gel TLC (60 F254, Merck) using isobutyric acid/ concentrated NHdOH/water 66:1:33 as a solvent. [rB-32PlNANDP with an initial specific activity of 144 000 cpml nmol was stored at -20 "C in methanol. Trapping of [PPINANDP on Scallop Myosin. NANDP was trapped on scallop myosin based on the
method of Goodno (1979). Filamentous scallop myosin was dissolved in 0.5 M KC1 and 20 mM Tris, pH 8.0, at a concentration of 4-5 mg/mL (300-600 mg of myosin was normally used in a photolabeling experiment). MnCl2 was added to a final concentration of 2 mM, [/3-32P]NANDP was added to a concentration of 2-4 times the number of ATP binding sites and Vi was added to a fiial concentration of 1mM. The solution was incubated for 25 min at 25 "C followed by a 3-4-h incubation on ice. Solid ammonium sulfate was added to 65% saturation and the precipitated myosin was collected by centrifugation a t l6000g for 20 min at 4 "C. The pellet was resuspended in a minimal volume of 30 mM KC1,20 mM Tris, pH 6.5, and 0.1 mM DTT, then dialyzed overnight against 6 L of the same buffer. The following day the dialyzate containing filamentous myosin was centrifuged at 7000g for 10 min at 4 "C and the resulting myosin*MnNANDP*Vipellet washed twice with low-salt buffer (30mM KC1 and 20 mM, Tris, pH 7.5). The final pellet was resuspended to a final concentration of approximately 5 mg/mL in the low-salt buffer (thiswas the final myosinMn-nucleotide-Vi complex used for photolysis). Photolysis of the M ~ O ~ ~ ~ M ~ [ ~ - ~ ~ P ] N A N D P Complex. Photolysis was based on the procedure of Cremo et al. (1991). Briefly, filamentous myosin (10-12 mL of 5 mg/mL solution) containing trapped [@-32PlNANDP was irradiated for 3 min on ice in a 9-cm Petri dish with a Pyrex cover using a 450-W medium-pressure Hg lamp (Hanovia, Ace Glass) a t a distance of approximately 9 cm. To determine the extent of photolabeling, aliquots of irradiated myosin (100-200 pL) were precipitated with 1mL of ice-cold 5% trichloroacetic acid and incubated for 2-3 h on ice. The precipitates were collected in a microcentrifuge tube, resuspended in 0.5 mL of 4% SDS/50 mM Tris-base, and incubated overnight a t room temperature prior to scintillation counting. SDS-PAGE Analysis. Myosin samples were analyzed by SDS-PAGE on 18X 20 cm 7-20 % gels accordingto the procedure of Laemmli (1970). Protein bands were visualized by staining with a solution of 0.05 % Coomassie Blue in45% methanol, 45% water, and 10% glacial acetic acid. Gel strips containing the bands of interest were excised, placed in 20-mL glass vials, and solubilized by heating for 2-4 h at 70 "C in 0.75 mL of 30% H202. After cooling, 18 mL of BCS scintillant (Amersham) was added and the radioactivity was determined in a scintillation counter. Scintillation Counting. Aliquots of samples (0.011.0 mL) were diluted to 4.5 mL with BCS scintillant and radioactively determined. Proteolytic Digestion of Photolabeled Myosin. Photolabeled myosin in low salt buffer was brought to 10 mM EDTA, pH 7.5, and incubated for 25 min on ice, then 5 min at 25 "C. Activated papain (Sigma, St, Louis, MO) (0.1 unita/mg of myosin) was added (Lowey et al., 1969) and the mixture incubated for 15 min at 25 "C. Papain was inactivated by addition of iodoacetic acid (0.1 M in 0.25 M Na2HP04) to 5 mM and the solution incubated an additional 5 min. MgC12 (1M) was added to 20 mM, and the reactions were incubated on ice for 20 min and then centrifuged a t 16000g for 10 min at 4 "C to remove aggregatedrods and undigested myosin. Solid ammonium sulfate was added to the supernatant to 70% saturation and the precipitated S1 collected by centrifugation as above. The S1 pellet was dissolved in 30 mM KC1 and 20 mM Tris, pH 7.5, and the concentration adjusted to 1-2 mg/mL based on = 8.0. CaCl2 (1M) was added to 1 mM, and urea (Ultrapure, ICN Biomedicals Inc., Cleveland, OH) added to 1M. The Sl(1-2 mg/mL) was digested N
330
Bioconjugate Chem., Voi. 3, No. 4, 1992
Table I. ATPase and NANTPase Activities of Scallop Myosin.
