Proteolysis and the domain organization of myosin subfragment 1.

Abstract

Because the proteolytic cleavage of a folded polypeptide depends not only on the specificity of the protease but on the nature of the folding, we investigated the cleavage of (chymotryptically produced) subfragment 1 (designated "S-1") or "head" segment of myosin by seven proteases with different specificities. All seven produced approximately the same three fragments of S-1-namely, fragments (from the NH2 terminus) of 27, 50, and 20 kilodaltons, suggesting that in intact S-1 these fragments are distinct domains. The same proteases were used to hydrolyze the MgADP complex of S-1. All failed to do so except trypsin, which, as found earlier [Hozumi, T. (1983) Biochemistry 22, 799-804], makes two additional cleavages. This result suggests that the conformational change induced by MgADP opens up only a small stretch of polypeptide chain, which stretch happens to be vulnerable to trypsin.Under specified conditions, the attack of chymotrypsin on myosin occurs at about 820 residues from the NH2 terminus, thus producing a particle (resistant to further chymotryptic digestion) known as subfragment 1, designated "S-1," bearing both the ATPase and actin-binding functions of the original myosin (1). Balint et al. (2) showed that limited trypsinolysis will, in turn, split S-1 into three major fragments of approximately 27, 50, and 20 kilodaltons (kDa) that have been named "20K," "50K," and "20K." In recent years one of us (D.M.) and colleagues in France have found that the actin-binding function of S-1 is associated with 50K and 20K, and we and others have succeeded in assigning various additional functions to one or another of these three fragments.Thinking that these two readily-made, but limited, trypsin cuts probably require that there be two stretches of relatively structureless heavy chain joining the three fragments, we entertained the possibility that these three fragments are actually domains of S-1, but until now we have made no effort to examine this hypothesis. A basic requirement for concluding that S-1 has a domain organization is to show that the location of these two cuts is not a peculiarity of trypsin, but that in fact enzymes of various specificity effect similar cuts. This paper supplies such evidence. The following experiments with seven different proteases also provided us with a convenient opportunity to test a second proposition-namely, that the domain organization (i.e., the conformation of S1iADP) is significantly different from that of S-1. As did Hozumi earlier (3), we found that under specified conditions, trypsin makes no additional cuts in the absence of MgADP and two additional cuts in the presence of MgADP. On the other hand, the other enzymes make no additional cuts without or with MgADP. These results show that MgADP induces conformational changes in the 27K and 50K regions that open up trypsin-vulnerable stretches of polypeptide chain, but that these stretches are not very long because they do not expose vulnerable regions to the other enzymes.Together with evidence in the compani...

Nucleotide trapping at the ATPase site of myosin subfragment 1 by a new interthiol crosslinking.

“…The split S-1 obtained by trypsin cleavage (S-1/trypsin weight ratio 1:25, at 25°C for 30 min in 0.05 M Tris Cl, pH 8.0) was purified as described by Mornet et al. (15). The concentration of the S-1 preparations was estimated by using All = 7.5 as described by Wagner and Weeds (16).…”

“…Rabbit skeletal myosin was prepared as described by Offer et al (13). S-1 was prepared by digestion of myosin filaments with a-chymotrypsin (14) and was purified as usually (15). The split S-1 obtained by trypsin cleavage (S-1/trypsin weight ratio 1:25, at 25°C for 30 min in 0.05 M Tris Cl, pH 8.0) was purified as described by Mornet et al.…”

Selective cleavage at lysine of the 50 kDa‐20 kDa connector loop segment of skeletal myosin S‐1 by endoproteinase Arg‐C

“…Because the 95 kDa heavy chain of the head contains the actin-and nucleotide-binding sites, knowledge of its substructure and functioning is required in order to provide a complete description of the molecular mechanism of muscle contraction. Proteolytic dissection of skeletal chymotryptic S-1 led to the first suggestion as to the existence of domain-like regions in the heavy chain, represented by the three major 25, 50 and 20 kDa tryptic fragments [2][3][4][5]. These are connected by two protease-vulnerable segments forming flexible loops at the surface of the protein [6,7].…”

On the mechanism of energy transduction in myosin subfragment 1.

“…The carbodiimide-mediated union of actin and S-1 has a natural character, since the Mg2+-ATPase of the complex is even more accelerated than that of the normal complex (29). Binding of nucleotide to the N site permits trypsin [but not other proteases tested (23)] to cleave off a 5-kDa NH2-terminal segment of 27K and a 5-kDa COOH-terminal segment of 50K (30). Experiments with photoaffinity labels (31,32) show that at least the adenosine moiety of ATP binds to 27K.…”