Synthesis and characterization of novel spin-labeled photoaffinity nonnucleoside analogues of ATP as structural and EPR probes for myosin

Two new spin-labeled photoreactive nonnucleoside ATP analogues, 1-(4-azido-2-nitrophenyl)amino-3-(1-oxyl-2,2,5, 5-tetramethylpyrrolidinyl-3-carbamido)-2-propyl triphosphate (SL-NANTP) and 2-(4-azido-2-nitrophenyl)amino-2,2-(1-oxyl-2,2,6, 6-tetramethyl-4-piperidylidene)di(oxymethylene) ethyl triphosphate (SSL-NANTP), were synthesized and characterized. This study aims to develop a second generation of NANTP-based analogues containing immobile spin labels that can be used to monitor conformational changes in myosin during the contractile cycle of muscle. Previous studies have shown that both a photoaffinity nonnucleoside ATP analogue, 2-[(4-azido-2-nitrophenyl)amino] ethyl triphosphate (NANTP) [Nakamaye et al. (1985) Biochemistry 24, 5226-5235], and a photoaffinity ATP analogue, 3'(2')-O-4-[4-oxo-(4-amino-2,2,6, 6-tetramethyl-piperidino-1-oxyl)-4-benzoyl] benzoyl adenosine 5'-triphosphate (SL-Bz(2)ATP) [Wang et al. (1999) J. Muscle Res. Cell Motil. 20, 743-753], behave like ATP in their interactions with myosin. Remarkably, photolabeled myosin recovers all of its normal enzymatic properties after treatment with actin in the presence of MgATP [Luo et al. (1995) Biochemistry 34, 1978-1987]. For SL-NANTP, the spin label moiety is attached to NANTP via an aminomethyl side chain. In SSL-NANTP, attachment is via a restricted spiro ring. The two new probes interact with myosin subfragment-1 (S1) in a manner analogous to ATP, and after photoincorporation, labeled S1 recovers full activity after treatment with actin and MgATP. The electron paramagnetic resonance (EPR) spectrum resulting from S1 photolabeled with SL-NANTP shows a very high degree of probe mobility. However, the EPR spectrum of S1 photolabeled with SSL-NANTP shows that the probe is highly immobilized with respect to S1, constrained to move within a cone of angle 52 degrees (full-width, half-max). Unlike the parent, NANTP, which photolabels on the 23 kDa tryptic fragment of S1, SSL-NANTP photolabels on the 20 kDa fragment. Its highly immobile nature means that it is potentially a useful reporter group to monitor cross-bridge motion in muscle fibers.     

Mechanics of glycerinated muscle fibers using nonnucleoside triphosphate substrates.

We have investigated the ability of the photoaffinity, nonnucleotide ATP analogues, 2-[(4-azido-2-nitrophenyl) amino] ethyl triphosphate (NANTP) and 2-[(4-azido-2-nitrophenyl) amino] propyl triphosphate (PrNANTP), to support active contraction in glycerinated rabbit psoas fibers. At millimolar concentrations, in the absence of calcium, both analogues relaxed fibers. In the presence of calcium, MgNANTP produced isometric tension and stiffness that were one-half to two-thirds the values obtained in MgATP. Maximum shortening velocity and the calcium-activated, myofibrillar catalyzed rate of hydrolysis were approximately the same for MgNANTP as for MgATP. With MgNANTP as the substrate, increasing concentrations of the diphosphate analogue, MgNANDP, inhibited shortening velocity but did not change isometric tension. The addition of increased concentrations of orthophosphate (P) decreased tension while shortening velocity increased. Thus, the effects of the hydrolysis products of NANTP were quite similar to those observed previously for ADP and P in the presence of MgATP. Taken together, these observations show that MgNANTP binds to, and functions in the active site of myosin in a manner quite analogous to MgATP. Thus, the aryl azido group should serve as a valid photoaffinity label for the purine portion of the active site. In contrast, MgPrNANTP, which differs from MgNANTP only in an extra CH2 spacer between the nitrophenyl ring and the triphosphate moiety did not support isometric tension or active shortening in the presence of calcium. Fiber stiffness increased in the presence of calcium and MgPrNANTP, with a calcium-activated, myofibrillar MgPrNANTPase which was about half that obtained with MgATP. Thus, in the presence of MgPrNANTP, cross-bridges appeared to be cycling through states that were attached to actin, but not producing force.     