Kerwin and Yount
ATPase activity was a factor of 10greater than the NANTPase for unknown reasons. rmol Pi /min per mg N A N T ~ ~ ~ ~ / Trapping of [B-32P]NANDPat the Active Site of assay system NANTPase ATPase ATPase Scallop Myosin. Previous studies have shown that Co2+ can be used in conjunction with Vi to trap nucleoside Ca2+/Mg2+(high salt) 0.074 f 0.002 0.078 f 0.009 0.95 diphosphates at the ATP binding site of both skeletal and Ca2+(high salt) 0.19 f 0.03 1.9 * 0.1 0.1 CaZ+/Mg2+(low salt) 0.18 f 0.03 0.12 f 0.01 1.5 smooth muscle myosin (Grammer et al., 1988; Cole & CaZ+/Mgz+/F-actin Yount, 1990;Garabedian & Yount, 1990,1991). However, 0.29 f 0.03 1.4 pCa 4 (low salt) 0.39 f 0.03 when 2 mM Co2+was added to scallopmyosin under normal pCa 9 (low salt) 0.058 f 0.011 0.044 f 0.007 1.3 trapping conditions the myosin formed a white flocculent pCa 4/pCa 9 6.6 6.8 precipitate (B. Kerwin, unpublished observations). PreA freshly prepared myosin stock solution of 4.3 mg/mL was vious studies have shown that Mn2+and Ni2+can also be assayed at 25 "Cwith substrate concentrations of 2 mM. The highused to trap nucleoside diphosphate and quench the phosalt CaZ+assay used 0.46 pM myosin, 0.5 M KC1, 20 mM Tris, pH toreaction of Vi with myosin (Grammer et al., 1988). Both 7.5, and 5 mM CaC12. The high-salt Ca2+/Mg2+assay used 0.92 pM of these metals effectively trapped nucleoside diphosphates myosin, 0.5 M KCl, 20 mM Tris, pH 7.6,0.11 mM CaC12, and 3 mM MgC12. The low-salt Ca*+/Mg2+assay was similar but with 30 mM on scallop myosin without precipitating the myosin, KC1 replacing 0.5 M KCl. The CaZ+/Mgz+/F-actin pCa 4 assay although the time course of trapping with the Mn2+was contained 0.46 pM myosin, 4.6 pM F-actin, 30 mM KCl, 20 mM Tris, much faster than that with Ni2+ (data not shown). pH 7.6,0.11 mM CaC12, and 3 mM MgC12. The comparable pCa 9 Trapping of [/3-32PlNANDPand [l4C1ADP with Vi and assay contained in addition 3.41 mM EGTA. Each activity value is Mn2+on scallop myosin in high salt was complete within expressed as the average and standard deviation calculated from the 5 min (data not shown). Approximately 62% of the ATP data from six experiments. binding sites were complexed with [/3-32PlNANDPusing Mn2+and Vi with a parallel decrease in the CaATPase at room temperature with TPCK-treated trypsin (in 1 activity to a value approximately 30% of that for untrapped mM HC1) (Sigma, St. Louis, MO) at a 150-1:lOO ratio of myosin. Trapping was done in high salt because these trypsin:Sl. Three additions of trypsin were made at 0,30, conditions produced the highest percent trapping. Trapand 60 min, and the solution was allowed to digest ping with [14C]ADP under identical conditions blocked overnight. approximately 73 % of the ATP binding sites. We were Purification of Peptides by HPLC. HPLC separaunable to increase the percentage trapping of the tions were performed at room temperature using a myo~in.Mn[@3~PlNANDP.Vicomplex through either microprocessor-controlledAltex/Beckman dual-pump setsubsequent additions of [/3-32PlNANDPafter 10 min of up connected to a Beckman 165dual-wavelength detector. incubation or readdition of Mn2+,Vi, and [fl-32PlNANDP Initial separations were carried out on a semipreparative after purification of the trapped myosin (datanot shown). (7.0 X 250 mM) 300-A pore C8 reversed-phase column Control experiments with skeletal muscle myosin S1 and (Aquapore RP-300, Brownlee Labs) equilibrated in 0.11% [@-32PlNANDP under identical conditions also resulted trifluoroacetic acid (TFA), pH 2, at a flow rate of 2.0 mL/ in only 60 % trapping. Following trapping in high salt the min. All subsequent separations were carried out on an my~sin.Mn[@-~~P] NANDP-Vi complex was dialyzed into analytical (4.6 X 220 mm) 300-1\ pore C8 reversed-phase a low-salt buffer to produce myosin filaments. The final column (Aquapore RP-300, Brownlee Labs) at a flow rate percent