Proteolytic approach to structure and function of actin recognition site in myosin heads

Mornet, Dominique; Bertrand, Raoul; Pantel, Pierre; Audemard, Etienne; Kassab, Ridha

doi:10.1021/bi00511a007

Cited by 172 publications

(141 citation statements)

References 71 publications

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“…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].…”

Section: Introductionmentioning

confidence: 99%

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

1989

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The reaction of endoproteinase Arg-C on the skeletal myosin head heavy chain was investigated through characterization of peptides and amino acid sequence analysis. The protease splits exclusively the 50 kDa-20 kDa junction at the lysine cluster spanning residues 639~i41 and does not affect any other protease-sensitive region of the entire myosin heavy chain. The sensitivity of the cleavage to actin and nucleotide binding makes this protease a very specific conformational probe of S-1. The nicked S-1 derivative, containing an intact NH2-terminal 75 kDa fragment, may serve as a tool for gaining further insights into the domain structure and function of the myosin head.

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Section: Introductionmentioning

confidence: 99%

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

1989

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“…Crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide shows that the first (NH2-terminal) 12 residues of actin (27), but possibly other regions as well (28), bind to either side of connector II. (It remains undecided whether in three dimensions the two binding regions of S-1 form a single site for binding one actin or two sites for different actins.)…”

mentioning

confidence: 99%

On the mechanism of energy transduction in myosin subfragment 1.

Botts¹,

Takashi²,

Torgerson³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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It is proposed that the myosin subfragment 1 moiety of the muscle contractile apparatus is a self-contained "engine" whose operational plan is based on the interactive nature of ATP (or degradation intermediate) binding and actin binding, made possible by an intersite communication system. It is suggested that the spatial information required for examining this engine can, at least provisionally, be derived from fluorescence resonance energy transfer measurements interpreted by the Forster equation and that the existence of an intersite communication system can be deduced from piecewise detection of interacting pairs of points. A Theory of How the Myosin Subfragment 1 (S-i) Engine WorksIt has been thought that myosin S-1 moieties in active muscle fibers, pivoting about their attachments to subfragment 2 (S-2) moieties, deliver mechanical impulses to adjacent actin filaments, thus producing the relative interfilament translation ("slide") that characterizes isotonic muscle contraction (see ref. 1 for a review). Since there is no evidence that the gross shape (as distinct from local conformation) of S-1 changes during interaction with nucleotides (2) or actin (3), it is plausible to think that in its "purposeful" motion, as in its thermal motion, the cross-bridge rotates about S-1/S-2 as a quasirigid body. Because S-1 is elongate, a natural positional parameter is the angle that its "long" axis makes with the filament axis (4). There are now investigations showing that during isometric activity (i) this angle fluctuates (5) (7). Furthermore, the motional frequencies observed in i and iii are what might be expected from independent biochemical (8) and mechanical (9) observations and are magnitudes slower than thermally activated frequencies. Thus points i-iii support the idea that during activity cross-bridges move. They also suggest that the motion is describable by angles that position S-1 relative to the fiber axis (10), but in what follows it is necessary only that positional parameters relating S-1 to actin change with time repetitively.It is known that each S-1 bears an ATPase (N) site separate from the site (A) at which it binds actin (11) and that binding at these sites is interactive (12, 13). So it is tempting to think that the observed motions of S-1 are not those of an appendage passively moved by another agency, but rather that S-1 is a self-contained "unitary engine" strategically placed in the muscle apparatus. Previously we have elaborated on this hypothesis (14). We have noted that to examine this hypothesis we must look at common knowledge about S-1 in an uncommon way. For example, instead of considering S-1 motion in a fiber-based coordinate system we should position ligands (actin, nucleotides) in an S-1-based system, and we should think of enzymatic intermediates (15) as sequentially bound N-site ligands. These simple reinterpretations suggest a plan for the S-1 "engine": Because of the intersite communication system, binding of a particular intermediate at the N site determines a parti...

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“…Because the LC2 extremity has been located [7] close to the neck of myosin, it is not unreasonable to view this interaction zone as being in the filament shaft. In the light of recent findings that LC2 interacts with the C-terminal region of S1 [8], it must be assumed that the ATPase site transconformation is conveyed through this segment, believed to be also involved in actin binding [21,22]. This suggests that the attribution of specialized functions to particular 'domains' delimited along the linear amino acid sequence is an oversimplified picture; the three tryptic peptides in S1 were previously shown to be maintained in a stable functional tertiary structure [23], evidence that these three domains present a considerable degree of entanglement.…”

Section: Discussionmentioning

confidence: 99%

Proteolysis rates of a myosin heavy chain site with papain Evidence for a combined LC2‐filament‐mediated mechanism

Cardinaud

1987

FEBS Letters

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In striated muscle myosin, a proteolysis site at the 25-50 kDa junction, susceptible in the filament and efficiently protected by nucleotides, is similarly protected when myosin is monomeric. Kinetic studies at low ionic strength show a close relationship between LC2 cleavage or degradation rate and cleavage of the 25-50 kDa heavy chain site. The myosin-[(T)-LC2'] species forms normal reconstituted filaments but its 25-50 kDa site susceptibility is closer to that of monomeric myosin, thus becoming practically ionic strength-independent. In this species the absence of the LC2 N-terminal segment induces a significantly greater susceptibility of the papain-sensitive site in LC1. In an LC2-depleted myosin the 25-50 kDa site susceptibility also becomes ionic strength-independent, however, the cleavage rates are then closer to that of filaments. Susceptibility in HMM and S1 is also much less dependent on ionic strength with rates intermediary between those of filament and monomer. These observations show that the maximum susceptibility to papain of the 25-50 kDa site requires both the integrity of the LC2 light chain and the filament structure and furthermore provide evidence that: (i) the LC2 N-terminus interacts specifically with some part of the filament; (ii) this interaction induces a specific transconformation in a region close to the ATPase active site; (iii) there is an interrelationship between LC 1 and LC2 light chain N-terminal extremities, at least in the filament structure. Studies related in the introductory part were presented at

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Proteolytic approach to structure and function of actin recognition site in myosin heads

Cited by 172 publications

References 71 publications

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

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

On the mechanism of energy transduction in myosin subfragment 1.

Proteolysis rates of a myosin heavy chain site with papain Evidence for a combined LC2‐filament‐mediated mechanism

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