Mapping of ATP-dependent trypsin-sensitive sites on the beta chain of outer-arm dynein from sea urchin sperm flagella.

The beta chain of sea urchin outer-arm dynein showed a peculiar tryptic digestion pattern in the presence of ATP (or ADP) plus Vi. Examination of the molecular mass of the products formed by photocleavage of tryptic fragments indicated that the trypsin-sensitive sites on the 165-kDa ATP-binding polypeptide in the presence of ATP and Vi are located 15 kDa apart from its amino-terminus, 2 kDa apart from its carboxy-terminus, and near the middle portion between the adenine- and gamma-Pi-binding sites. On the other hand, the carboxy-terminal region of the beta chain, the 135-kDa polypeptide, was cleaved into a 96-kDa polypeptide by tryptic digestion in the presence of ATP and Vi. Peptide mapping of 135-kDa, 96-kDa, and carboxy-terminally truncated polypeptides of the 135-kDa polypeptide revealed that the 96-kDa region is located at the amino-terminal portion of the 135-kDa region. These results indicate that the changes of trypsin susceptibility of dynien beta chain caused by binding ADP and Vi occur not in local region but over an extensive region on the beta chain.     

Dynamic conformational changes of 21 S dynein ATPase coupled with ATP hydrolysis revealed by proteolytic digestion

Conformational changes of 21 S dynein ATPase from sea urchin sperm flagella were examined by tryptic digestion under physiological conditions. In the presence of 2 mM ATP or ADP plus 100 microM inorganic vanadate (Vi), dynein heavy chains were digested by trypsin into quite different polypeptides from those obtained in other cases (no addition, 2 mM ATP, 4 mM adenosine 5'-(beta,gamma-imido)triphosphate, 4 mM adenosine 5'-(beta,gamma-methylene)triphosphate, 2 mM ADP, 100 microM Vi). In the presence of 4 mM adenosine 5'-O-(3-thiotriphosphate), however, the digestion pattern was similar to that in the presence of ATP (ADP) and Vi, to a certain extent. In all conditions other than the presence of ATP (ADP) and Vi, 165- and 135-kDa polypeptides were the main products, whereas in the presence of ATP (ADP) and Vi, 200-, 150/148-, and 105/96-kDa peptides were produced and 320-kDa peptide became rather inaccessible to trypsin. The latter digestion pattern was not observed in the absence of divalent cations. These results suggest that, in the ATP hydrolysis cycle, dynein changes its conformation remarkably in the dynein-ADP-Pi state, which is presumably responsible for force generation.     

Two states of the conformation of 21S outer arm dynein coupled with ATP hydrolysis

Tryptic digestion of 21S outer arm dynein from sea urchin sperm flagella in the presence of ATP (or ADP) and vanadate produced quite different polypeptides from those obtained in the absence of ATP (ADP) and/or vanadate (Inaba and Mohri (1989) J. Biol. Chem. 264, 8384-8388). The 21S dynein heavy chains were consistently digested into 165- and 135-kDa polypeptides in the absence of both ATP (ADP) and vanadate. In the presence of 2 mM ADP and 100 microM vanadate, 300-kDa polypeptide, which appeared to be a precursor of 165- and 135-kDa polypeptides, became less accessible to trypsin, and 165- and 135-kDa polypeptides were digested into 150-/148-kDa and 96-kDa polypeptides, respectively. Quantitative analysis of the degradation of 165- and 135-kDa polypeptides showed that the conformations of these polypeptides change remarkably in the presence of ATP (ADP) and vanadate, and slightly in the presence of ATP gamma S. Photoaffinity labeling with 8-azidoadenosine 5'-triphosphate and vanadate-mediated photocleavage of dynein heavy chains revealed that both adenine- and gamma-Pi-binding sites were located on 165- and 150-/148-kDa polypeptides, but not on 135-kDa polypeptide. These results suggest that the conformational change occurring in the 165-kDa region on binding ATP spreads to the 135-kDa region and causes the conformational change of the 135-kDa region.     

