Interacting-heads Motif Research Articles

Improved Imaging, 3D Reconstruction and Homology Modeling of Tarantula Thick Filaments

Tarantula muscle is an outstanding model system for elucidating thick filament molecular structure. In past studies we showed by cryo-EM, single particle reconstruction, and atomic fitting with a hybrid myosin head structure, that relaxed tarantula filaments are characterized by intramolecular interaction between heads (J-motif) that is thought to inhibit myosin activity by blocking actin binding in one head and ATPase activity in the other (Woodhead et al., 2005). We have been pursuing improvements in technique to obtain a higher resolution reconstruction. A key problem in reconstructing myosin filaments is intrinsic flexibility of the heads, which reduces the resolution we can achieve. We have used staurosporine to minimize RLC phosphorylation and thus head disordering, and blebbistatin to stabilize the heads. Automated data collection has been used to obtain images on a field emission cryo-electron microscope (Titan Krios). Preprocessing of images has enabled the selection of a data set as homogeneous as possible. Both SPIDER and RELION were used for 3D reconstruction, and yielded a similar resolution of ∼13 A, 2-fold better than our original reconstruction, but presumably still limited by some head flexibility. The J-motif is consistent with the original findings, but the structure of the S2 segment of the myosin tail is much more robust and some details of the paramyosin core are now visible. We have built an homology model of the interacting-heads motif (using the tarantula sequence for both heavy and light chains), that includes all surface loops, and we are using this for molecular dynamics flexible fitting (MDFF). Preliminary results suggest the need for repositioning of several loops from their positions in the crystal structure, with possible implications for filament assembly and stability.

The Myosin Inhibitor Blebbistatin Stabilizes the Super-Relaxed State in Skeletal Muscle

The super-relaxed state of myosin (SRX), in which the myosin ATPase activity is strongly inhibited, has been observed in a variety of muscle types. It has been proposed that myosin heads in this state are inhibited by binding to the core of the thick filament in a structure known as the interacting-heads motif. The myosin inhibitor blebbistatin has been shown in structural studies to stabilize the binding of myosin heads to the thick filament, and here we have utilized measurements of single ATP turnovers to show that blebbistatin also stabilizes the SRX in both fast and slow skeletal muscle, providing further support for the proposal that myosin heads in the SRX are also in the interacting-heads motif. We find that the SRX is stabilized using blebbistatin even in conditions that normally destabilize it, e.g., rigor ADP. Using blebbistatin we show that spin-labeled nucleotides bound to myosin have an oriented spectrum in the SRX in both slow and fast skeletal muscle. This is to our knowledge the first observation of oriented spin probes on the myosin motor domain in relaxed skeletal muscle fibers. The spectra for skeletal muscle with blebbistatin are similar to those observed in relaxed tarantula fibers in the absence of blebbistatin, demonstrating that the structure of the SRX is similar in different muscle types and in the presence and absence of blebbistatin. The mobility of spin probes attached to nucleotides bound to myosin shows that the conformation of the nucleotide site is closed in the SRX.

Different Head Environments in Tarantula Thick Filaments Support a Cooperative Activation Process

Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). Structural analysis of relaxed tarantula thick filaments shows that the RLCs of the interacting free and blocked myosin heads are in different environments. This and other data suggested a phosphorylation mechanism in which Ser-35 of the free head is exposed and constitutively phosphorylated by protein kinase C, whereas the blocked head is hidden and unphosphorylated; on activation, myosin light chain kinase phosphorylates the monophosphorylated free head followed by the unphosphorylated blocked head, both at Ser-45. Our goal was to test this model of phosphorylation. Mass spectrometry of quickly frozen, intact muscles showed that only Ser-35 was phosphorylated in the relaxed state. The location of this constitutively phosphorylated Ser-35 was analyzed by immunofluorescence, using antibodies specific for unphosphorylated or phosphorylated Ser-35. In the relaxed state, myofibrils were labeled by anti-pSer-35 but not by anti-Ser-35, whereas in rigor, labeling was similar with both. This suggests that only pSer-35 is exposed in the relaxed state, while in rigor, Ser-35 is also exposed. In the interacting-head motif of relaxed filaments, only the free head RLCs are exposed, suggesting that the constitutive pSer-35 is on the free heads, consistent with the proposed mechanism.