Photoaffinity labeling of human sex hormone-binding globulin using 17alpha-alkylamine derivatives of 3beta-androstanediol substituted with azidonitrophenylamido, azidonitrophenylamino, or trifluoroazidonitrophenylamino chromophores. Localization of Trp-84 in the vicinity of the steroid-binding site.

Purified human SHBG was photoaffinity labeled with 17alpha-aminomethyl (M), 17alpha-aminoethyl (E), and 17alpha-aminopropyl (P) derivatives of [3alpha-(3)H]-5alpha-androstane-3beta,17beta-diol coupled to 5-azido-2-nitrobenzoylamido (ANB), 4-azido-2-nitrophenylamino (ANP), and 5-azido-2-nitro-3,4,6-trifluorophenylamino (ANTFP) chromophores. Successful labeling was achieved in all cases except for the two photoreagents with the shortest side chains, namely, ANP-M and ANTFP-M derivatives. Edman sequencing and mass spectrometry of immunopurified photolabeled tryptic fragments revealed that radioactivity was present either on the sequence of residues 73-94, uniquely at the level of Trp-84 (stable covalent labeling), or on one of the two overlapping sequences of residues 126-134 and 126-135, at the level of Pro-130 (labile labeling) and Lys-134 (either stable or partially labile labeling), respectively. The same Trp-84 was photolabeled with the three ANB derivatives of increasing lengths, and by the ANP-P photoreagent. This residue was the exclusive target for the shortest [(3)H]ANB-M photoreagent but was a minor site for the longest [(3)H]ANB-P photoreagent, essentially recovered at the level of Pro-130. The [(3)H]ANB-E photoreagent of intermediate size also labeled exclusively Trp-84, except in some experiments in which photolabeling was recovered predominantly at the level of Pro-130. The [(3)H]ANP-P photoreagent with an overall length similar to that of the ANB-P photoreagent labeled simultaneously Trp-84 (minor site) and Lys-134. The other [(3)H]ANP-E, [(3)H]ANTFP-E, and [(3)H]ANTFP-P derivatives labeled in all cases Lys-134. These findings indicate that the conserved Trp-84 and the two Pro-130 and Lys-134 residues are all located in the vicinity of the D ring of steroid ligands and remain freely accessible from the C17alpha position, thus providing biochemical data delineating the corresponding region of the steroid-binding site.     

2-[(4-Azido-2-nitrophenyl)amino]ethyl triphosphate, a novel chromophoric and photoaffinity analogue of ATP. Synthesis, characterization, and interaction with myosin subfragment 1

A facile and high-yield synthesis of a new ATP analogue, 2-[(4-azido-2-nitrophenyl)amino]ethyl triphosphate (NANTP), is described. NANTP and ATP are hydrolyzed by skeletal myosin subfragment 1 (SF1) at comparable rates in the presence of Ca2+, Mg2+, or NH4+-EDTA. NANTP is also cleaved but less readily by mitochondrial F1-ATPase and by (Na+ + K+)-ATPase from dog brain and hog kidney. F-Actin markedly activates NANTP cleavage by SF1 in the presence of Mg2+, suggesting that the diphosphate product NANDP is slow to be released from the enzyme. [alpha-32P]NANDP binds to a single site on SF1 (KA = 1 X 10(6) M-1) with an affinity identical with that of ADP. The absorption maximum of NANDP was shifted from 474 to 467 nm upon binding to SF1, suggesting that the purine binding site has a dielectric constant of about 45. NANDP was trapped in nearly stoichiometric amounts at the active site by cross-linking SH1 and SH2 with N,N'-p-phenylenedimaleimide (pPDM) or by chelation with cobalt (III) phenanthroline [Wells, J., & Yount, R. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966]. The trapped [beta-32P]NANDP X SF1 complex, like the comparable ADP X SF1 complex, was stable for days at 0 degree C and could be purified free of extraneous analogue by ammonium sulfate precipitation and gel filtration. Photolysis of the purified complex gave greater than 50% covalent incorporation of the trapped NANDP into the 95-kilodalton (kDa) heavy chain of SF1. Limited trypsinization and analysis by gel electrophoresis showed that greater than 95% of the bound label was associated with the 25-kDa NH2-terminal peptide. Without trapping, NANDP labeling of SF1 was nonspecific and was not prevented by addition of a large excess of ATP. This new approach of trapping photoaffinity analogues by cross-linking agents before photolysis may prove to be of general usefulness in increasing the specificity and extent of labeling of enzymes that undergo substrate-induced conformation changes.     