Proteolytic analysis of domain structure in the beta heavy chain of dynein from sea urchin sperm flagella.

The stability of different regions of the beta heavy chain of dynein has been investigated by examining the perturbing effects of methanol, temperature, salt, and nucleotide on the pattern of tryptic digestion. In standard low-salt medium, tryptic proteolysis cleaves the beta heavy chain into three principal polypeptides of 130, 215, and 110 kDa, with the 215-kDa central peptide containing the ATP binding site as well as the vanadate and iron photocleavage sites (Mocz, G., Tang, W.-J. Y., & Gibbons, I. R. (1988) J. Cell Biol. 106, 1607-1614). The 130-kDa peptide is the most stable, and its susceptibility to trypsin appears unaffected by methanol concentrations up to 25% or temperatures up to 45 degrees C, although a 5-kDa region at one end is lost in the presence of salt (greater than 20 mM NaCl). The 215-kDa tryptic peptide contains two regions of different stability: its 123-kDa portion adjoining the 130-kDa peptide is destabilized by mild heat (37 degrees C) or by 25% methanol and becomes digested away to leave the more stable region of 92 kDa that is located toward the 110-kDa peptide and retains the V1 photocleavage site and most of the ATP binding site. The 110-kDa peptide is the least stable and at 37 degrees C, or in the presence of low concentrations of methanol or salt, it rapidly digested to small peptides. The presence of ATP during digestion of the beta heavy chain retards the formation of the 130- and 215-kDa peptides and also protects the 215-kDa peptide from further digestion at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific cleavage of dynein heavy chains by ultraviolet irradiation in the presence of ATP and vanadate

Irradiation of soluble dynein 1 from sea urchin sperm flagella at 254 nm in the presence of 50 microM ATP and 100 microM inorganic vanadate (Vi) cleaves the alpha and beta heavy chains into approximately equal quantities of two polypeptides of Mr 228,000 and 200,000, with a conversion efficiency of about 63%. A similar cleavage occurs in the presence of Vi and either ADP or 8-azidoadenosine 5'-triphosphate (8-N3ATP); in the latter case, 8-N3ATP becomes covalently bound principally to the Mr 228,000 polypeptide. No detectable amount of these fragments is formed if either the Vi or the nucleotide is omitted or in the presence of Vi and 50 microM AMP. These results emphasize the basic similarity of the two ATPases associated with the alpha and beta heavy chain subunits of dynein 1 and give a mean Mr of 428,000 for the intact heavy chains.     

Photosensitized cleavage of dynein heavy chains. Cleavage at the V2 site by irradiation at 365 NM in the presence of oligovanadate

Irradiation of the outer-arm dynein ATPase from sea urchin sperm flagella at 365 nm in the presence of 50-200 microM vanadate (Vi) and 1 mM manganese acetate, in the absence of ATP, cleaves the alpha and beta heavy chains at a specific site, termed the V2 site, to form discrete peptides of Mr approximately 260,000 and 170,000 from the alpha chain and of Mr approximately 255,000 and 175,000 from the beta chain, with a yield of 80%. This cleavage at the V2 site is not correlated with any direct effect on the dynein ATPase activity. In the presence of 100 microM Vi, the half-times for cleavage of the alpha and beta chains are about 12 and 50 min, respectively. The rate of heavy chain cleavage shows a sigmoidal dependence upon Vi concentration, with half-maximal rate occurring at 58 +/- 7 microM, consistent with the chromophore responsible for cleavage being tri-vanadate. Addition of 10 microM ATP or ADP, or of 100 microM CTP or UTP, to the irradiation medium inhibits cleavage at the V2 site, and results in a slow cleavage occurring at the V1 site described previously. The peptides produced by sequential cleavage at the V2 and then the V1 sites indicate that the sites are separated by about 100,000 Da along the length of each heavy chain. Photoaffinity labeling with [alpha-32P] 8-azidoadenosine 5'-triphosphate (8-N3ATP) gives specific incorporation of 32P into both the Mr 255,000 and 175,000 peptides of the beta chain but into only the Mr 260,000 peptide of the alpha chain. These results suggest that V2 cleavage occurs on a loop of the heavy chain that forms part of the ATP-binding site, close to the locus of 8-N3ATP attachment.     

