Assembly of Acanthamoeba Myosin-II Minifilaments. Definition of C-terminal Residues Required to Form Coiled-coils, Dimers, and Octamers

Turbedsky, Kirsi; Pollard, Thomas D.

doi:10.1016/j.jmb.2004.10.049

Cited by 11 publications

(12 citation statements)

References 28 publications

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“…4B). This is in agreement with previous experiments in which removing the negatively charged region of the tailpiece from smooth muscle myosin II had no effect on filament assembly properties (24,46). Nevertheless the negatively charged Tailpiece 1968 -2000 was sufficient to determine NMII paracrystal morphology as seen in chimeric NMII isoforms in which only the negatively charged region was swapped among the isoforms (Fig.…”

Section: Discussionsupporting

confidence: 93%

The Positively Charged Region of the Myosin IIC Non-helical Tailpiece Promotes Filament Assembly

Rönen¹,

Rosenberg

Shalev

et al. 2010

Journal of Biological Chemistry

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The motor protein, non-muscle myosin II (NMII), must undergo dynamic oligomerization into filaments to participate in cellular processes such as cell migration and cytokinesis. A small non-helical region at the tail of the long coiled-coil region (tailpiece) is a common feature of all dynamically assembling myosin II proteins. In this study, we investigated the role of the tailpiece in NMII-C self-assembly. We show that the tailpiece is natively unfolded, as seen by circular dichroism and NMR experiments, and is divided into two regions of opposite charge. The positively charged region (Tailpiece 1946(Tailpiece -1967 ) starts at residue 1946 and is extended by seven amino acids at its N terminus from the traditional coiled-coil ending proline (Tailpiece 1953(Tailpiece -1967 ). Pull-down and sedimentation assays showed that the positive Tailpiece 1946 -1967 binds to assembly incompetent NMII-C fragments inducing filament assembly. The negative region, residues 1968 -2000, is responsible for NMII paracrystal morphology as determined by chimeras in which the negative region was swapped between the NMII isoforms. Mixing the positive and negative peptides had no effect on the ability of the positive peptide to bind and induce filament assembly. This study provides molecular insight into the role of the structurally disordered tailpiece of NMII-C in shifting the oligomeric equilibrium of NMII-C toward filament assembly and determining its morphology.Myosin II is a hexameric protein (Fig. 1A) of a family of actinbased molecular motors that are involved in cellular activities such as muscle contraction, cell migration, and cytokinesis (1-4). Each hexamer comprises two identical heavy chains and two sets of light chains. The N-terminal region of myosin II heavy chain is a globular head containing the actin binding and ATPase domains, followed by a large coiled-coil rod, which enables independent myosin II hexamers to assemble into large filaments (1). Non-muscle myosin II (NMII) 4 is a ubiquitous protein expressed by all cell types that mediates cellular processes that require contractility (2-4). In contrast to skeletal myosin II, NMII undergoes dynamic filament assembly-disassembly cycles to facilitate its diverse range of activities (5). NMII can only function when in filament form. Therefore, the process of filament assembly is an important regulatory step controlling NMII action (1). Several crucial factors for NMII filament assembly have been identified, including charge periodicity in the amino acid sequence along the coiled-coil rod and the presence of two small assembly competence domains located at the C terminus of the coiled-coil (6 -11). Additionally myosin II light chain phosphorylation activates the motor domain and promotes filament assembly (12)(13)(14).Three isoforms of NMII (NMII-A, NMII-B, and NMII-C) have been identified in mammals (15-17), each with specific tissue distributions and functions (18 -23). The C terminus of NMII contains a non-helical tailpiece (tailpiece) of 33-47 amino acids depe...

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Section: Discussionsupporting

confidence: 93%

The Positively Charged Region of the Myosin IIC Non-helical Tailpiece Promotes Filament Assembly

Rönen¹,

Rosenberg

Shalev

et al. 2010

Journal of Biological Chemistry

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“…It should be noted that the presence of hydrophobic residues at the a and d positions (Fig. 1B) is not necessarily a good predictor of coiled coil formation, because segments of myosin 2 tails up to about 100 residues long can be monomeric and largely unstructured in solution (28).…”

Section: Discussionmentioning

confidence: 98%

The Predicted Coiled-coil Domain of Myosin 10 Forms a Novel Elongated Domain That Lengthens the Head

Knight

Thirumurugan

et al. 2005

Journal of Biological Chemistry

141

186

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Myosin 10 contains a region of predicted coiled coil 120 residues long. However, the highly charged nature and pattern of charges in the proximal 36 residues appear incompatible with coiled-coil formation. Circular dichroism, NMR, and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single ␣-helix (SAH) domain in solution and does not dimerize to form a coiled coil even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin 10 containing the motor, the three calmodulin binding domains, and the fulllength predicted coiled coil showed that it was mostly monomeric at physiological protein concentration. In dimers the molecules were joined only at their extreme distal ends, and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin 5 heavy meromyosin obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus the predicted coiled coil of myosin 10 forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins 6 and 7a and MyoM and could function to increase the size of the working stroke.Myosins make up a diverse superfamily of motor proteins (1). The human genome alone contains about 40 myosin genes (2). Of these, about one-third are "conventional" myosins (i.e. the well studied myosin 2), and the rest fall into about 10 different classes. The structure, properties and functions of the majority of myosin classes are poorly characterized and have largely been inferred from sequence comparisons rather than direct experiments on purified proteins (1-3).Muscle myosin 2 dimerizes through its ␣-helical coiled-coil tail. Therefore, it is commonly assumed that any myosin will also be dimeric if it contains a region predicted to be coiled coil. This assumption is dependent on the accuracy of coiled-coil prediction programs, such as COILS (4), PAIRCOIL, or MULTICOIL (5), which are also used by protein-fold prediction sites on the Web such as SMART (6).Although myosin 10 contains a region of predicted coiled coil (Fig. 1A), and is predicted to dimerize, this has not been determined experimentally. We noticed that part of the predicted coiled-coil domain of myosin 10 is highly enriched in charged residues (Fig. 1B). The proximal region consisting of 36 residues is particularly enriched in both positively and negatively charged residues, including the a and d positions of the heptad repeat (a-g) that are canonically hydrophobic residues in coiled coils (Fig. 1B). We suspected that this highly charged sequence is unlikely to form a coiled coil (7), suggesting that this part of myosin 10 may not be able to dimerize....

