Myosin chaperones.

The folding and assembly of myosin motor proteins is essential for most movement processes at the cellular, but also at the organism level. Importantly, myosins, which represent a very diverse family of proteins, require the activity of general and specialized folding factors to develop their full motor function. The activities of the myosin-specific UCS (UNC-45/Cro1/She4) chaperones range from assisting acto-myosin dependent transport processes to scaffolding multi-subunit chaperone complexes, which are required to assemble myofilaments. Recent structure-function studies revealed the structural organization of TPR (tetratricopeptide repeat)-containing and TPR-less UCS chaperones. The observed structural differences seem to reflect the specialized and remarkably versatile working mechanisms of myosin-directed chaperones, as will be discussed in this review.

Current Opinion in Structural Biology 2014, 25: 9–15

This review comes from a themed issue on Macromolecular machines

Edited by Karl-Peter Hopfner and Tom Smith

For a complete overview see the and the

Available online 3rd December 2013

0959-440X/$ – see front matter, © 2014 The Authors. Published by Elsevier Ltd. All rights reserved.

Introduction

Myosins are cytoskeletal motor proteins that use the energy of ATP hydrolysis to move along actin filaments. The molecular force generated by acto-myosin complexes is critical for a plethora of biological processes including cell motility, adhesion, endocytosis and neuron growth [1]. Moreover, myosin molecules are essential to transport molecular cargo along specific actin tracks to various locations within the cell [2]. The extraordinary strength of the myosin motor becomes evident in the sarcomeres of our skeletal muscles, when myosin (thick) filaments pull on actin (thin) filaments leading ultimately to muscle contraction [3,4]. To this end, the molecular motor of myosin represents an intricately folded structural motif that is pieced together by different protein domains including a 50 kDa ATPase entity, a composite actin-binding motif, an SH3 (Src homology 3)-like domain and the converter region (Figure 1a). The myosin motor is followed by the so-called neck region that harbors the landing sites, the IQ (Ile, Gln) motifs, for regulatory partner proteins, the so-called light chains. The C-terminal tail region is most diverged among the 35 different myosin classes containing coiled-coil dimerization domains and binding sites for cargo and adaptor proteins [5].

Figure 1

Organization of myosin. (a) Structure of the myosin motor domain. Cartoon representation of the myosin V head domain (PDB code: 1w7i). The conserved domain structure of myosin proteins is indicated with the 50 kDa ATPase domain shown in green, the N-terminal (often SH3-like) domain in blue, the converter in magenta and the neck in yellow. The N-termini and C-termini of the molecule are labeled pointing to the highly intertwined tertiary structure of the myosin head domain. (b) Schematic representation of a sarcomeric unit. Actin filaments (grey) interdigitate with myosin II filaments (green). Myosin heads protrude as dimeric motifs from the filament backbone to interact with their actin counterparts. (c) Schematic representation of myosin mediated cargo transport in S. cerevisiae. Myosin V dimers (green), loaded with vesicular cargo, walk along actin filaments (grey).

In this review, we will discuss the folding and assembly of myosin proteins belonging to class II and V, illustrating distinct features of myosin-specific chaperones. The class II proteins comprise the largest myosin family, being expressed in non-muscle cells, where they are critical to assemble the cytokinetic ring during cell division, and in muscle cells forming the characteristic thick filaments of cardiac and skeletal muscle cells (Figure 1b) [3,5]. The basic building block of the thick filament is a hexamer, which is composed of two myosin heavy chains, two essential and two regulatory light chains. Formation of myofilaments is believed to proceed by the staggered association of adjacent hexamers via their coiled-coil regions. Electron microscopic studies of resultant thick filaments illustrate that the ATPase heads of myosin protrude in a helical fashion from the backbone of the filament having an axial spacing of about 145 Å [6]. Importantly, the correct patterning of myosin thick filaments is essential for the functional interplay with actin. Only precisely aligned myosin head domains can interact with their actin partner proteins in the so-called ‘crossbridge cycle’ and convert the chemical energy derived from ATP hydrolysis into mechanical work thereby mediating muscle contraction [4]. Class V proteins belong to the unconventional myosins found in a variety of different cell types. They are involved in organizing the cellular environment by transporting cellular contents ranging from protein complexes to whole organelles (Figure 1c) [2]. With regards to oligomer structure, these myosins do not form higher-order oligomers, but walk as dimers along actin tracks.