Iron(III)-mediated photolysis of outer arm dynein ATPase from sea urchin sperm flagella

Irradiation of outer arm dynein ATPase from sea urchin sperm tail flagella at 365-410 nm in the presence of Fe(III)-gluconate complex and ATP produces photolytic cleavage at two distinct sites on the beta heavy chain, located approximately 250 and approximately 230 kDa from its amino terminus. The former cut is close to or identical with the V1 site of the vanadate-mediated photocleavage (Gibbons, I.R., Lee-Eiford, A., Mocz, G., Phillipson, C. A., Tang, W.-J.Y., and Gibbons, B.H. (1987) J. Biol. Chem. 262, 2780-2786. The rate of photolysis shows a hyperbolic dependence on Fe(III)-gluconate concentration with half-maximal rate occurring at 23 microM at pH 6.3. In the presence of 0.1-0.5 mM Fe(III)-gluconate-ATP, approximately 58% of the beta chain becomes cleaved with a half-time of about 34 s; the remainder of the beta chain and almost all of the alpha chain are resistant to cleavage. This photolytic cleavage of the beta chain is accompanied by an approximately parallel loss of the dynein latent ATPase activity, whereas the Triton-activated ATPase is lost to a somewhat greater extent. Mg2+ concentrations above approximately 3 mM inhibit photolysis. Substitution of ADP for ATP changes the pattern of cleavage so that both the alpha and beta heavy chain undergo scission but at the 250-kDa site only. AMP, adenyl-5'-yl imidodiphosphate and Fe(II) do not support cleavage at either site. Trivalent rhodium-ATP complexes, as models of MgATP, can also catalyze photolysis of the beta chain at the 250-kDa site. These results suggest that photolysis results from the activation of an Fe(III)-ATP complex bound to the hydrolytic ATP binding site of the beta chain and that both Fe(III) cleavage sites are located close to the nucleotide binding site in the tertiary folding of the beta heavy chain. The cleavage reaction possibly involves initial photoreduction of Fe(III) bound at the Mg2+ binding site in the dynein.Fe.ATP complex, followed by covalent modification of an amino acid side chain that leads to eventual peptide scission.     

DNA and nucleotide-induced conformational changes in the Escherichia coli Rep and helicase II (UvrD) proteins