Photoaffinity labeling of scallop myosin with 2-[(4-azido-2-nitrophenyl)amino]ethyl diphosphate: identification of an active site arginine analogous to tryptophan-130 in skeletal muscle myosin.

The ADP photoaffinity analogue 2-[(4-azido-2-nitrophenyl)amino]ethyl 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 irradiation. 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 Arg128. 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, 1575-1580]. Scallop and rabbit skeletal muscle myosin display a high degree of sequence similarity in this region with Arg128 in an equivalent position as Trp130. 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.     

Effect of nucleotide on interaction of the 567-578 segment of myosin heavy chain with actin

To probe the effect of nucleotide on the formation of ionic contacts between actin and the 567-578 residue loop of the heavy chain of rabbit skeletal muscle myosin subfragment 1 (S1), the complexes between F-actin and proteolytic derivatives of S1 were submitted to chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. We have shown that in the absence of nucleotide both 45 kDa and 5 kDa tryptic derivatives of the central 50 kDa heavy chain fragment of S1 can be cross-linked to actin, whereas in the presence of MgADP.AlF4, only the 5 kDa fragment is involved in cross-linking reaction. By the identification of the N-terminal sequence of the 5-kDa fragment, we have found that trypsin splits the 50 kDa heavy chain fragment between Lys-572 and Gly-573, the residues located within the 567-578 loop. Using S1 preparations cleaved with elastase, we could show that the residue of 567-578 loop that can be cross-linked to actin in the presence of MgADP.AlF4 is Lys-574. The observed nucleotide-dependent changes of the actin-subfragment 1 interface indicate that the 567-578 residue loop of skeletal muscle myosin participates in the communication between the nucleotide and actin binding sites.     

Ionic interaction of myosin loop 2 with residues located beyond the N-terminal part of actin probed by chemical cross-linking

To probe ionic contacts of skeletal muscle myosin with negatively charged residues located beyond the N-terminal part of actin, myosin subfragment 1 (S1) and actin split by ECP32 protease (ECP-actin) were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). We have found that unmodified S1 can be cross-linked not only to the N-terminal part, but also to the C-terminal 36 kDa fragment of ECP-actin. Subsequent experiments performed on S1 cleaved by elastase or trypsin indicate that the cross-linking site in S1 is located within loop 2. This site is composed of Lys-636 and Lys-637 and can interact with negatively charged residues of the 36 kDa actin fragment, most probably with Glu-99 and Glu-100. Cross-links are formed both in the absence and presence of MgATP.P(i) analog, although the addition of nucleotide decreases the efficiency of the cross-linking reaction.     

A short hydrophobic segment next to tryptophan-130 in myosin heavy chain is close to the ribose ring of ADP bound in the adenosinetriphosphatase site

The ATPase site of rabbit skeletal myosin was covalently labeled by an ADP analogue that carried a biotin moiety on its adenine ring and a photoreactive phenyl azide group on its ribose ring [Sutoh, K., Yamamoto, K., & Wakabayashi, T. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 212-216]. The ADP analogue was tightly trapped into the ATPase site in the presence of vanadate ions and then covalently cross-linked to the site by UV irradiation. The N-terminal 23,000-dalton tryptic fragment of the heavy chain was selectively labeled with the analogue. Further mapping of the labeled segment along the 23-kDa fragment was carried out by "end-label fingerprinting" which employed site-directed antibodies against both ends of the N-terminal heavy chain fragment. The mapping revealed that a hydrophobic segment of approximately 10 residues next to Trp-130, which was reported to be in proximity to the adenine ring of ADP bound to the ATPase site [Okamoto, Y., & Yount, R. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1575-1579], was the site of covalent labeling with the ADP analogue. The result indicates that the hydrophobic segment is close to the ribose ring of ADP bound to the ATPase site.     

