Journal of Molecular Biology
Volume 345, Issue 2, 14 January 2005, Pages 351-361
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Assembly of Acanthamoeba Myosin-II Minifilaments. Definition of C-terminal Residues Required to Form Coiled-coils, Dimers, and Octamers

https://doi.org/10.1016/j.jmb.2004.10.049Get rights and content

Acanthamoeba myosin-II forms bipolar octamers by three successive steps of dimerization of the C-terminal, coiled-coil tail. In this study, we generated N-terminal and C-terminal truncation constructs and point mutants of the Acanthamoeba myosin-II tail to delineate the structural requirements for assembly of bipolar mini-filaments. By the use of light-scattering, CD spectroscopy, analytical ultracentrifugation, and tryptophan fluorescence experiments, we determined that: (1) the C-terminal 14 heptad repeats plus most of the tailpiece (residues 1381–1509) are required to form antiparallel dimers of coiled-coils; (2) amino acid residues within heptads 23–32 (residues 1254–1325) are required to form tetramers; (3) the C-terminal 32 heptad repeats suffice to assemble octameric minifilaments; (4) A1378 is outside of the interaction interface; (5) the mutation L1475W inhibits dimerization; and (6) F1443 is involved in the dimerization interface but is exposed to the solvent. We propose that the tailpiece (residues 1483–1509) interacts with two heptads (13 and 14, residues 1381–1393), which are important for dimerization and coiled-coil formation. These results support a model in which hydrophobic as well as electrostatic interactions control the register between myosin-II coiled-coils and guide sequential steps of dimerization that generate stable, octameric mini-filaments.

Introduction

Acanthamoeba myosin-II provides an attractive system to study the assembly of bipolar filaments, because these minifilaments consist of only eight monomers and the assembly pathway is well characterized.1, 2, 3 Two myosin monomers bind very rapidly (k+>108 M−1 s−1) and with high affinity (Kd<10−11 M) to form antiparallel dimers with an overlap of ∼15 nm (Figure 1). Two antiparallel dimers associate with another ∼15 nm stagger to form an antiparallel tetramer. Finally, two tetramers bind with a ∼30 nm stagger to form octamers. In millimolar concentrations of divalent cations or at acid pH, minifilaments associate laterally to form thick filaments. Both minifilaments and thick filaments have been observed in fixed4 and in live cells.5

The Acanthamoeba myosin-II tail, like other type-II myosins, contains extensive heptad repeats of hydrophobic residues characteristic of coiled-coils (Figure 2),6 as well as 28 residue charge repeats implicated in defining the ∼14.3 nm stagger of molecules within myosin filaments.6, 7, 8 The middle of the Acanthamoeba myosin-II tail has a helix-breaking proline residue (Pro1244) surrounded by ∼20 residues with low propensity to form α-helix. This hinge region results in a statistically significant bend in electron micrographs of Acanthamoeba myosin-II monomers.6 A second proline residue (Pro1483) initiates the non-helical tailpiece, which consists of the last 27 residues and contains multiple serine and glycine residues instead of the coiled-coil heptad repeat.9

Previous work identified the critical role of the C-terminal end of the myosin-II tail in assembly. Studies using 25 different monoclonal antibodies with mapped epitopes and nine C-terminal truncations identified regions of the coiled-coil tail involved in each step of Acanthamoeba myosin-II assembly.10, 11 For example, antibodies that bind tightly in the last 37 nm of the tail or deletion of more than 100 residues at the C terminus precluded assembly. In electron micrographs of Acanthamoeba myosin-II at high concentrations of salt, the tailpiece of one myosin occasionally associates with another myosin tail ∼15.0 nm from the C terminus.12 Constructs missing as few as 15 residues from the tailpiece do not form antiparallel dimers but are capable of making parallel dimers with a stagger similar to that observed for tetramers. In addition, monoclonal antibodies that bind near residue 1380, ∼15.0 nm from the C terminus, inhibit the antiparallel dimerization of myosin-II.10 Therefore, the tailpiece (residues 1483–1509) is clearly essential for step 1 of assembly. Further experiments using monoclonal antibodies show that residues between 1380 and 1481 are involved in formation of the ∼15 nm stagger in step 2, and that residues between 1280 and 1380 are important in step 3.

Here, we performed biophysical measurements of recombinant N-terminal and C-terminal constructs to define the regions within the myosin-II tail that are required for assembly. We tested N-terminal truncation constructs and point mutations to determine the minimal sequence required to form specific assembly intermediates. By the use of light-scattering, CD spectroscopy, analytical ultracentrifugation, and tryptophan fluorescence, we determined that the C-terminal 32 heptad repeats suffice to assemble octamers, while the C-terminal 14 heptad repeats plus most of the tailpiece are required to form dimers. In addition, we show that residues 1381–1393 are involved in dimerization and coiled-coil formation.

Section snippets

Design and purification of constructs

We designed a series of N-terminal and C-terminal truncation constructs of the Acanthamoeba myosin-II tail on the basis of assembly and stability criteria to examine the structural requirements for assembly of bipolar filaments. Computational algorithms13, 14 predicted that the coiled-coil begins after Pro847 and ends at Pro1483, encompassing a total of 90 heptad repeats. Our longest construct with 32 heptads (32T) begins at residue 1254 and was predicted to form dimers and tetramers. This 34 nm

Discussion

In this study, we generated N-terminal and C-terminal truncation constructs and point mutants of the Acanthamoeba myosin-II tail to delineate the structural requirements for formation of the coiled-coil and the assembly of the coiled-coil into dimers, tetramers and octamers. CD spectroscopy revealed the α-helical content of the oligomers, and analytical ultracentrifugation studies documented the size of the products. Lateral interactions between dimers were examined by fluorescence spectroscopy

Cloning

Truncation constructs were obtained by PCR with the Elongase Enzyme Mix (Life Technologies, Inc.) using Acanthamoeba myosin-II cDNA as a template with primers generating various N and C termini (Figure 2). We designed a total of 13 N-terminal primers to produce constructs ranging in length from six to 32 heptads (Figure 2, Figure 3). Three C-terminal primers were designed to produce variable ends based on protein domains and proteolytic sensitive sites. The three C-terminal primers created

Acknowledgements

We thank Mark Yeager for helpful discussions and a critical reading of the manuscript. This work was supported by NIH research grant GM26132.

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    Present address: T. D. Pollard, Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA

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