The domain structures of the Escherichia coli Rep and Helicase II proteins and their ligand-dependent conformational changes have been examined by monitoring the sensitivity of these helicases to proteolysis by trypsin and chymotrypsin. Limited treatment of unliganded Rep protein (73 kDa) with trypsin results in cleavage at a single site in its carboxyl-terminal region, producing a 68-kDa polypeptide which is stabilized in the presence of ATP, ADP, or adenosine 5'-O-thiotriphosphate) (ATP gamma S). The purified 68-kDa Rep tryptic polypeptide retains single-stranded (ss) DNA binding, DNA unwinding (helicase), and full ATPase activities. When bound to ssDNA, the Rep protein can be cleaved by trypsin at an additional site in its carboxyl-terminal region, producing a 58-kDa polypeptide that also retains ssDNA binding and ATPase activities. This 58-kDa Rep tryptic polypeptide can also be produced by further tryptic treatment of the 68-kDa Rep tryptic polypeptide when the latter is bound to ssDNA. This 58-kDa polypeptide displays a lower affinity for ssDNA indicating that the 10-kDa carboxyl-terminal peptide facilitates Rep protein binding to ssDNA. The 58-kDa Rep tryptic polypeptide is also stabilized in the presence of nucleotides. Based on these and previous studies that showed that the 68-kDa Rep tryptic polypeptide cannot support DNA replication in a system that is dependent upon the phi X174 cis-A protein (Arai, N. & Kornberg, A. (1981) J. Biol. Chem. 256, 5294-5298), we conclude that the carboxyl-terminal end (approximately 5 kDa) of the Rep protein is not required for its helicase or ATPase activities. However, we suggest that this region of the Rep protein is important for its interactions with the phi X174 cis-A protein. Limited treatment of unliganded Helicase II protein (82 kDa) with chymotrypsin results in cleavage after Tyr254, producing a 29-kDa amino-terminal polypeptide and a 53-kDa carboxyl-terminal polypeptide, which remain associated under nondenaturing conditions. This chymotrypsin cleavage reduces the ssDNA binding activity and eliminates the ssDNA-dependent ATPase and helicase activities of the Helicase II protein. The binding of ATP, ADP, ATP gamma S, and/or DNA to Helicase II protein results in protection of this site (Tyr254) from cleavage by chymotrypsin. Limited treatment of Helicase II protein with trypsin results in cleavage near its carboxyl-terminal end producing two polypeptides with apparent Mr = 72,000, in a manner similar to that observed with the Rep protein; these polypeptides are also stabilized by binding ATP or single-stranded DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Photosensitized cleavage of dynein heavy chains. Cleavage at the "V1 site" by irradiation at 365 nm in the presence of ATP and vanadate

Irradiation of soluble dynein 1 from sea urchin sperm flagella at 365 nm in the presence of MgATP and 0.05-50 microM vanadate (Vi) cleaves the alpha and beta heavy chains (Mr 428,000) at their V1 sites to give peptides of Mr 228,000 and 200,000, without the nonspecific side effects produced by irradiation at 254 nm as described earlier (Lee-Eiford, A., Ow, R. A., and Gibbons, I. R. (1986) J. Biol. Chem. 261, 2337-2342). The decrease in intact heavy chain material is biphasic; in 10 microM Vi, approximately 80% occurs with a half-time of 7 min and the remainder with a half-time of about 90 min, and the yield of cleavage peptides is better than 90%. Loss of dynein ATPase activity appears to be a direct result of the cleavage process and is not significantly affected by the presence of up to 0.1 M cysteamine (CA, 60-23-1) or 2-aminoethyl carbamimidothioic acid dihydrobromide (CA, 56-10-0) as free radical trapping agents. The concentration of Vi required for 50% maximal initial cleavage rate is 4.5 microM, while that for 50% ATPase inhibition is 0.8 microM, both in a 0.6 M NaCl medium. In the presence of 20 microM Vi, CTP and UTP support cleavage at about half the rate of ATP, whereas GTP and ITP support cleavage only if the Vi concentration is raised to about 200 microM. Substitution of any of the transition metal cations Cr2+, Mn2+, Fe2+, or Co2+ for the usual Mg2+ suppresses the photocleavage, presumably by quenching the excited chromophore prior to scission of the heavy chain. The photocleaved dynein 1 binds to dynein-depleted flagella similarly to intact dynein 1, but upon reactivation of the flagella with 1 mM ATP their motility is partially inhibited, rather than being augmented as with intact dynein. These results indicate that Vi acts as a photosensitizing catalyst and suggest that the cleavage proceeds through excitation of Vi bound to dynein at the hydrolytic ATP binding site on each heavy chain, probably in a dynein X MgADP X Vi complex. The exquisite specificity of Vi-sensitized photocleavage will aid the peptide mapping of dynein heavy chains and may be of broader use in studies of protein structure.     

