Proteolytic digestions of myosin. subfragment 1 (S-i) with elastase, subtilisin, papain, thermolysin, and Staphylococcmu aurew protease reveal that the two trypsin-sensitive regions in S-1 have broad protease susceptibility. The cleavage of S-I by these enzymes yields products that correspond within 1-2 kilodaltons (kDa) to the 25-, 50-, and 20-kDa fragments produced by trypsin. Papain and thermolysin cut preferentially at the 26-kDA/70-kDa junction, whereas elastase, subtilisin, and S. aureus protease cleave both the 26-kDa/70-kDa and 75-kDa/22-kDa junctions in S-1. Binding of actin to S-1 decreases the rate of all proteolytic reactions in the 95-kDa heavy chain. The protection of the 26-kDa/70-kDa junction by actin is greatest against papain and thermolysin attack. The reaction times of elastase, subtilisin, and S. aureus protease with S-i increase 2-fold in the presence of actin. However, in contrast to similar reactions with trypsin, they proceed at both junctions and lead to formation of the 50-and 22-kDa fragments. The cleavage of the 22-kDa/50-kDa junction by elastase increases the K. value for the actin-activated ATPase.The presence of the two protease-sensitive regions in S-1 is consistent with a three-domain structure of the myosin head and may have important implications to the mode of intersite communication in this protein.The contractile process in a muscle cell requires the functional coupling between two distinct sites on the myosin subfragment 1 (S-1), the actin-binding site and the active site for ATP hydrolysis. The "communication" between these sites finds its most striking and direct expression in the effect of actin on ATP hydrolysis by S-1 and, conversely, in the effect of nucleotides on the binding of actin to S-1. The mechanism and the pathway for this intersite communication are still unknown. One approach taken in the past to clarify the relationship between the actin and nucleotide sites has been to modify specific residues on S-1 [e.g., the reactive cysteine groups SH-1 and SH-2; for a recent review see Morales et al. (1)] and monitor the consequent changes at the above sites.More recently, important progress has been made in the topological study of S-1 by using the method of limited tryptic proteolysis. The 95-kilodalton (kDa) heavy chain of S-1 is cleaved by trypsin to produce three discrete fragments (25-, 50-, and 20-kDa), which remain associated under nondenaturing conditions. With the assignment of the active site to the 25-kDa peptide (2) and identification of two separate actin-binding sites, one on the 50-kDa and the other on the 20-kDa peptide (3-5), the tryptic fragments of S-1 have become a useful tool and convenient framework for examination of the intersite communication on S-1.The tryptic cleavage of S-1 into three fragments has led Mornet et al. (6) to suggest a three-domain structure for the myosin head, in which the 25-, 50-, and 20-kDa peptides are covalently connected by two protease-sensitive regions. Irrespective of whether this description will coinci...
The crystal structure of a recombinant ␣ E C domain from human fibrinogen-420 has been determined at a resolution of 2.1 Å. The protein, which corresponds to the carboxyl domain of the ␣ E chain, was expressed in and purified from Pichia pastoris cells. Felicitously, during crystallization an amino-terminal segment was removed, apparently by a contaminating protease, allowing the 201-residue remaining parent body to crystallize. An x-ray structure was determined by molecular replacement. The electron density was clearly defined, partly as a result of averaging made possible by there being eight molecules in the asymmetric unit related by noncrystallographic symmetry (P1 space group). Virtually all of an asparagine-linked sugar cluster is present. Comparison with structures of the -and ␥-chain carboxyl domains of human fibrinogen revealed that the binding cleft is essentially neutral and should not bind Gly-Pro-Arg or Gly-His-Arg peptides of the sort bound by those other domains. Nonetheless, the cleft is clearly evident, and the possibility of binding a carbohydrate ligand like sialic acid has been considered.