Amino acid sequence of the amino-terminal 24 kDa fragment of the heavy chain of chicken gizzard myosin

Chicken gizzard myosin was modified with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine (IAEDANS) in the presence of ATP and in 0.15 M KCl, where the myosin assumed 10S conformation. From the tryptic digest of the modified myosin, a fluorescent fragment (24 kilodaltons) was isolated by gel filtration on a Sephadex G-100 column followed by chromatography on a CM 52 column. The amino acid sequence of the fragment was analyzed by conventional methods, and was: (S,Z)K-P-L-S-D-D-E-K-F-L-F-V-D-K-N-F-V-N-N-P-L-A-Q-A-D-W-S-A-K-K- L-V-W-V-P-S-E-K-H-G-F-E-A-A-S-I-K-E-E-K-G-D-E-V-T-V-E-L-Q-E-N-G-K-K- V-T-L-S-K-D-D-I-Q-K-M-N-P-P-K-F-S-K-V-E-D-M-A-E-L-T-C-L-N-E-A-S-V-L- H-N-L-R-E-R-Y-F-S-G-L-I-Y-T-Y-S-G-L-F-C-V-V-I-N-P-Y-K-Q-L-P-I-Y-S-E-K-I- I-D-M-Y-K-G-K-K-R-H-E-M-P-P-H-I-Y-A-I-A-D-T-A-Y-R-S-M-L-Q-D-R-E-D-Q- S-I-L-C-T-G-E-S-G-A-G-K-T-E-N-T-K-K-V-I-Q-Y-L-A-V-V-A-S-S-H-K-G-K. The amino-terminus was blocked, and the fragment was assigned as an amino-terminal part of the heavy chain of gizzard myosin. Position 127 was occupied by epsilon-N-trimethyllysine. Trp-130 of rabbit skeletal myosin heavy chain, which was reported to cross-link to an azide derivative of ATP by Okamoto and Yount (Proc. Natl. Acad. Sci. U.S. 82, 1575-1579 (1985], was replaced by glutamine in gizzard myosin. Cys-93 of the fragment is the amino acid residue whose reaction with IAEDANS alters the ATPase activity of gizzard myosin (Onishi, H. (1985) J. Biochem. 98, 81-86).     

Actin-binding site of pig cardiac myosin

An actin-binding site is also present in the tryptic 20 kDa peptide fragment of the subfragment-1 heavy chain of pig cardiac myosin. As previously reported for skeletal myosin (Katoh, T., Katoh, H., and Morita, F. (1985) J. Biol. Chem. 260, 6723-6727), the site was further narrowed down to the 10 kDa peptide containing the reactive SH1 and SH2 groups. Thus it appears that the actin-binding site around the two thiols found in skeletal myosin is common to different types of myosin.     

Preparation of subfragment-1 from abalone smooth muscle myosin

Myosin subfragment-1 (S1), which has one heavy chain (HC) (93 kDa) and two light chains (LC1 and LC2), was prepared by papain digestion of myosin from abalone-smooth muscle in the presence of Ca2+. The Ca-sensitivity of abalone S1 itself was not lost completely (about 30%). The tryptic digestion of S1 showed that in the presence of EDTA, S1 HC was split into 68, 55, and 23 kDa fragments, as in the presence of Ca2+, but 23 kDa was further degraded into 19 kDa. In contrast to the result in the presence of Ca2+, LCs disappeared in the early stage of reaction and Ca-ATPase activity decreased rapidly to about 70% of that of intact S1. This rapid decrease of Ca-ATPase activity seemed to be accompanied with the digestion of LCs. Therefore, LCs contribute to the protection of 23 kDa fragment from further digestion, to the maintenance of Ca-ATPase activity by stabilizing the structure of S1 to some extent in the presence of Ca2+. Since F-actin suppressed the cleavage of S1 HC to 68 and 23 kDa during tryptic digestion, it might be that 23 and 68 kDa corresponded to 20 kDa (C-terminal fragment) and to 50 + 25 kDa (N-terminal fragment) of skeletal myosin S1, respectively.     

Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed mutagenesis.

In order to identify molecular features of the calmodulin (CaM) activated adenylate cyclase of Bordetella pertussis, a truncated cya gene was fused after the 459th codon in frame with the alpha-lacZ' gene fragment and expressed in Escherichia coli. The recombinant, 604 residue long protein was purified to homogeneity by ion-exchange and affinity chromatography. The kinetic parameters of the recombinant protein are very similar to that of adenylate cyclase purified from B.pertussis culture supernatants, i.e. a specific activity greater than 2000 mumol/min mg of protein at 30 degrees C and pH 8, a KmATP of 0.6 mM and a Kd for its activator, CaM, of 0.2 nM. Proteolysis with trypsin in the presence of CaM converted the recombinant protein to a 43 kd protein with no loss of activity; the latter corresponds to the secreted form of B.pertussis adenylate cyclase. Site-directed mutagenesis of residue Trp-242 in the recombinant protein yielded mutants expressing full catalytic activity but having altered affinity for CaM. Thus, substitution of an aspartic acid residue for Trp-242 reduced the affinity of adenylate cyclase for CaM greater than 1000-fold. Substitution of a Gln residue for Lys-58 or Lys-65 yielded mutants with a drastically reduced catalytic activity (approximately 0.1% of that of wild-type protein) but with little alteration of CaM-binding. These results substantiated, at the molecular level, our previous genetic and biochemical studies according to which the N-terminal tryptic fragment of secreted B.pertussis adenylate cyclase (residues 1-235/237) harbours the catalytic site, whereas the C-terminal tryptic fragment (residues 235/237-399) corresponds to the main CaM-binding domain of the enzyme.     

The interaction and photolabeling of myosin subfragment 1 with 3'(2')-O-(4-benzoyl)benzoyladenosine 5'-triphosphate

The photoprobe 3'(2')-O-(4-benzoyl)benzoyladenosine 5'-triphosphate (Bz2ATP) was used to characterize the nucleotide-binding site of myosin subfragment 1 (SF1). Improved synthesis and purification of Bz2ATP are reported. 1H NMR and ultraviolet spectroscopy show that Bz2ATP is a 60:40 mixture of the 3'(2')-ribose isomers and that the epsilon M261 is 41,000 M-1 cm-1. Bz2ATP is hydrolyzed by SF1 comparably to ATP in the presence of actin or K+, NH4+, or Mg2+ ions; and the product, Bz2ADP, has a single binding site on SF1 (K'a = 3.0 X 10(5) M-1). [3H]Bz2ATP was photoincorporated into SF1 with concomitant loss of K+-EDTA-ATPase activity. Analysis of photolabeled SF1 showed that the three major tryptic peptides (23, 50, and 20 kDa) of the heavy chain fragment and the alkali light chains were labeled. The presence of ATP during irradiation protected only the 50-kDa peptide, indicating that the other peptides were nonspecifically labeled. If Bz2ATP was first trapped on SF1 by cross-linking the reactive thiols, SH1 and SH2, with p-phenylenedimaleimide, only the 50-kDa tryptic peptide was labeled. These results confirm and extend previous observations that [3H]Bz2ATP trapped on SF1 by cobalt(III) phenanthroline photolabeled the same 50-kDa peptide (Mahmood, R., and Yount, R.G. (1984) J. Biol. Chem. 259, 12956-12959). Thus, the 50-kDa peptide is labeled with or without thiol cross-linking, indicating that the relative position of SH1 and SH2 does not affect the labeling pattern.     