The Mr 78,000 intermediate chain of Chlamydomonas outer arm dynein interacts with alpha-tubulin in situ

We have used the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to examine protein-protein associations within purified outer arm dynein and axonemes from Chlamydomonas flagella. When axonemes were treated with 0.5-1 mM EDC in either the presence or absence of ATP/vanadate, a polypeptide band of Mr 127,000 recognized by monoclonal antibody 1878A (specific for the Mr 78,000 intermediate chain (IC78) of outer arm dynein) was generated. This conjugate was not obtained when purified dynein was treated with EDC. Further immunological analysis demonstrated that this complex also contained alpha- (but not beta-) tubulin. These results indicate that IC78 interacts with alpha-tubulin in situ in an ATP-insensitive manner. Identification of this interface between dynein and tubulin suggests that IC78, which probably is located at the base of the dynein particle (King, S. M., and Witman, G. B. (1990) J. Biol. Chem. 265, 19807-19811), contributes to the structural attachment of the dynein arms to the A-tubules of the outer doublet microtubules. Analysis of the cross-linked products from the purified dynein revealed several additional interactions involving the intermediate chains; these adducts provide further evidence for an intermediate chain/light chain complex within dynein and confirm that IC78 and IC69 associate directly.     

The motile beta/IC1 subunit of sea urchin sperm outer arm dynein does not form a rigor bond

We used in vitro translocation and cosedimentation assays to study the microtubule binding properties of sea urchin sperm outer arm dynein and its beta/IC1 subunit. Microtubules glided on glass-absorbed sea urchin dynein for a period of time directly proportional to the initial MgATP2- concentration and then detached when 70-95% of the MgATP2- was hydrolyzed. Detachment resulted from MgATP2- depletion, because (a) perfusion with fresh buffer containing MgATP2- reconstituted binding and gliding, (b) microtubules glided many minutes with an ATP-regenerating system at ATP concentrations which alone supported gliding for only 1-2 min, and (c) microtubules detached upon total hydrolysis of ATP by an ATP-removal system. The products of ATP hydrolysis antagonized binding and gliding; as little as a threefold excess of ADP/Pi over ATP resulted in complete loss of microtubule binding and translocation by the beta/IC1 subunit. In contrast to the situation with sea urchin dynein, microtubules ceased gliding but remained bound to glass-absorbed Tetrahymena outer arm dynein when MgATP2- was exhausted. Cosedimentation assays showed that Tetrahymena outer arm dynein sedimented with microtubules in an ATP-sensitive manner, as previously reported (Porter, M.E., and K. A. Johnson. J. Biol. Chem. 258: 6575-6581). However, the beta/IC1 subunit of sea urchin dynein did not cosediment with microtubules in the absence of ATP. Thus, this subunit, while capable of generating motility, lacks both structural and rigor-type microtubule binding.     

ADP-dependent microtubule translocation by flagellar inner-arm dyneins

Previous studies have shown that the motility of flagellar and ciliary axonemes in many organisms are influenced by the concentration of both ATP and ADP. Detergent-extracted cell models of Chlamydomonas oda1, a mutant lacking flagellar outer-arm dynein, displayed slightly lower flagellar beating frequencies when reactivated with ATP in the presence of an ATP-regenerating system, composed of creatine phosphate and creatine phosphokinase, than when reactivated with ATP alone. Thus, presence of a low concentration of ADP may somehow stimulate axonemal motility. To see if this motility stimulation is due to a direct effect on dynein, we analyzed the effect of ADP on the in vitro microtubule translocation caused by isolated inner-arm dyneins in the presence of ATP. Of the seven inner-arm dyneins (species a-g) fractionated by ion-exchange chromatography, most species translocated microtubules at faster speed in the presence of 0.1 mM ATP and 0.1 mM ADP than in the presence of 0.1 mM ATP alone. Most notably, species a and e did not translocate microtubules at all in the presence of the ATP-regenerating system, indicating that a trace amount of ADP is necessary for their motility. This regulation may be effected through binding of ADP to some of the four nucleotide binding sites in each dynein heavy chain.     

