Function of the head–tail junction in the activity of myosin II

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Highlights

  • All conventional myosins have the conserved Pro-Leu-Leu at their head–tail junctions.

  • We examined the conserved amino acid sequence by site-directed mutagenesis.

  • The first Leu is important for actin-activated ATPase activity while the Pro and the second Leu are not.

Abstract

All class II myosins have the conserved amino acid sequence Pro-Leu-Leu at their head–tail junctions. We systematically altered this sequence in smooth muscle heavy meromyosin (HMM) by site-directed mutagenesis and examined the effects of these mutations on actin–myosin interactions. Deletion of the proline and second leucine did not cause any noticeable change in either actin-activated ATPase activity or actin-sliding velocity. In contrast, deletion of the two leucine residues and substitution of the first leucine with alanine resulted in a 14-fold and 5-fold decrease, respectively, in actin-activated ATPase activity. However, both these mutations did not appreciably affect actin-sliding velocity, which was consistent with a result that there was no considerable change in the ADP release rate from acto-HMM in the deletion mutant. In contrast to double-headed HMM, a single-headed subfragment-1 (S1) with a Leu-Leu deletion mutation exhibited actin activated ATPase activity similar to that by wild type S1. Our results suggest that the first leucine of the conserved Leu-Leu sequence at the head–tail junction profoundly affects the cooperativity between the two heads involved in the actin activated ATPase activity of myosin II.

Introduction

Myosin II is composed of a pair of heavy chains, essential light chains and regulatory light chains. The N-terminus of each heavy chain forms a globular structure called S-1 or head, which contains an actin binding site, an ATPase site and two light chains. A long α-helical light chain-binding site together with two light chains forms a rigid bar, which acts as a lever arm to generate movement [1], [2], [3]. The remainder of the heavy chain forms a long tail consisting of an α-helical coiled coil structure, which confers the two-headed structure of myosin II.

Recent studies have suggested that one head of myosin II assists the other head by enabling its favorable orientation and optimal interaction with actin, thus enabling its maximal performance. Binding of one head to actin increases the second head’s probability of binding to actin [4]. A single-headed myosin II constructed by coexpressing the tail portion of Dictyostelium myosin II heavy chain together with the entire heavy chain of myosin II exhibited only half the Vmax value of the actin-activated ATPase activity per head and half the sliding velocity compared to those with native double-headed myosin II [5]. Tyska et al. proteolytically produced single-headed skeletal and smooth muscle myosins and compared their actin displacement and force generation with those of double-headed ones. Single molecular analysis of these myosins revealed that a double-headed myosin produced twice the unitary displacement and twice the force of a single-headed myosin [6]. Because the difference in the unitary displacement was not due to a successive stroke of the two heads, they suggested that one head merely led the other head to its optimal interaction with actin. A mutation in the nucleotide binding site (E470A) locked myosin in a weak actin-binding state [7], [8]. A heterodimeric myosin in which one heavy chain had the E470A mutation and the other heavy chain was that of the wild type exhibited the same step size as wild-type myosin. This suggested that the presence of the first, weakly bound head was necessary to produce maximal displacement of the motion-generating second, strongly bound head [9].

The mechanism of how the first head helps the second head to optimally perform its full function and the nature of head–head interaction are not well understood. Head–head interaction at the N-terminal segment is known to occur when the two heads are bound to actin [10]. The head–tail junction also appears to be critical for both head orientation and the efficient transmission of force produced during the mechanochemical cycle of interaction between myosin heads with actin and ATP.

All myosin II molecules have a highly conserved amino acid sequence (Pro-Leu-Leu) at their head–tail junctions. We systematically altered this region in smooth muscle heavy meromyosin (HMM) by PCR-based mutagenesis and examined the actin activated ATPase activity and the actin-sliding velocity of these mutants. We found that the actin-activated ATPase activity of some of these mutants was considerably lower than that of the wild type, suggesting the importance of the head–tail junction in actin–myosin interactions.

Section snippets

HMM constructs

Chicken smooth muscle heavy meromyosin heavy chain in pFastBac (pFastBac his-HMM-myc) was a gift from Dr. Onishi [11]. This construct contains a His-tag at the N-terminus followed by chicken smooth muscle myosin heavy chain from positions 1–1315 and a myc tag. Mutant constructs were generated by PCR based site directed mutagenesis (ExSite, Stratagene). Primers used to generate AL, LA, del-LL, and del-QV were CTCTACAGGTCACCCGCC and CCGGCTTCACTTTGGTG, CGCAGGTCACCCGCC and

Mutant HMMs

The C-terminal half of a myosin II heavy chain has a heptad repeating sequence, which allows this region to dimerize in the form of the α-helical coiled-coil tail [24]. The COILS program provided by Lupas et al. (http://www.ch.embnet.org/software/COILS_form.html, [25]) predicted that the head of chicken smooth muscle myosin II would terminate at Pro-849 and the coiled-coil tail would begin at Leu-850. The Pro-Leu-Leu sequence at the head–tail junction is highly conserved in myosin II (Fig. 1;

Discussion

Both actin activated ATPase activity and the affinity for actin of a double-headed smooth muscle myosin HMM are higher than those of a single-headed myosin S1 [6]. Actin sliding velocity and the unitary step of a double-headed myosin II are also higher than those of a single-headed myosin II molecules produced by genetic engineering [5] or by the proteolytically method [30]. It has been suggested that one head of myosin II helps the other head of the same molecule to bind to actin [9]. The

Acknowledgments

We thank Dr. Hirofumi Onishi for providing pFastBac-chicken smooth muscle myosin heavy chain with a His-tag, pFastBac-chicken smooth muscle myosin essential light chain, and pFastBac-chicken smooth muscle myosin regulatory light chain constructs. We also thank Dr. Kazuo Sutoh for allowing the use of the stopped-flow apparatus.

References (30)

  • S.S. Rosenfeld et al.

    The ATPase mechanism of skeletal and smooth muscle acto-subfragment 1

    J. Biol. Chem.

    (1984)
  • H.L. Sweeney et al.

    Kinetic tuning of myosin via a flexible loop adjacent to the nucleotide binding pocket

    J. Biol. Chem.

    (1998)
  • I. Rayment et al.

    Three-dimensional structure of myosin subfragment-1: a molecular motor

    Science

    (1993)
  • T.Q.P. Uyeda et al.

    The neck region of the myosin motor domain acts as a lever arm to generate movement

    Proc. Natl. Acad. Sci. USA

    (1996)
  • K.M. Trybus et al.

    Spare the rod, spoil the regulation: necessity for a myosin rod

    Proc. Natl. Acad. Sci. USA

    (1997)
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    These authors contributed equally to this work.

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