Myosin folding and assembly

Given the apparent importance of myosin activity for life on earth, folding of myosin represents an equally important process, which, however, is only little understood. To this end, the myosin motor domain comprises a relatively complicated protein fold that requires several folding factors to reach its functional state, and consequently, to interact with its partner proteins. In particular, the folding of muscle myosin II asks for a specialized chaperone mechanism. Notably, the assembly of the myosin filament and its organization within the muscle sarcomere has to be coordinated — spatially and temporally — with the folding of the motor domain. A recent genetic screen has implicated more than 100 assembly factors to be involved in this process [7], however, only very few of them have been characterized so far. Among the characterized factors is the CCT chaperonin that is implicated in promoting the coiled-coil interactions required for heavy chain dimerization [8]. Moreover, it has been shown that the functionality of the head domain of skeletal muscle myosin relies on several accessory proteins [9,10] including the general folding factors Hsp70 and Hsp90 and a myosin-specific chaperone having a characteristic UCS (UNC-45/Cro1/She4) domain.

Hsp70 and Hsp90 are ATP-dependent chaperones that maintain protein homeostasis in all prokaryotic and eukaryotic cells. The most prominent roles of Hsp70 and Hsp90 include the folding of newly synthesized proteins as well as quality-controlling cellular proteins during diverse stress situations [11-13]. Also in the case of myosin, the two general chaperones have been proposed to interact with premature proteins and promote the folding of myosin molecules during the formation of thick filaments in striated muscle cells [10]. The role of Hsp90 in myosin folding and assembly has furthermore been established by genetic studies. A comprehensive analysis of Hsp90 in Caenorhabditis elegans revealed that mutations that reduce its ATPase activity lead to aggregation of myosin [14]. Myofibrillar disorganization and associated lack of skeletal muscle contraction has also been observed upon Hsp90 knockdown and mutations reducing the ATPase activity in zebrafish embryos [15,16].

In contrast to Hsp70 and Hsp90, UCS proteins function as specialized chaperones acting exclusively on the myosin substrate. UCS proteins, which share a highly conserved ARM (armadillo repeat motif) domain of about 400 residues (the UCS domain), are expressed in all eukaryotes and exhibit a remarkably large spectrum of chaperone activities (Table 1). Fungal UCS proteins comprise Podospora anserina Cro1, Saccharomyces cerevisiae She4 and Schizosaccharomyces pombe Rng3 and are involved in a variety of acto-myosin dependent processes such as cytokinesis, mRNA transport and endocytosis. Though not all are essential for myosin function, the fungal UCS proteins can interact with different myosin classes and contribute to their stability [17-19]. UCS proteins of metazoans, to which we refer as UNC-45 proteins, are essential for cell proliferation, cytokinesis and for organizing thick filaments in muscle sarcomeres (Table 1). In contrast to the fungal proteins, UNC-45 proteins have an additional N-terminal TPR (tetratricopeptide repeat) domain allowing for collaboration with Hsp70 and Hsp90 [20,21]. Moreover, the substrate specificity of UNC-45 proteins appears to be more pronounced as an interaction has been shown only for myosin type II forms [20,22].

Table 1

UCS chaperones and their functions

UCS protein		Function
Fungal/TPR-less UCS chaperones	P. anserina Cro1	• Sexual reproduction (generation of functional myosin?) [23]
	S. cerevisiae She4	• Proper localization of mRNAs (myosin-dependent transport) [24]
		• Endocytosis [25]
		• Myosin stabilization and proper localization [17–19]
		• Organization of actin cyctoskeleton [25]
		• Establishing acto-myosin interaction [19]
		• Determining step size of myosin walking along actin filaments [26••]
	S. pombe Rng3	• Cytokinesis (establishing acto-myosin interaction in the cytokinetic ring) [19,27]
		• Co-translational myosin folding [28]
UNC-45 proteins/TPR-containing UCS chaperones	C. elegans UNC-45	• Cytokinesis (proper folding of non-muscle myosin) [22,29]
		• Sarcomere formation in the body wall muscles (muscle myosin folding and thick filament assembly) [20,30,31]
	Drosophila melanogaster UNC-45	• Sarcomere formation in skeletal and heart muscles (muscle myosin folding and thick filament assembly) [32,33]
	Vertebrate UNC-45a	• Non-muscle myosin folding [34]
		• Cell proliferation [35]
		• Aortic arch development [36]
		• Conferring resistance to histone deacetylase inhibitors [37]
		• Assisting Hsp90 dependent folding of the progesterone receptor [38]
	Vertebrate UNC-45b	• Sarcomere formation in skeletal and heart muscles (muscle myosin folding and thick filament assembly) [35,39–41]
		• Ensuring myosepta and myofibre integrity (by interplay with Apo2) [42]
		• Muscle myosin maintenance [43••]