A map of photolytic and tryptic cleavage sites on the beta heavy chain of dynein ATPase from sea urchin sperm flagella

NH2-terminal analysis of the alpha and beta heavy chain polypeptides (Mr greater than 400,000) from the outer arm dynein of sea urchin sperm flagella, compared with that of the 230,000- and 200,000-Mr peptides formed upon photocleavage of dynein by irradiation at 365 nm in the presence of vanadate and ATP, shows that the NH2 termini of the intact chains are acetylated and that the 230,000- and 200,000 Mr peptides constitute the amino- and carboxy-terminal portions of the heavy chains, respectively. Tryptic digestion of the beta heavy chain is known to separate it into two particles, termed fragments A and B, that sediment at 12S and 6S (Ow, R. A., W.-J. Y. Tang, G. Mocz, and I. R. Gibbons, 1987. J. Biol. Chem. 262:3409-3414). Immunoblots against monoclonal antibodies specific for epitopes on the beta heavy chain, used in conjunction with photoaffinity labeling, show that the ATPase-containing fragment A is derived from the amino-terminal region of the beta chain, with the two photolytic sites thought to be associated with the purine-binding and the gamma-phosphate-binding areas of the ATP-binding site spanning an approximately 100,000 Mr region near the middle of the intact beta chain. Fragment B is derived from the complementary carboxy-terminal region of the beta chain.     

The photoaffinity probe 8-azidoadenosine 5'-triphosphate selectively labels the heavy chain of Chlamydomonas 12 S dynein

Chlamydomonas 12 S dynein, which makes up part of the outer arm of the flagellar axoneme, consists of three polypeptides of 330,000, 22,000, and 18,000 daltons. We have used 8-azidoadenosine 5'-triphosphate (8-N3ATP), a photoaffinity analog of ATP, to investigate which of the dynein polypeptides contains the site of ATP hydrolysis. 8-N3ATP is a competitive inhibitor of the hydrolysis of ATP by 12 S dynein and is hydrolyzed by 12 S dynein in an ATP- and vanadate-sensitive fashion, indicating that it binds to the 12 S dynein hydrolytic site in the same way as ATP. When dynein was incubated with [gamma-32P]- or [alpha-32P]8-N3ATP in the presence of UV light to activate the azido moiety, the analog was incorporated into 12 S dynein's heavy polypeptide chain, but not its light chains. The incorporation was UV-dependent, was blocked by addition of ATP or vanadate plus ADP to the reaction mixture, and did not occur in heat-denatured dynein. These results strongly suggest that the hydrolytic site of 12 S dynein is contained in its heavy chain.     

Labeling of Chlamydomonas 18 S dynein polypeptides by 8-azidoadenosine 5'-triphosphate, a photoaffinity analog of ATP

The 18 S dynein from the outer arm of Chlamydomonas flagella is composed of an alpha subunit containing an alpha heavy chain (Mr = approximately 340,000) and an Mr = 16,000 light chain, and a beta subunit containing a beta heavy chain (Mr = approximately 340,000), two intermediate chains (Mr = 78,000 and 69,000), and seven light chains (Mr = 8,000-20,000). Both subunits contain ATPase activity. We have used 8-azidoadenosine 5'-triphosphate (8-N3 ATP), a photoaffinity analog of ATP, to investigate the ATP-binding sites of intact 18 S dynein. 8-N3ATP is a competitive inhibitor of 18 S dynein's ATPase activity and is itself hydrolyzed by 18 S dynein; moreover, 18 S dynein's hydrolysis of ATP and 8-N3ATP is inhibited by vanadate to the same extent. 8-N3ATP therefore appears to interact with at least one of 18 S dynein's ATP hydrolytic sites in the same way as does ATP. When [alpha- or gamma-32P]8-N3ATP is incubated with 18 S dynein in the presence of UV irradiation, label is incorporated primarily into the alpha, beta, and Mr = 78,000 chains; a much smaller amount is incorporated into the Mr = 69,000 chain. The light chains are not labeled. The incorporation is UV-dependent, ATP-sensitive, and blocked by preincubation of the enzyme with vanadate plus low concentrations of ATP or ADP. These results suggest that the alpha heavy chain contains the site of ATP binding and hydrolysis in the alpha subunit. In the beta subunit, the beta heavy chain and one or both intermediate chains may contain ATP-binding sites.     

