Dianne W. Taylor

Three-dimensional image reconstruction of dephosphorylated smooth muscle heavy meromyosin reveals asymmetry in the interaction between myosin heads and placement of subfragment 2

Regulation of the actin-activated ATPase of smooth muscle myosin II is known to involve an interaction between the two heads that is controlled by phosphorylation of the regulatory light chain. However, the three-dimensional structure of this inactivated form has been unknown. We have used a lipid monolayer to obtain two-dimensional crystalline arrays of the unphosphorylated inactive form of smooth muscle heavy meromyosin suitable for structural studies by electron cryomicroscopy of unstained, frozen-hydrated specimens. The three-dimensional structure reveals an asymmetric interaction between the two myosin heads. The ATPase activity of one head is sterically “blocked” because part of its actin-binding interface is positioned onto the converter domain of the second head. ATPase activity of the second head, which can bind actin, appears to be inhibited through stabilization of converter domain movements needed to release phosphate and achieve strong actin binding. When the subfragment 2 domain of heavy meromyosin is oriented as it would be in an actomyosin filament lattice, the position of the heads is very different from that needed to bind actin, suggesting an additional contribution to ATPase inhibition in situ.

Recreation of the terminal events in physiological integrin activation

In vitro analysis confirms talin binding is sufficient for activation and extension of membrane-embedded integrin.

Three-dimensional structure of the myosin V inhibited state by cryoelectron tomography

Unconventional myosin V (myoV) is an actin-based molecular motor that has a key function in organelle and mRNA transport, as well as in membrane trafficking. MyoV was the first member of the myosin superfamily shown to be processive, meaning that a single motor protein can ‘walk’ hand-over-hand along an actin filament for many steps before detaching. Full-length myoV has a low actin-activated MgATPase activity at low [Ca2+], whereas expressed constructs lacking the cargo-binding domain have a high activity regardless of [Ca2+] (refs 5–7). Hydrodynamic data and electron micrographs indicate that the active state is extended, whereas the inactive state is compact. Here we show the first three-dimensional structure of the myoV inactive state. Each myoV molecule consists of two heads that contain an amino-terminal motor domain followed by a lever arm that binds six calmodulins. The heads are followed by a coiled-coil dimerization domain (S2) and a carboxy-terminal globular cargo-binding domain. In the inactive structure, bending of myoV at the head–S2 junction places the cargo-binding domain near the motor domains ATP-binding pocket, indicating that ATPase inhibition might occur through decreased rates of nucleotide exchange. The actin-binding interfaces are unobstructed, and the lever arm is oriented in a position typical of strong actin-binding states. This structure indicates that motor recycling after cargo delivery might occur through transport on actively treadmilling actin filaments rather than by diffusion.

The 3D Structure of Villin as an Unusual F-Actin Crosslinker

Villin is an F-actin nucleating, crosslinking, severing, and capping protein within the gelsolin superfamily. We have used electron tomography of 2D arrays of villin-crosslinked F-actin to generate 3D images revealing villins crosslinking structure. In these polar arrays, neighboring filaments are spaced 125.9 +/- 7.1 A apart, offset axially by 17 A, with one villin crosslink per actin crossover. More than 6500 subvolumes containing a single villin crosslink and the neighboring actin filaments were aligned and classified to produce 3D subvolume averages. Placement of a complete villin homology model into the average density reveals that full-length villin binds to different sites on F-actin from those used by other actin-binding proteins and villins close homolog gelsolin.

Structure of myosin filaments from relaxed Lethocerus flight muscle by cryo-EM at 6 Å resolution

Cryo-EM of relaxed myosin filament reveals unique molecular motor conformation and unprecedented details of the filament backbone. We describe a cryo–electron microscopy three-dimensional image reconstruction of relaxed myosin II–containing thick filaments from the flight muscle of the giant water bug Lethocerus indicus. The relaxed thick filament structure is a key element of muscle physiology because it facilitates the reextension process following contraction. Conversely, the myosin heads must disrupt their relaxed arrangement to drive contraction. Previous models predicted that Lethocerus myosin was unique in having an intermolecular head-head interaction, as opposed to the intramolecular head-head interaction observed in all other species. In contrast to the predicted model, we find an intramolecular head-head interaction, which is similar to that of other thick filaments but oriented in a distinctly different way. The arrangement of myosin’s long α-helical coiled-coil rod domain has been hypothesized as either curved layers or helical subfilaments. Our reconstruction is the first report having sufficient resolution to track the rod α helices in their native environment at resolutions ~5.5 Å, and it shows that the layer arrangement is correct for Lethocerus. Threading separate paths through the forest of myosin coiled coils are four nonmyosin peptides. We suggest that the unusual position of the heads and the rod arrangement separated by nonmyosin peptides are adaptations for mechanical signal transduction whereby applied tension disrupts the myosin heads as a component of stretch activation.