Identification of the site of photocross-linking formed in the absence of magnesium nucleotide from SH2 (Cys-697) in myosin subfragment 1 labeled with 4'-maleimidylbenzophenone

The site of photocross-linking between Cys-697 (SH2), prelabeled with 4'-[14C]maleimidylbenzophenone, and the 50-kDa segment of myosin S1 on irradiation in the absence of nucleotide has been determined by isolation of the 20-50-kDa adduct and subsequent tryptic proteolysis. Isolation and partial sequencing of the radioactively labeled peptide indicate that the site of cross-linking is Arg-239. This result indicates that, in the absence of nucleotide, Arg-239 resides at about 1.0 nm from SH2 and, on the basis of the recent work of Sutoh and Hiratsuka (Sutoh, K. and Hiratsuka T. (1988) Biochemistry 27, 2964-2969) places Arg-239 at no more than 1.45 nm from either Lys-184 or Lys-189 of the nucleotide-binding "glycine-rich" loop prior to the binding of nucleotide.     

Photoaffinity labeling of human serum vitamin D binding protein and chemical cleavages of the labeled protein: identification of an 11.5-kDa peptide containing the putative 25-hydroxyvitamin D3 binding site.

In this paper, we describe photoaffinity labeling and related studies of human serum vitamin D binding protein (hDBP) with 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether (25-ANE) and its radiolabeled counterpart, i.e., 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitro-[3,5-3H]phenyl)amino]propyl ether (3H-25-ANE) (Ray et al., 1986, 1991). We have carried out studies to demonstrate that (1) 25-ANE competes with 25-OH-D3 for the binding site of the latter in hDBP and (2) 3H-25-ANE is capable of covalently labeling the hDBP molecule when exposed to UV light. Treatment of a sample of purified hDBP, labeled with 3H-25-ANE, with BNPS-skatole produced two Coomassie Blue stained peptide fragments, and the majority of the radioactivity was associated with the smaller of the two peptide fragments (16.5 kDa). On the other hand, cleavage of the labeled protein with cyanogen bromide produced a peptide (11.5 kDa) containing most of the covalently attached radioactivity. Considering the primary amino acid structure of hDBP, this peptide fragment (11.5 kDa) represents the N-terminus through residue 108 of the intact protein. Thus, our results tentatively identify this segment of the protein containing the binding pocket for 25-OH-D3.     

Thermal denaturation of the alkali light chain-20 kDa fragment complex obtained from myosin subfragment 1.

The thermal denaturation of the myosin subfragment 1 (S1) from rabbit skeletal muscle and of its derivatives obtained by tryptic digestion has been studied by means of differential scanning calorimetry. Two distinct thermal transitions were revealed in the isolated complex of the C-terminal 20 kDa fragment of the S1 heavy chain with the alkali light chain. These transitions were identified by means of a thermal gel analysis method. It has been shown that the thermal denaturation of the 20 kDa fragment of the S1 heavy chain correlates with the melting of the most thermostable domain in the S1 molecule. It is concluded that this domain is located in the C-terminal 20 kDa segment of the S1 heavy chain.     

Comparative structure of the protease-sensitive regions of the subfragment-1 heavy chain from smooth and skeletal myosins

The heavy chain fragments generated by restricted proteolysis of the smooth chicken gizzard myosin subfragment-1 (S-1) with trypsin, Staphylococcus aureus V8 protease, and chymotrypsin were isolated and submitted to partial amino acid sequencing. The comparison between the smooth and striated muscle myosin sequences permitted the unambiguous structural characterization of the two protease-vulnerable segments joining the three putative domain-like regions of the smooth head heavy chain. The smooth carboxyl-terminal connector is a serine-rich region located around positions 632-640 of the rabbit skeletal sequence and would represent the "A" site that is conformationally sensitive to the myosin 10 S-6 transition and to its interaction with actin (Ikebe, M., and Hartshorne, D. J. (1986) Biochemistry 25, 6177-6185). A third site which undergoes a nucleotide-dependent chymotryptic cleavage which inactivates the Mg2+-ATPase (Okamoto, Y., and Sekine, T. (1981) J. Biochem. (Tokyo) 90, 833-842, 843-849) was identified at Trp-31/Ser-32. It is vicinal to Lys-34 that is monomethylated in the skeletal heavy chain but not at all in the smooth sequence. However, the two trimethyl lysine residues present in the skeletal sequence are conserved in the same regions of the smooth S-1 and may play a general functional role in myosin. The smooth central 50-kDa segment could be selectively destroyed by a mild tryptic digestion in the absence of any unfolding agent, with a concomitant inhibition of the ATPase activities. This feature is in line with the proposed domain structure of the S-1 heavy chain and also suggests a relationship between the specific biochemical properties of the smooth S-1 and the particular conformation of its 50-kDa region.     

