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Electron cryomicroscopy reveals the three-dimensional structure of F-actin at a resolution of 3.7 A in complex with tropomyosin at a resolution of 6.5 A; the stabilizing interactions and the effects of disease-causing mutants are also investigated. Filamentous actin (F-actin) — a main component of the cytoskeleton — is the major protein of thin filaments in the muscle. The binding of the motor protein myosin to F-actin is mediated by another protein called tropomyosin, which also binds to F-actin in smooth muscle and in non-muscle cells, stabilizing and regulating these filaments. Using electron cryomicroscopy, Stefan Raunser and colleagues have obtained the first high-resolution, three-dimensional structure of F-actin in complex with tropomyosin. The structure reveals the interactions that stabilize the F-actin and sheds light on the possible effect of prominent disease-causing mutations. Comparison of the F-actin structure with the crystal structure of monomeric (G)-actin reveals conformational changes associated with filament formation. Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss1, familial thoracic aortic aneurysms and dissections2, and multiple variations of myopathies3. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin4,5. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin6. Although crystal structures for monomeric actin (G-actin) are available7, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7 A in complex with tropomyosin at a resolution of 6.5 A, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg2+ and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin–tropomyosin with its position in our previously determined F-actin–tropomyosin–myosin structure8 reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted development of drugs.

Cytoplasmic β-actin supports fundamental cellular processes in healthy and diseased cells including cell adhesion, migration, cytokinesis and maintenance of cell polarity. Mutations in ACTB, the gene encoding cytoplasmic β-actin, lead to severe disorders with a broad range of symptoms. The two dominant heterozygous gain-of-function β-actin mutations p.R183W and p.E364K were identified in patients with developmental malformations, deafness and juvenile-onset dystonia (p.R183W) and neutrophil dysfunction (p.E364K). Here, we report the recombinant production and functional characterization of the two mutant proteins. Arg183 is located near the nucleotide-binding pocket of actin. Our results from biochemical studies and molecular dynamics simulations show that replacement by a tryptophan residue at position 183 establishes an unusual stacking interaction with Tyr69 that perturbs nucleotide release from actin monomers and polymerization behavior by inducing a closed state conformation. The replacement of Glu364 by a lysine residue appears to act as an allosteric trigger event leading to the preferred formation of the closed state. Thus, our approach indicates that both mutations affect interdomain mobility and nucleotide interactions as a basis for the formation of disease phenotypes in patients.

Inherited cardiomyopathies are caused by point mutations in sarcomeric gene products, including alpha-cardiac muscle actin (ACTC1). We examined the biochemical and cell biological properties of the alpha-cardiac actin mutations Y166C and M305L identified in hypertrophic cardiomyopathy (HCM). Untagged wild-type (WT) cardiac actin, and the Y166C and M305L mutants were expressed by the baculovirus/Sf9-cell system and affinity purified by immobilized gelsolin G4-6. Their correct folding was verified by a number of assays. The mutant actins also displayed a disturbed intrinsic ATPase activity and an altered polymerization behavior in the presence of tropomyosin, gelsolin, and Arp2/3 complex. Both mutants stimulated the cardiac beta-myosin ATPase to only 50 % of WT cardiac F-actin. Copolymers of WT and increasing amounts of the mutant actins led to a reduced stimulation of the myosin ATPase. Transfection of established cell lines revealed incorporation of EGFP- and hemagglutinin (HA)-tagged WT and both mutant actins into cytoplasmic stress fibers. Adenoviral vectors of HA-tagged WT and Y166C actin were successfully used to infect adult and neonatal rat cardiomyocytes (NRCs). The expressed HA-tagged actins were incorporated into the minus-ends of NRC thin filaments, demonstrating the ability to form hybrid thin filaments with endogenous actin. In NRCs, the Y166C mutant led after 72 h to a shortening of the sarcomere length when compared to NRCs infected with WT actin. Thus our data demonstrate that a mutant actin can be integrated into cardiomyocyte thin filaments and by its reduced mode of myosin interaction might be the basis for the initiation of HCM.

Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.

SummaryRegulation of myosin and filamentous actin interaction by tropomyosin is a central feature of contractile events in muscle and nonmuscle cells. However, little is known about molecular interactions within the complex and the trajectory of tropomyosin movement between its “open” and “closed” positions on the actin filament. Here, we report the 8 Å resolution structure of the rigor (nucleotide-free) actin-tropomyosin-myosin complex determined by cryo-electron microscopy. The pseudoatomic model of the complex, obtained from fitting crystal structures into the map, defines the large interface involving two adjacent actin monomers and one tropomyosin pseudorepeat per myosin contact. Severe forms of hereditary myopathies are linked to mutations that critically perturb this interface. Myosin binding results in a 23 Å shift of tropomyosin along actin. Complex domain motions occur in myosin, but not in actin. Based on our results, we propose a structural model for the tropomyosin-dependent modulation of myosin binding to actin.

Phalloidin and fluorescently labeled phalloidin analogs are established reagents to stabilize and mark actin filaments for the investigation of acto-myosin interactions. In the present study, we employed transient and steady-state kinetic measurements as well as in vitro motility assays to show that phalloidin perturbs the productive interaction of human non-muscle myosin-2A and -2C1 with filamentous actin. Phalloidin binding to F-actin results in faster dissociation of the complex formed with non-muscle myosin-2A and -2C1, reduced actin-activated ATP turnover, and slower velocity of actin filaments in the in vitro motility assay. In contrast, phalloidin binding to F-actin does not affect the interaction with human non-muscle myosin isoform 2B and Dictyostelium myosin-2 and myosin-5b.