19Striated muscle thick filaments are composed of myosin II and several non-myosin proteins. 20Myosin II's long α-helical coiled-coil tail forms the dense protein backbone of filaments while 21 its N-terminal globular head containing the catalytic and actin binding activities extends outward 22 from the backbone. Here we report the structure of thick filaments of the flight muscle of the 23 fruit fly Drosophila melanogaster at 7 Å resolution. Its myosin tails are arranged in curved 24 molecular crystalline layers identical to flight muscles of the giant waterbug Lethocerus indicus. 25Four non-myosin densities are observed, three of which correspond to ones found in Lethocerus; 26 one new density, possibly stretchin-Mlck, is found on the backbone outer surface. Surprisingly, 27the myosin heads are disordered rather than ordered along the filament backbone. Our results 28show striking myosin tail similarity within flight muscle filaments of two insect orders separated 29 by several hundred million years of evolution. 30 31 Significance Statement 32 Myosin thick filaments are one of striated muscle's key structures, but also one of its least 33 understood. A key question is how the myosin a-helical coiled-coil tail is arranged in the 34 backbone. At 7Å resolution, sufficient to resolve individual a-helices, the myosin tail arrangement 35 in thick filaments from the flight muscle of the fruit fly Drosophila melanogaster is strikingly 36 similar to the myosin tail arrangement in flight muscles of the giant waterbug Lethocerus indicus. 37 -2 -Nearly every other thick filament feature is different. Drosophila and Lethocerus evolved 38 separately >245 million years ago suggesting myosin tail packing into curved molecular crystalline 39 layers forms a highly conserved thick filament building block and different properties are obtained 40 by alterations in non-myosin proteins. 41 42 43 -3 -Introduction:
Myosin II, composed of two heavy chains and two light chains, is the main component of bipolar thick filaments of striated muscle. Compared to thin filaments, sarcomeric thick filaments are poorly understood, especially in the backbone. The Drosophila flight muscle myosin rod sequence compared to human cardiac β-myosin (MYH7) is 56% identical, 74% similar with no insertions or deletions between sequences of the two species. Here we report a ~7 Å resolution structure of isolated thick filaments from asynchronous flight muscle with disordered heads. Drosophila thick filaments are 3.2 µm in length and 250 Å in diameter and have a helical structure with C4 rotational symmetry and a helical rise of 145 Å and a helical angle of 3.86°-33.92°. The 145 Å repeat is generally referred to as a "crown". Relaxed myosin filaments from multiple species are observed to fold in an asymmetric conformation called Interacting Heads Motif (IHM). IHM shows two myosin heads interacting asymmetrically with each other which inhibits their interaction with actin filaments. Our reconstruction surprisingly failed to show densities resembling the IHM of relaxed myosin. Thick filaments of Lethocerus indicus, the only other thick filament structure reported at comparable resolution had well resolved myosin heads in a modified IHM positioned very differently from all previously-reported structures. Instead of being positioned against the proximal S2 via an interaction with the "so-called" blocked head, the free head was positioned against the thick filament backbone and the "blocked" head appearing to pin the free head against the backbone. In Drosophila densities were found in the expected axial position of myosin heads but unconnected to the filament surface.
Recent structural studies of ex vivo amyloid filaments extracted from human patients demonstrated that the ex vivo filaments associated with different disease phenotypes adopt diverse molecular conformations distinct from those in vitro amyloid filaments. A very recent cryo-EM structural study also revealed that ex vivo α-synuclein filaments extracted from multiple system atrophy (MSA) patients adopt quite distinct molecular structures from those of in vitro α-synuclein filaments, suggesting the presence of co-factors for α-synuclein aggregation in vivo. Here, we report structural characterizations of α-synuclein filaments derived by a potential co-factor, tau, using cryo-EM and solid-state NMR. Our cryo-EM structure of the tau-promoted α-synuclein filament at 4.0 Å resolution is somewhat similar to one of the polymorphs of in vitro α-synuclein filaments. However, the N- and C-terminal regions of the tau-promoted α-synuclein filament have different molecular conformations. Our structural studies highlight the conformational plasticity of α-synuclein filaments, requiring additional structural investigation of not only more ex vivo α-synuclein filaments, but also in vitro α-synuclein filaments formed in the presence of diverse co-factors to better understand molecular basis of diverse molecular conformations of α-synuclein filaments.
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