Abstract. To identify regulatory mechanisms potentially involved in formation of actomyosin structures in smooth muscle cells, the influence of F-actin on smooth muscle myosin assembly was examined. In physiologically relevant buffers, AMPPNP binding to myosin caused transition to the soluble 10S myosin conformation due to trapping of nucleotide at the active sites. The resulting 10S myosin-AMPPNP complex was highly stable and thick filament assembly was suppressed. However, upon addition to F-actin, myosin readily assembled to form thick filaments. Furthermore, myosin assembly caused rearrangement of actin filament networks into actomyosin fibers composed of coaligned F-actin and myosin thick filaments. Severin-induced fragmentation of actin in actomyosin fibers resulted in immediate disassembly of myosin thick filaments, demonstrating that actin filaments were indispensable for mediating myosin assembly in the presence of AMPPNP. Actomyosin fibers also formed after addition of F-actin to nonphosphorylated 10S myosin monomers containing the products of ATP hydrolysis trapped at the active site. The resulting fibers were rapidly disassembled after addition of millimolar MgATP and consequent transition of myosin to the soluble 10S state. However, reassembly of myosin filaments in the presence of MgATP and F-actin could be induced by phosphorylation of myosin P-light chains, causing regeneration of actomyosin fiber bundles. The results indicate that actomyosin fibers can be spontaneously formed by F-actin-mediated assembly of smooth muscle myosin. Moreover, induction of actomyosin fibers by myosin light chain phosphorylation in the presence of actin filament networks provides a plausible hypothesis for contractile fiber assembly in situ. ~.Iow smooth muscle cells form contractile actomyosin fibrils is unknown. Studies to date have focused ,L on the assembly properties of isolated smooth muscle myosin. These investigations have consistently shown that, unlike skeletal muscle myosin, the assembly and enzymatic properties of smooth muscle myosin are closely linked (5,7,16,27,34,36,38,39,40). In particular, binding and hydrolysis of MgATP promotes depolymerization of nonphosphorylated smooth muscle myosin filaments in vitro (5,7,16,27,34,36,40). The effect is intimately linked to stabilization of the folded 10S myosin monomeric conformation resulting from active site trapping of ATP hydrolysis products (ADP.Pi) (7,8). Phosphorylation of myosin P-light chains, which is required for actin-activated MgATPase of myosin (reviewed in 29, 35), destabilizes the 10S conformation (8) and thereby promotes myosin assembly in the presence of MgATP (5,16,27,36,38,39).The presence of myosin thick filaments in both relaxed and contracting smooth muscle (32) excludes regulated assembly of myosin as a primary factor governing smooth muscle contractility. Nevertheless, the reversible assembly of smooth muscle myosin in vitro has prompted speculation that a pool of soluble 10S myosin may be present in smooth muscle ceils (8). Activation ...
The cross-linking of actin to myosin subfragment 1 (S-1) with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide was reexamined by using two cross-linking procedures [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Nature (London) 292, 301-306; Sutoh, K. (1983) Biochemistry 22, 1579-1585] and two independent methods for quantitating the reaction products. In the first approach, the cross-linked acto-S-1 complexes were cleaved with elastase at the 25K/50K and 50K/22K junctions in S-1. This enabled direct measurements of the cross-linked and un-cross-linked fractions of the 50K and 22K fragments of S-1. We found that in all cases actin was preferentially cross-linked to the 22K fragment and that the overall stoichiometry of the main cross-linked products was that of a 1:1 complex of actin and S-1. In the second approach, actin was cross-linked to tryptically cleaved S-1, and the course of these reactions was monitored by measuring the decay of the free 50K and 20K fragments and the formation of cross-linked products. After selecting the optimal cross-linking procedure and conditions, we determined that the rate of actin cross-linking to the 20K fragment of S-1 was 3-fold faster than the reaction with the 50K peptide. The overall rate of cross-linking actin to S-1 corresponded to the sum of the individual reactions of the 50K and 20K fragments, indicating their mutually exclusive cross-linking to actin. Thus, the reactions with tryptically cleaved S-1 were consistent with the 1:1 stoichiometry of actin and S-1 in the main cross-linked products and verified the preferential cross-linking of actin to the 20K fragment of S-1. These results are discussed in the context of the binding of actin to S-1.
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