Regulation of 22S dynein by a 29-kD light chain

Previously, a 29-kD axonemal polypeptide (p29) that copurifies with 22S dynein has been shown to be phosphorylated in a cAMP- and Ca(2+)- sensitive manner, consistent with a role for this molecule in the signal transduction cascade leading to fast forward swimming in Paramecium tetraurelia (Hamasaki, T., K. Barkalow, J. Richmond, and P. Satir. 1991. Proc. Natl. Acad. Sci. USA. 88:7912-7922). This study demonstrates the nature of the relationship between p29 and 22S dynein. Chaotropic agents can be used to separate p29 fractions from 22S dynein. When extracted p29 is exchanged into physiological buffers, it regains the ability to recombine with 22S dynein with an apparent dissociation constant of 25 nM; no recombination is seen with 14S dynein or with unrelated control proteins. p29 from Paramecium will also recombine with Tetrahymena 22 but not 14S dynein. After chymotryptic digestion of 22S dynein, p29 preferentially binds to a single-headed fragment, homologous to the alpha H chain of Tetrahymena 22S dynein. 22S dynein treated in vitro by Paramecium protein kinase A in the presence of cAMP and ATP to phosphorylate p29 translocates bovine brain microtubules significantly (1.53x; p < 0.001) faster than before phosphorylation. Similarly, 22S dynein reconstituted in vitro with thiophosphorylated p29 translocates microtubules significantly (1.31x; p < 0.001) faster than controls reconstituted with nonthiophosphorylated p29. p29 is the only moiety thiophosphorylated in the reconstituted dynein. We conclude that p29 functions as a 22S dynein regulatory light chain in that it alone is sufficient to control the rate of microtubule translocation by changes in its phosphorylation state.     

Photolabeling of the 6 and 10 S conformations of gizzard myosin with 3'(2')-O-(4-Benzoyl)benzoyl-ATP. Proline 324 is near the active site

3'(2')-O-(4-Benzoyl)benzoyl-ATP (Bz2ATP) was used as a photoaffinity label of the ATP binding site of unphosphorylated chicken gizzard myosin. Specific photolabeling of the active site of 6 S myosin was assured by forming a stable myosin.Co(II)Bz2ADP.orthovanadate complex (termed trapping) prior to irradiation. Co2+ was used in place of Mg2+ to prevent the known photoreaction of vanadate with myosin which destabilizes the trapped complex. [3H] Bz2ADP.Pi was also stably trapped on gizzard myosin by forming the 10 S folded conformation of the protein in the presence of [3H]Bz2ATP and Mg2+. Irradiation of 6 S myosin containing orthovanadate trapped [3H] Bz2ADP or 10 S trapped [3H]Bz2ADP.Pi gave 32 and 30% covalent incorporation, respectively. The 50-kDa and precursor 68-kDa tryptic peptides of the subfragment-1 heavy chain derived from both forms of myosin were found to contain essentially all of the covalently attached [3H]Bz2ADP. Parallel experiments with untrapped [3H]Bz2ADP showed extensive nonspecific labeling of all of the major tryptic peptides and the light chains. Eight labeled peptides, isolated from 6 and 10 S photolabeled myosin, contained the sequence G319-H-V-P-I-X-A-Q326, where X corresponds to labeled proline 324. [14C]Bz2ADP was previously shown to label serine 324 in skeletal subfragment-1 (Mahmood, R., Elzinga, M., and Yount, R. G. (1989) Biochemistry 28, 3989-3995), which corresponds to alanine 325 in the gizzard sequence. Thus, this region of the 50-kDa tryptic fragment, near the nucleotide binding site, in both skeletal and smooth muscle myosins, must fold in essentially the same manner.     