The myosin interacting-heads motif is present in the relaxed thick filament of the striated muscle of scorpion

Electron microscopy (EM) studies of 2D crystals of smooth muscle myosin molecules have shown that in the inactive state the two heads of a myosin molecule interact asymmetrically forming a myosin interacting-heads motif. This suggested that inactivation of the two heads occurs by blocking of the actin-binding site of one (free head) and the ATP hydrolysis site of the other (blocked head). This motif has been found by EM of isolated negatively stained myosin molecules of unregulated (vertebrate skeletal and cardiac muscle) and regulated (invertebrate striated and vertebrate smooth muscle) myosins, and nonmuscle myosin. The same motif has also been found in 3D-reconstructions of frozen-hydrated (tarantula, Limulus, scallop) and negatively stained (scallop, vertebrate cardiac) isolated thick filaments. We are carrying out studies of isolated thick filaments from other species to assess how general this myosin interacting-heads motif is. Here, using EM, we have visualized isolated, negatively stained thick filaments from scorpion striated muscle. We modified the iterative helical real space reconstruction (IHRSR) method to include filament tilt, and band-pass filtered the aligned segments before averaging, achieving a 3.3 nm resolution 3D-reconstruction. This reconstruction revealed the presence of the myosin interacting-heads motif (adding to evidence that is widely spread), together with 12 subfilaments in the filament backbone. This demonstrates that conventional negative staining and imaging can be used to detect the presence of the myosin interacting-heads motif in helically ordered thick filaments from different species and muscle types, thus avoiding the use of less accessible cryo-EM and low electron-dose procedures.

Slow Myosin ATP Turnover in the Super-Relaxed State in Tarantula Muscle

We measured the nucleotide turnover activity of myosin in tarantula leg-muscle fibers by observing single turnovers of the fluorescent nucleotide analog, mantATP, as monitored by the decrease in fluorescence when mantATP is replaced by ATP in a chase experiment. We find a multi-exponential process, with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant, ∼30 minutes. This slow turnover state is termed the super-relaxed state (SRX) and is a highly novel adaptation for energy conservation in an animal that spends extremely long periods of time in a quiescent state (days) employing a lie-in-wait hunting strategy. If fibers are incubated in mantADP and chased with ADP, the SRX is not seen, indicating that relaxed myosins are responsible for the SRX. Phosphorylation of the myosin regulatory light chain eliminates the fraction of myosin with the very long lifetime. The presence of the SRX measured here correlates well with the binding of myosin to the core of the thick filament in a structure known as the interacting-head motif (or J-motif) observed previously by electron microscopy. Both the structural array and the long-lived SRX require ATP, both are lost upon myosin phosphorylation, and both appear to be more stable in tarantula than in skeletal or cardiac preparations. EPR spectroscopy of a spin-labeled nucleotide bound to the motor domain of myosin in relaxed tarantula fibers likewise shows orientation that is lost when the myosin is phosphorylated. Together, the data support the hypothesis that the SRX myosin and the myosin seen in the EM of the order helical array in tarantula filaments are the same.

Slow Myosin ATP Turnover in the Super-Relaxed State in Tarantula Muscle

We measured the nucleotide turnover rate of myosin in tarantula leg muscle fibers by observing single turnovers of the fluorescent nucleotide analog 2′-/3′- O-( N′-methylanthraniloyl)adenosine-5′- O-triphosphate, as monitored by the decrease in fluorescence when 2′-/3′- O-( N′-methylanthraniloyl)adenosine-5′- O-triphosphate (mantATP) is replaced by ATP in a chase experiment. We find a multiexponential process with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant (∼ 30 min). This slow-turnover state is termed the super-relaxed state (SRX). If fibers are incubated in 2′-/3′- O-( N′-methylanthraniloyl)adenosine-5′- O-diphosphate and chased with ADP, the SRX is not seen, indicating that trinucleotide-relaxed myosins are responsible for the SRX. Phosphorylation of the myosin regulatory light chain eliminates the fraction of myosin with a very long lifetime. The data imply that the very long-lived SRX in tarantula fibers is a highly novel adaptation for energy conservation in an animal that spends extremely long periods of time in a quiescent state employing a lie-in-wait hunting strategy. The presence of the SRX measured here correlates well with the binding of myosin heads to the core of the thick filament in a structure known as the “interacting-heads motif,” observed previously by electron microscopy. Both the structural array and the long-lived SRX require relaxed filaments or relaxed fibers, both are lost upon myosin phosphorylation, and both appear to be more stable in tarantula than in vertebrate skeletal or vertebrate cardiac preparations.