trapping under these conditions was approxiof 1.0 mL/min. Prior to separation on the analytical NANDP'Vi mately 52 % and 54% for the my~sin.Mn[fl-~~P] column, samples were acidified with TFA to approximately complex and the myosin.Mn[l4C1ADP.Vi complex, repH 2 (by litmus), and 0-mercaptoethanol was added to 10 spectively, with concomitant increases in the CaATPase mM. The samples were then passed through a 0.45" activities. Nylon-66 filter syringe (Rainin) before loading via a 7.5mL sample loop. Volumesgreater than 6.0 mL were loaded Further evidence that [/3-32PlNANDPwas trapped at on the column by multiple loadings. Following each the ATP binding site of scallop myosin came from trapping separation samples which were radioactive and absorbed inhibition studies. Figure 2 shows that ADP, but not AMP, inhibited the trapping of [@-32PlNANDPon scallop at 320 nm were concentrated to one-half their volume on myosin. Inhibition occurred in a 1:l manner (Figure 2, a SpeedVac (Savant, Farmingdale, NY) to remove acetonitrile and then diluted with the appropriate equiliinset) such that 50 % inhibition of trapping occurred when brating buffer and brought to 1.0 mM with @-mercapthe concentration of ADP equaled that of [fl-32Pl NANDP. toethanol before further HPLC chromatographic sepThese resultsdemonstrate that NANDP and ADP compete arations. for the same binding site with nearly identical binding constants. Photoincorporation of [B-32P]NANDPinto Scallop RESULTS Myosin. As seen in Figure 3 maximum photoincorporation of [/3-32P]NANDP into myosin filaments occurred NANTP as a Substrate for Scallop Myosin. The within 2-3 min of irradiation. Approximately 37 % of the ability of scallop myosin to utilize NANTP as a substrate total trapped [fl-32PlNANDPwas covalently incorporated was investigated using a variety of assay conditions. As as determined by trichloroacetic acid precipitation of the shown in Table I the ATPase and NANTPase activities protein. In order to determine if the photoincorporated of scallop myosin differ by less than a factor of 2 for a number of assay conditions. Comparison of the degree of adducts were stable in the low-saltbuffer and to determine if nonspecific dark reactions were occurring (Staros & Baycalcium activation for the actin activated NANTPase and ATPase activities at pCa 4 and 9 in the presence of Mg2+ ley, 19841,a photolyzed sample of [/3-32PlNANDP-trapped demonstrated no significant difference between the two scallop myosin was stored on ice in the dark and aliquots activities, suggesting that the rate-limiting steps for hywere precipitated with trichloroacetic acid to determine drolysis of these two substrates is the same with or without the extent of covalent modification. Figure 4 shows that Ca2+. The only activity which differed significantly were the covalently incorporated [/3-32PlNANDPwas stable over a period of 5 h. In addition, precipitable counts did those measured in 5 mM Ca2+and high salt. Here the
Bioconlugete Chem., Voi. 3, No. 4, 1992 331
Photoafflnlty Labeling of Scallop Myosin
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Figure 2. ADP-dependent inhibition of [j3-32P]NANDPtrapping. Aliquots (1 mL) of freshly prepared scallop myosin in 0.5 M KCl, 20 mM Tris, pH 8.0, [@-32P]NANDPat a 2 times excess over active sites and either ADP or AMP were added to 2 mM MnC12 and 1 mM Vi and incubated for 20 min at 25 “C. The reactions were quenched by the addition of ATP to 25 times excess over the [j3J2P]NANDP and excess nucleotides were removed by centrifugation through columns of Sephadex G-50 (Penefsky, 1977). Radioactivity was determined by counting duplicate 200-pL aliquots as described under Experimental Procedures. Protein concentratioins were measured by the absorbance at 280 nm. Increasing concentrations of ADP (0) or AMP (+) were added to each reaction mixture. The percent NANDP trapped (-62%) was normalized to 100%.