The Structure of a Full-length Membrane-embedded Integrin Bound to a Physiological Ligand

Background: Integrins undergo large conformational changes when ligand-bound. Results: Using nanodisc technology and EM, we observed Mn2+-activated αIIbβ3 integrin both alone and fibrin-bound. Conclusion: MnCl2-activated αIIbβ3 integrin alone has a compact conformation, becoming fully upright and open when fibrin-bound. Significance: The first structure of membrane-embedded integrins bound to physiological substrate reveals the importance of integrin extension in macromolecular ligand binding. Increased ligand binding to integrin (“activation”) underpins many biological processes, such as leukocyte trafficking, cell migration, host-pathogen interaction, and hemostasis. Integrins exist in several conformations, ranging from compact and bent to extended and open. However, the exact conformation of membrane-embedded, full-length integrin bound to its physiological macromolecular ligand is still unclear. Integrin αIIbβ3, the most abundant integrin in platelets, has been a prototype for integrin activation studies. Using negative stain electron microscopy and nanodisc-embedding to provide a membrane-like environment, we visualized the conformation of full-length αIIbβ3 in both a Mn2+-activated, ligand-free state and a Mn2+-activated, fibrin-bound state. Activated but ligand-free integrins exist mainly in the compact conformation, whereas fibrin-bound αIIbβ3 predominantly exists in a fully extended, headpiece open conformation. Our results show that membrane-embedded, full-length integrin adopts an extended and open conformation when bound to its physiological macromolecular ligand.

3-D Structure of Z-Disks Isolated from the Flight Muscle of Lethocerus Indicus

The reconstruction of the recent publication of a 3-D image of isolated Z-disk from Apis mellifera flight muscle [1] is the first of its kind utilizing isolated Z-disks, confirmed many details of the previously published 3-D image obtained from plastic sections. However, they used relatively harsh treatment of KCl/KI to remove both thick and thin filaments in a single step that regardless produced a specimen suitable for cryoelectron tomography. Here, we use a different approach for Z-disk isolation from flight muscle from the large waterbug Lethocerus indicus that combines 1.4M NaCl and 10 mM pyrophosphate to remove the thick filaments and gelsolin to remove the thin filaments. Myofibrils are suspended in the high salt-pyrophosphate buffer. I-Z-I brushes are then centrifuged gently to remove the dissolved thick filaments. The I-Z-I brushes are placed on the grid bar side of a QuantifoilTM grid and treated with a calcium insensitive gelsolin construct for 4.5 min after which the broken up thin filaments are washed from the grid and the 3 μm diameter Z-disk are visualized over holes. Tilt series are collected on a Titan Krios electron microscope, with a DE-20 camera and merged using PROTOMO [6]. Fourier transforms of the Z-disks show spots from a hexagonal lattice with a spacing of 520Å extending to 87Å.

Coupling between myosin head conformation and the thick filament backbone structure

The recent high-resolution structure of the thick filament from Lethocerus asynchronous flight muscle shows aspects of thick filament structure never before revealed that may shed some light on how striated muscles function. The phenomenon of stretch activation underlies the function of asynchronous flight muscle. It is most highly developed in flight muscle, but is also observed in other striated muscles such as cardiac muscle. Although stretch activation is likely to be complex, involving more than a single structural aspect of striated muscle, the thick filament itself, would be a prime site for regulatory function because it must bear all of the tension produced by both its associated myosin motors and any externally applied force. Here we show the first structural evidence that the arrangement of myosin heads within the interacting heads motif is coupled to the structure of the thick filament backbone. We find that a change in helical angle of 0.16° disorders the blocked head preferentially within the Lethocerus interacting heads motif. This observation suggests a mechanism for how tension affects the dynamics of the myosin heads leading to a detailed hypothesis for stretch activation and shortening deactivation, in which the blocked head preferentially binds the thin filament followed by the free head when force production occurs.

Identification of interfaces involved in weak interactions with application to F-actin-aldolase rafts

Macromolecular interactions occur with widely varying affinities. Strong interactions form well defined interfaces but weak interactions are more dynamic and variable. Weak interactions can collectively lead to large structures such as microvilli via cooperativity and are often the precursors of much stronger interactions, e.g. the initial actin-myosin interaction during muscle contraction. Electron tomography combined with subvolume alignment and classification is an ideal method for the study of weak interactions because a 3-D image is obtained for the individual interactions, which subsequently are characterized collectively. Here we describe a method to characterize heterogeneous F-actin-aldolase interactions in 2-D rafts using electron tomography. By forming separate averages of the two constituents and fitting an atomic structure to each average, together with the alignment information which relates the raw motif to the average, an atomic model of each crosslink is determined and a frequency map of contact residues is computed. The approach should be applicable to any large structure composed of constituents that interact weakly and heterogeneously.

The Structure of the Relaxed Thick Filaments from Lethocerus Flight Muscle

Flight muscle thick filaments consist of myosin II and several types of non-myosin proteins. Myosin II consists of two heavy chains and two pairs of light chains, an essential light chain (ELC) and a regulatory light chain (RLC). The first ~850 residues make up the head which possesses the catalytic and actin binding activity and the remaining ~1100 residues forming a long α-helical coiled-coil, which is essential for thick filament formation. Non-myosin proteins likely to be visible in the flight muscle thick filament reconstruction are paramyosin [1], flightin [2], myofilin [3]. How these proteins are organized in thick filaments is largely unknown as is the detailed myosin rod structure. Our cryoEM structure of relaxed Lethocerus flight muscle thick filaments at 5.5Å resolution shows the myosin rod in unprecedented detail and suggests the arrangement of at least three non-myosin proteins.