Effect of actin on the tryptic digestion of myosin subfragment 1 in the weakly attached state.

The structure of myosin subfragment 1 (S1) in the weakly attached complex with actin was studied at three specific sites, at the 50-kDa/20-kDa and 27-kDa/50-kDa junctions, and at the N-terminal region, using tryptic digestion as a structure-exploring tool. The structure of S1 at the vicinity of the 50-kDa/20-kDa junction is pH dependent in the weakly attached state because the tryptic cleavage at this site was fully protected by actin at pH 6.2, but the protection was only partial at pH 8.0. Since the actin protection is complete in rigor at both pH values, the results indicate that the structure of S1 at the 50-kDa/20-kDa junction differs in the two states at pH 8.0, but not at pH 6.2. Actin restores the ADP-suppressed tryptic cleavage after Lys213 at the 27-kDa/50-kDa junction in the strongly attached state, but not in the weakly attached state, which indicates structural difference between the two states at this site. ATP and ADP open a new site for tryptic cleavage in the N-terminal region of the S1 heavy chain between Arg23 and Ile24. Actin was found to suppress this cleavage in both weakly and strongly attached states, which shows that, in the vicinity of this site, the structure of S1 is similar in both states. The results indicate that the binding of S1 to actin induces localized changes in the S1 structure, and the extent of these changes is different in the various actin-S1 complexes.     

Properties of the full-length heavy chains of Tetrahymena ciliary outer arm dynein separated by urea treatment

An important challenge is to understand the functional specialization of dynein heavy chains. The ciliary outer arm dynein from Tetrahymena thermophila is a heterotrimer of three heavy chains, called alpha, beta and gamma. In order to dissect the contributions of the individual heavy chains, we used controlled urea treatment to dissociate Tetrahymena outer arm dynein into a 19S beta/gamma dimer and a 14S alpha heavy chain. The three heavy chains remained full-length and retained MgATPase activity. The beta/gamma dimer bound microtubules in an ATP-sensitive fashion. The isolated alpha heavy chain also bound microtubules, but this binding was not reversed by ATP. The 19S beta/gamma dimer and the 14S alpha heavy chain could be reconstituted into 22S dynein. The intact 22S dynein, the 19S beta/gamma dimer, and the reconstituted dynein all produced microtubule gliding motility. In contrast, the separated alpha heavy chain did not produce movement under a variety of conditions. The intact 22S dynein produced movement that was discontinuous and slower than the movement produced by the 19S dimer. We conclude that the three heavy chains of Tetrahymena outer arm dynein are functionally specialized. The alpha heavy chain may be responsible for the structural binding of dynein to the outer doublet A-tubule and/or the positioning of the beta/gamma motor domains near the surface of the microtubule track.     

Solubilization of dynein from Tetrahymena ssp. axonemes using phosphate analogues

One major protein was selectively solubilized when phosphate analogues, such as inorganic vanadate (Vi), beryllium fluoride (BeFx) or aluminum fluoride (AlFx), were added to ciliary axonemes of Tetrahymena ssp. (T. pyriformis or T. thermophila) in the presence of ATP. This protein contains three high molecular weight polypeptides, characteristic of an outer arm dynein. Electron microscopic observation of the axonemes after solubilization using ATP and Vi revealed axonemes partially lacking outer arm dyneins. These results suggest that the solubilized protein is an outer arm dynein and also that a dynein-ADP-phosphate complex decreases its affinity with the adjacent microtubules within axonemes. Limited digestion with chymotrypsin revealed that each solubilized dynein has a similar conformation, but it is markedly different from that of dynein in the absence of ATP or a phosphate analogue. The solubilized dynein obtained by the addition of Vi and ATP to axonemes was digested by UV irradiation to yield at least five new polypeptides (240, 230, 225, 180 and 160 kDa) but the dyneins solubilized by BeFx (or AlFx) in the presence of ATP did not produce any photocleavage products under the same conditions.