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“…The assembly of full-length Acanthamoeba myosin II was studied extensively by Pollard and colleagues (12)(13)(14)(15)(16). They found that minifilaments (octamers) were formed in three steps when polymerized in ≤100 mM KCl and no ATP or MgCl 2 : Two monomers formed an antiparallel dimer with an ∼15-nm overlap of their tails and a bare zone of ∼160 nm; two dimers formed an antiparallel tetramer with a bare zone of ∼140 nm and an ∼15-nm stagger between the dimers; and two tetramers associated to form an antiparallel octamer with a bare zone of ∼117 nm and a stagger between tetramers of ∼30 nm.…”

Section: Discussionmentioning

confidence: 99%

“…Also, it had been concluded that only filamentous AMII has actin-activated MgATPase activity (7,8), and it was inferred that the ATPase activity is regulated by a change in the conformation of the bipolar minifilaments (9)(10)(11). However, detailed studies by the Pollard laboratory found no significant differences in either the polymerization properties or electron microscopic images of minifilaments of phosphorylated and dephosphorylated myosins (12)(13)(14)(15)(16).…”

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confidence: 96%

Regulation of the filament structure and assembly ofAcanthamoebamyosin II by phosphorylation of serines in the heavy-chain nonhelical tailpiece

Liu

Hong

Shu

et al. 2012

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

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Acanthamoeba myosin II (AMII) has two heavy chains ending in a 27-residue nonhelical tailpiece and two pairs of light chains. In a companion article, we show that five, and only five, serine residues can be phosphorylated both in vitro and in vivo: Ser639 in surface loop 2 of the motor domain and serines 1489, 1494, 1499, and 1504 in the nonhelical tailpiece of the heavy chains. In that paper, we show that phosphorylation of Ser639 down-regulates the actin-activated MgATPase activity of AMII and that phosphorylation of the serines in the nonhelical tailpiece has no effect on enzymatic activity. Here we show that bipolar tetrameric, hexameric, and octameric minifilaments of AMII with the nonhelical tailpiece serines either phosphorylated or mutated to glutamate have longer bare zones and more tightly clustered heads than minifilaments of unphosphorylated AMII, irrespective of the phosphorylation state of Ser639. Although antiparallel dimers of phosphorylated and unphosphorylated myosins are indistinguishable, phosphorylation inhibits dimerization and filament assembly. Therefore, the different structures of tetramers, hexamers, and octamers of phosphorylated and unphosphorylated AMII must be caused by differences in the longitudinal stagger of phosphorylated and unphosphorylated bipolar dimers and tetramers. Thus, although the actin-activated MgATPase activity of AMII is regulated by phosphorylation of Ser639 in loop 2 of the motor domain, the structure of AMII minifilaments is regulated by phosphorylation of one or more of four serines in the nonhelical tailpiece of the heavy chain.A s summarized in the accompanying paper (1), Acanthamoeba myosin II (AMII) is a typical class II myosin with two heavy chains and two pairs of light chains (2, 3). The coiled-coil helical tails of the heavy chains terminate with a 27-residue nonhelical tailpiece, 1483PSSRGGSTRGASARGASVRAGSARAEE1509, which has a pattern of four contiguous repeats of XXSXR (4). Previous work had shown that phosphorylation of two or more of these serines (residues 1489, 1494, 1499, and 1504) correlates with, and was assumed to be responsible for, inactivation of AMII's actin-activated MgATPase activity (5, 6). Also, it had been concluded that only filamentous AMII has actin-activated MgATPase activity (7, 8), and it was inferred that the ATPase activity is regulated by a change in the conformation of the bipolar minifilaments (9-11). However, detailed studies by the Pollard laboratory found no significant differences in either the polymerization properties or electron microscopic images of minifilaments of phosphorylated and dephosphorylated myosins (12-16).The earlier studies were carried out with purified endogenous myosin. To avoid possible complications in enzymatic and structural studies caused by the partial phosphorylation of purified endogenous myosin and incomplete dephosphorylation by phosphatase, we initiated studies on the enzymatic activity and structure of expressed recombinant wild-type, truncated, and mutant myosins before and after phos...

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Assembly of Acanthamoeba Myosin-II Minifilaments. Definition of C-terminal Residues Required to Form Coiled-coils, Dimers, and Octamers

Cited by 11 publications

References 28 publications

The Positively Charged Region of the Myosin IIC Non-helical Tailpiece Promotes Filament Assembly

The Positively Charged Region of the Myosin IIC Non-helical Tailpiece Promotes Filament Assembly

The Predicted Coiled-coil Domain of Myosin 10 Forms a Novel Elongated Domain That Lengthens the Head

Regulation of the filament structure and assembly ofAcanthamoebamyosin II by phosphorylation of serines in the heavy-chain nonhelical tailpiece

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