Figure 4. Stability of photoincorporated [j3-32P]NANDP. Scallop myosin was trapped with [fl-32P]NANDP,transferred into low-salt buffer, and 10 mL was photolyzed as described under Experimental Procedures. Following photolysis three 150-pL aliquots were removed at each of the indicated times and precipitated with trichloroacetic acid, and the amount of radioactivity present was determined as described under Experimental Procedures. 20
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Figure 3. Photoincorporation of [j3-32P]NANDP. Scallop myosin was trapped with [j3-32P]NANDPand transferred into the low-salt buffer as described under Experimental Procedures. Trapped myosin (10 mL, 54% trapped) was irradiated as described under Experimental Procedures. Three 100-pL aliquota were removed at each of the indicated times and precipitated with trichloroacetic acid (ExperimentalProcedures). The Y-axis is defined as the percent of the originally trapped myosin that became covalently labeled with [j3-32P]NANDP.
not increase over time, indicating that dark reactions were not occurring. Furthermore, if the active site of unmodified scallop myosin was blocked with the MnADP-Vi complex followed by addition of free [/3-32PlNANDP (equal to one-half of the total number of ATP binding sites) immediately prior to irradiation, photoincorporation appeared to be nonspecific. As seen in Figure 5A no distinct radiolabeled peptides were evident following photolysis in the presence of uncomplexed [/3-32PlNANDP. When the [8-32PlNANDPwas trapped at the active site with Vi and Mn2+,though, only a discrete number of peptides were labeled (Figure 5B). Taken together these results indicate that [j3-32PlNANDPmust be trapped at the ATP binding site for specific photoincorporation to occur and that long-lived reactive intermediates were not labeling the scallop myosin following irradiation.
60
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Figure 5. Photolabeling of ADP-blocked scallop myosin with free [8-32P]NANDP. Scallop myosin (120 mg) was inactivated by complexation with ADP, Mn2+, and Vi and transferred into low-salt buffer as described for trapping of [j3-32PlNANDP under Experimental Procedures. To 10 mL of MnADPmVi trapped myosin (5.1 mg/mL), [j3-32P]NANDPwas added equal to onehalf the total number of ATP binding sites. The sample was photolyzed for 3 min, then S1 was prepared and digested with trypsin (1:100,trypsin:myosin) as described under Experimental Procedures. The tryptic digest was filtered, chromatographed, and analyzed on a RP-300 semipreparative HPLC column as described in the legend for Figure 7. Aliquota (1.0 mL) were analyzed for 32P as described under Experimental Procedures. Panel A, radioactive profile of HPLC separation of tryptic peptides from ADP-blocked scallop myosin photolabeled with free [@-32P]NANDP.Panel B, radioactiveprofile of HPLC separation of tryptic peptides from S C ~ O myosin P photolabeledwith trapped [fl-32P]NANDP(see Figure 7). Arrows indicate the beginning of the elution gradient as described in the legend to Figure 7. The two initial peaks in “A” and three early peaks in “B” resulted from loading the tryptic digestion mixture of S1 in two steps and three steps, respectively,and presumably is free photolyzed [32PlNANDP.
The photoincorporation was limited to the S1 heavy chain. As seen in Figure 6, less than 2 7% of the total photoincorporated counts were in the light chains or the rod. SDS-PAGE analysis of a limited tryptic digestion of photolabeled Sl indicated that the photolabel was present in the NH2-terminal75 and 63 kDa tryptic fragments (data not shown). These results indicate that [/3-32PlNANDP
332 B~Or&p?t8 Chem., Vol. 3,No. 4, 1992
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