Structural organization of UNC-45 proteins

Only recently, structural analysis of three UCS proteins provided first mechanistic insight into the function of a myosin-specific chaperone [21]. As seen in the crystal structure of C. elegans ceUNC-45 [21], UCS proteins have a rigid protein backbone, which is established by the central and the neck domain that fold into a series of irregular ARM repeats. TPR and UCS domains are attached as functional entities that mediate the binding of partner chaperones and myosin substrates, respectively (Figure 2a). In addition to orienting the functional domains to each other, the central-neck backbone is critical for oligomer formation, as this motif promotes the assembly of linear ceUNC-45 chains. The TPR domain at the N-terminus consists of three TPR repeats that fold into a compact protein–protein interaction moiety. Co-crystal structures with Hsp70 and Hsp90 peptides revealed that the two general chaperones compete for the same binding site on the concave face of the TPR domain, with Hsp90 having a 10-fold higher affinity. Opposite of the TPR domain lies the C-terminal UCS domain that is composed of eight regular ARM repeats assembling a right-handed superhelix. The UCS domain is penetrated in its entire length by a deep canyon. Given the structural homology to beta-catenins, the UCS canyon is well-suited for binding a polypeptide stretch of myosin in an unfolded, extended conformation. The crystal structure of S. cerevisiae She4 highlights the distinct domain orientation of the TPR-less UCS chaperones (Figure 2b) [26]. Most notably, the neck region of the yeast protein is tilted by 90° against the central domain such that it can directly participate in dimer formation. In the resultant Z-shaped molecule, the relative orientation of the UCS domains, which are related by two-fold symmetry, is strikingly different compared to the tandemly arranged UCS repeats of the ceUNC-45 chaperone chain (Figure 2).

Figure 2

Structures of UCS proteins. (a) Structure of C. elegans UNC-45 (PDB code: 4i2z). Upper panel: Cartoon representation of an UNC-45 protomer with the TPR, central, neck and UCS domains shown in green, orange, yellow and grey, respectively. Lower panel: UNC-45 chain (three protomers are shown) as observed in the crystal lattice. The co-crystallized Hsp90 peptide (magenta) marks the TPR interaction site for partner chaperones. Interaction of the UCS domain with the myosin substrate (blue) was modeled on the basis of the beta-catenin/E-cadherin co-crystal structure (PDB code: 1i7x). (b) Structure of S. cerevisiae She4 (PDB code: 3opb). Upper panel: Cartoon representation of a She4 molecule. Domain colors as in (a). Lower panel: The She4 dimer as observed in the crystal packing. The central and neck domains constitute the dimerization interface.

UCS proteins — versatile myosin chaperones

On the basis of the recent structural data, two concrete models have been put forward describing different chaperone functions of UCS proteins that may reflect the common roles of TPR-containing and TPR-less homologues. Consistent with their different structural organization, S. cerevisiae She4 and C. elegans UNC-45 seem to exhibit highly specialized functions in the cell regulating specific myosin-based processes. Moreover, the recent structural, biochemical and in vivo data highlight the remarkable versatility of UCS proteins in folding and activating their myosin substrates by different means.

Fungal UCS proteins are organized as three-domain proteins lacking the TPR entity. S. cerevisiae She4 folds into 16 helical repeats that assemble an L-shaped superhelix. Strikingly, in the crystal lattice, She4 associates with a neighboring molecule yielding a Z-shaped dimer (Figure 2b). Moreover, biochemical data suggest that the She4 UCS domain accommodates a 27-residue epitope of a yeast myosin V protein, which is located in close proximity to the nucleotide-binding and actin-binding sites of the myosin motor domain [26]. On the basis of these findings, it was proposed that the She4 dimer may physically link two myosin motor domains thereby determining the step size of myosin molecules walking along actin filaments (Figure 3).

Figure 3

UCS proteins employ distinct mechanisms to exert their chaperone activity. Left panel: TPR-containing UCS chaperones function as oligomers in myosin folding and thick filament assembly. UNC-45 chains assemble a platform to foster myosin and partner chaperone (Hsp70, Hsp90) interactions. In parallel this assembly line should help to arrange myosin II head domains along thick filaments. Right panel: TPR-less UCS proteins work as dimers. In addition to promoting myosin folding, the She4 dimer can also interact with folded myosin, determining the step-size of myosin V when walking along actin filaments.