Structural and immunological characterization of the myosin-like 110-kD subunit of the intestinal microvillar 110K-calmodulin complex: evidence for discrete myosin head and calmodulin-binding domains

The actin bundle within each microvillus of the intestinal brush border is tethered laterally to the membrane by spirally arranged bridges. These bridges are thought to be composed of a protein complex consisting of a 110-kD subunit and multiple molecules of bound calmodulin (CM). Recent studies indicate that this complex, termed 110K-CM, is myosin-like with respect to its actin binding and ATPase properties. In this study, possible structural similarity between the 110-kD subunit and myosin was examined using two sets of mAbs; one was generated against Acanthamoeba myosin II and the other against the 110-kD subunit of avian 110K-CM. The myosin II mAbs had been shown previously to be cross-reactive with skeletal muscle myosin, with the epitope(s) localized to the 50-kD tryptic fragment of the subfragment-1 (S1) domain. The 110K mAbs (CX 1-5) reacted with the 110-kD subunit as well as with the heavy chain of skeletal but not with that of smooth or brush border myosin. All five of these 110K mAbs reacted with the 25-kD, NH2-terminal tryptic fragment of chicken skeletal S1, which contains the ATP-binding site of myosin. Similar tryptic digestion of 110K-CM revealed that these five mAbs all reacted with a 36-kD fragment of 110K (as well as larger 90- and 54-kD fragments) which by photoaffinity labeling was shown to contain the ATP-binding site(s) of the 110K subunit. CM binding to these same tryptic digests of 110K-CM revealed that only the 90-kD fragment retained both ATP- and CM-binding domains. CM binding was observed to several tryptic fragments of 60, 40, 29, and 18 kD, none of which contain the myosin head epitopes. These results suggest structural similarity between the 110K and myosin S1, including those domains involved in ATP- and actin binding, and provide additional evidence that 110K-CM is a myosin. These studies also support the results of Coluccio and Bretscher (1988. J. Cell Biol. 106:367-373) that the calmodulin-binding site(s) and the myosin head region of the 110-kD subunit lie in discrete functional domains of the molecule.     

Spatial proximity of the glycine-rich loop and the SH2 thiol in myosin subfragment 1

Subfragment 1 (S1) prepared from rabbit skeletal muscle myosin was digested with trypsin to cleave the 95K heavy chain into three pieces, i.e., the 23K, 50K, and 20K fragments. The trypsin-treated S1 was then cross-linked with p-nitrophenyl iodoacetate. The cross-linker bridged one of the reactive thiols (SH2) in the 20K fragment and a lysine residue in the 23K fragment [Hiratsuka, T. (1987) Biochemistry 26, 3168-3173]. Location of the lysine residue was mapped along the 23K fragment by "end-label fingerprinting", which employed site-directed antibodies against the N-terminus of the 23K fragment and against the C-terminus of the 24K fragment (the 23K fragment plus nine extra residues at its C-terminus). The mapping revealed that Lys-184 or Lys-189 was the residue cross-linked with SH2. Since the cross-linker used here spans only several angstroms, the result indicates that Lys-184 or Lys-189 is very close to SH2 in the three-dimensional structure of myosin head. Examination of the primary structure of the 23K fragment has revealed that these lysine residues are in and very close to the so-called "glycine-rich loop", whose sequence is highly homologous to those of nucleotide-binding sites of various nucleotide-binding proteins.     

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.