Identification of polyphosphate recognition sites communicating with the actin sites on the skeletal myosin subfragment-1 heavy chain

Abstract

Using the thrombin-cut [68-30 kilodalton (kDa)] myosin subfragment 1 (S-1) whose heavy chain has been selectively split within the central 50-kDa region, at Lys-560, with concomitant specific alterations of the ATPase and actin binding properties [Chaussepied, P., Mornet, D., Audemard, E., Derancourt, J., & Kassab, R. (1986) Biochemistry 25, 1134-1140; Chaussepied, P., Mornet, D., Barman, T., Travers, F., & Kassab, R. (1986) Biochemistry 25, 1141-1149], we have isolated and renatured the COOH-terminal 30-kDa fragment associated with the alkali light chains by the procedure recently described [Chaussepied, P., Mornet, D., Audemard, E., Kassab, R., Goodearl, J., Levine, B., & Trayer, I. P. (1986) Biochemistry 25, 4540-4547]. The 30-kDa peptide preparation was found to exhibit a crucial feature of the native S-1; namely, it interacts with F-actin in an adenosine 5'-triphosphate (ATP)-dependent manner. Studies by ultracentrifugation, turbidity measurements, and chemical cross-linking experiments showed that the acto-30-kDa peptide complex was dissociated almost completely by the gamma-phosphoryl group containing ligands ATP, 5'-adenylyl imidodiphosphate, and pyrophosphate, to a lesser extent by ADP, and not at all by AMP and inorganic phosphate. The maximal dissociating effect is operating with the thrombic 30-kDa entity, whereas the 22-kDa fragment produced by staphylococcal protease is only slightly dissociated. In contrast, the tryptic 20-kDa fragment binds irreversibly to actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping of the actomyosin interfaces.

“…(iii) The S1-HC C bound to G-actin in the same way as to F-actin following the same ionic dependence. (iv) The ATP dependence of the actin interaction with the S1-HC C was similar to that observed with otherwise prepared C-like fragments; i.e., 26-and 30-kDa fragments (34,35). All the functional similarities between recombinant and "natural" MHC fragments thus suggest that most of the observed interactions do not depend on the preparation method.…”

“…The observed effect of polyglutamic acid and the lack of direct ATP binding on S1-HC C suggest that the ATP effect was probably due to the charge of its polyphosphate moiety. The ATP dependence of all the actin binding previously observed on isolated natural S1-HC (1,35,38) and the recombinant S1-HC C probably result from the electrostatic charges of the ATP molecule. S1-HC N was designed to test its interaction with actin and ATP (1).…”

“…The functional S1-HC C was designed to center around the well-documented, very flexible loop (residues 625-640), which has been shown to play a crucial role both in connecting the functional parts of myosin heads (7,8) and in F-actin activation of S-1 ATP hydrolysis (6,(35)(36)(37). Sequence analyses revealed that this junctional region differs substantially throughout the myosin isoform family, suggesting that it is a hypervariable "connector," a critical modulator of the actomyosin interface.…”

“…S1-HC N was designed to test its interaction with actin and ATP (1). S1-HC M was designed to test the role of the central MHC fragment in communications between the two putative distal actin binding sites and the ATP binding site within the myosin head (9,35,39). The present observations showing cosedimentation of S1-HC N with F-actin are consistent with the previous direct measurements of actin interactions with the N-terminal 23-kDa fragment of S1-HC (1).…”

Decavanadate Binding to a High Affinity Site near the Myosin Catalytic Centre Inhibits F-Actin-Stimulated Myosin ATPase Activity

“…The large effect of KCl on the dissociation constant of V 10 from S1 indicates that electrostatic interactions plays a major role in V 10 (a polyanion) binding to myosin. The presence of a binding site for polyvalent anions in the head of myosin has been well documented (14,(54)(55)(56)(57), and it was proposed that they could account for the inhibition of the actomyosin ATPase activity by polyvalent anions (54). Although the surface electrostatic charge contour of S1 reveals the presence of a protein region positively charged from residues 627 to 646, containing five Lys (loop 2), which makes part of the actin-binding interface (50), it is very unlikely that the V 10 polyanion high-affinity binding site we are probing at the present work is located at this protein region because V 10 binding to S1 is not affected by actin.…”