In higher eukaryotes, which evolved muscle tissues containing arrays of highly ordered myosin filaments, UCS proteins seem to have co-evolved a more sophisticated version of a myosin chaperone. In addition to the myosin-binding UCS domain, the corresponding UNC-45 proteins employ an N-terminal TPR domain that is instrumental to bind Hsp70/Hsp90 chaperones thus allowing to establish multi-chaperone complexes [21]. The crystal structure of C. elegans UNC-45 did not only reveal the overall organization of TPR, central, neck and UCS domains in forming a mouth-like chaperone structure, but, most importantly, uncovered the capability of UNC-45 proteins to form linear, polar protein filaments. Accordingly, UNC-45 is able to adopt a similar filamentous structure as its native myosin substrate. The backbone of the observed UNC-45 filament is formed by the central and neck domains leaving the functional TPR and UCS domains free to interact with partner chaperones and myosin substrates (Figure 2a). Accordingly, UNC-45 assembles a multimeric scaffolding complex that offers binding sites for Hsp70 and Hsp90 chaperones in a precisely defined pattern to work on the array of myosin head domains protruding from prearranged myofilaments (Figure 3). In fact, the periodicity of UNC-45 molecules within the chaperone chain is similar to the spacing of adjacent myosin heads along the thick filaments. The crystallized UNC-45 filament could be also observed in solution by employing a directed photo-crosslinking methodology. This approach demonstrated the transient and concentration dependent formation of short UNC-45 chains, having 2–5 subunits. Strikingly, the relevance of UNC-45 chains to serve as a multimeric patterning complex during muscle development could be confirmed in vivo. Upon expressing UNC-45 mutants that specifically impede UNC-45 chain formation, a dominant-negative effect was observed in wild-type worms. The expressed mutants disturbed thick filament formation and compromised sarcomere integrity [21].

Conclusion and future directions/open questions

Recent structure–function analyses highlight the versatility of UCS proteins in coordinating myosin folding with the formation of specialized, high-molecular weight complexes underlying fundamental biological processes such as cytokinesis, transport of cargo and muscle contraction. In fungi, UCS chaperones act on monomeric and dimeric myosin molecules [17,18]. The newly identified dimerization properties of yeast She4 led to a model — which still has to be confirmed — that the She4 chaperone can physically link two myosin motors coordinating their activity when moving along actin tracks [26]. She4 could thus have a direct role in modulating the myosin step-size and/or the interaction between actin and myosin molecules (Figure 3). In addition, studies of TPR-containing UCS proteins revealed a so far undescribed chaperone concept that may be of broad relevance for the folding and assembly of filamentous proteins. As seen for C. elegans UNC-45, chaperones themselves can be organized in molecular chains and thus build up filamentous folding machineries that mimic industrial assembly lines [21]. Accordingly, UCS proteins extend the function of multi-TPR proteins teaming up Hsp70 and Hsp90 chaperones over long distances in a precise pattern. By offering multiple substrate binding sites in parallel, UNC-45 could simultaneously function as a patterning chaperone that organizes myosin head domains along the developing thick filaments (Figure 3).

Myosin proteostasis requires UCS chaperones to function in de novo protein folding and assembly, but also to carry out protein quality control under cellular stress conditions. Indeed UNC-45 proteins have been shown to stay associated with completed sarcomeric structures, getting recruited to damaged myosin molecules during stress situations [43]. Moreover, a comprehensive analysis of UNC-45 in the fly heart muscle demonstrated the fundamental role of the chaperone in maintaining cardiac contractility during remodeling of the myocardium [32]. Notably, UNC-45 knockdown in Drosophila heart leads to conditions similar to cardiomyopathies. Whether the ability to form UNC-45 chains is also required at later stages of development, in the process of muscle maintenance or even during cytokinesis, is still elusive. The modular architecture of oligomeric UNC-45 assemblies certainly offers the possibility that different oligomeric states hold different chaperone activities. Affecting the equilibrium between monomers and oligomers would be a powerful way of directing the chaperone to execute a certain task. Interestingly, recent in vivo data indicated that it is important to carefully control the intracellular levels of UCS proteins [40,45-47]. The developmental regulation of UNC-45 by the ubiquitin–proteasome system is critical for proper sarcomere assembly [45,47]. The overall level of UNC-45 decreases at later developmental stages when the scaffolding role of UNC-45 is possibly not required anymore or even counterproductive. Sustained or increased levels of UNC-45 are associated with inclusion body myophathies and have been observed in heart failure patients [47,48]. Addressing the molecular mechanisms of regulating UNC-45 levels in the cell may thus reveal novel strategies in dealing with myosin-based muscle diseases. In addition, it would be interesting to delineate the specific roles of UCS chaperones — and their oligomeric assemblies — in various myosin-dependent processes. In fact, vertebrates always express two specialized UNC-45 isoforms (UNC-45a and UNC-45b) that carry out general and muscle-specific chaperone functions, respectively (Table 1) [35,49]. Therefore, studying the isoform-specific activities of UNC-45 and their regulation will be critical to enhance our understanding of myosin chaperone function in health and disease.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

•• of outstanding interest