Molecular Basis for the Dual Function of Eps8 on Actin Dynamics: Bundling and Capping

The unusual dual functions of the actin-binding protein EPS8 as an actin capping and actin bundling factor are mapped to distinct structural features of the protein and to distinct physiological activities in vivo

The structure of the C-terminal actin-binding domain of talin

Talin is a large dimeric protein that couples integrins to cytoskeletal actin. Here, we report the structure of the C-terminal actin-binding domain of talin, the core of which is a five-helix bundle linked to a C-terminal helix responsible for dimerisation. The NMR structure of the bundle reveals a conserved surface-exposed hydrophobic patch surrounded by positively charged groups. We have mapped the actin-binding site to this surface and shown that helix 1 on the opposite side of the bundle negatively regulates actin binding. The crystal structure of the dimerisation helix reveals an antiparallel coiled-coil with conserved residues clustered on the solvent-exposed face. Mutagenesis shows that dimerisation is essential for filamentous actin (F-actin) binding and indicates that the dimerisation helix itself contributes to binding. We have used these structures together with small angle X-ray scattering to derive a model of the entire domain. Electron microscopy provides direct evidence for binding of the dimer to F-actin and indicates that it binds to three monomers along the long-pitch helix of the actin filament

The R403Q Myosin Mutation Implicated in Familial Hypertrophic Cardiomyopathy Causes Disorder at the Actomyosin Interface

Mutations in virtually all of the proteins comprising the cardiac muscle sarcomere have been implicated in causing Familial Hypertrophic Cardiomyopathy (FHC). Mutations in the beta-myosin heavy chain (MHC) remain among the most common causes of FHC, with the widely studied R403Q mutation resulting in an especially severe clinical prognosis. In vitro functional studies of cardiac myosin containing the R403Q mutation have revealed significant changes in enzymatic and mechanical properties compared to wild-type myosin. It has been proposed that these molecular changes must trigger events that ultimately lead to the clinical phenotype.Here we examine the structural consequences of the R403Q mutation in a recombinant smooth muscle myosin subfragment (S1), whose kinetic features have much in common with slow beta-MHC. We obtained three-dimensional reconstructions of wild-type and R403Q smooth muscle S1 bound to actin filaments in the presence (ADP) and absence (apo) of nucleotide by electron cryomicroscopy and image analysis. We observed that the mutant S1 was attached to actin at highly variable angles compared to wild-type reconstructions, suggesting a severe disruption of the actin-myosin interaction at the interface.These results provide structural evidence that disarray at the molecular level may be linked to the histopathological myocyte disarray characteristic of the diseased state

Comparison between actin decorated with wild-type smooth muscle myosin S1 and actin decorated with a R403Q smooth muscle myosin mutant.

<p>A: Wild-type smooth muscle actomyosin in the presence of ADP. The top row shows a 4-nm thick central slice perpendicular to the filament axis. The second row shows a 4-nm thick central slice parallel to the filament axis (cutting plane indicated by the left line in top row). The pointed end is to the top of the figure in the two lower rows. The bottom row shows an adjacent 4-nm thick slice (cutting plane indicated by right line in top row). This slice goes primarily through the S1 molecules. B: R403Q mutant smooth muscle actomyosin in the presence of ADP. Organization of rows as in (A). C: Wild-type smooth muscle actomyosin in the absence of nucleotide (apo). The approximate location of the motor domain (MD) and the light chain (LC) regions are labeled in each view. Organization of rows as in A. The approximate outline of the actin portion of the filament is indicated. D: Apo R403Q mutant smooth muscle actomyosin. Organization of rows as in (A). Bar:10 nm.</p

Proximity of residue Arg⁴⁰⁶ (Arg⁴⁰³ in cardiac myosin) to the actin interface.

<p>Arg⁴⁰⁶ (red spheres) is immediately adjacent to residues of the cardiomyopathy loop that were previously implicated in actin binding by docking studies (407–414; green). While the conformation of the Arg⁴⁰⁶ in the smooth muscle myosin crystal structure points away from the interface (A), it can easily reach actin by simple, stereochemically permitted bond angle rotations (B). The resulting conformation does not generate serious clashes with other myosin residues. Myosin is shown in blue, the interacting actin filament subunits in grey. Residue Pro³³³ of actin, the closest to myosin Arg⁴⁰⁶, is shown as spheres.</p

Surface representations of smooth muscle actomyosin constructs.

<p>The pointed end of the filaments is towards the top of the figure. A: Wild-type smooth muscle actomyosin in the presence of ADP. B: Wild-type smooth muscle actomyosin in the apo state. The contour level for A and B is chosen to represent the correct molecular mass. Note the well defined density and angle of the light-chain region (LC) C: R403Q mutant smooth muscle actomyosin in the presence of ADP. D: R403Q mutant smooth muscle actomyosin in the apo state. The contour level for C and D is chosen to show as much of the light chain domains as possible without completely obscuring the shape of the motor domain. E: Overlay of the maps in A–D. Color code and contouring as in A–D. F, G: Watershed segmentation of the maps in C (F) and D (G). These results reconfirm the orientation of the light-chain domains that correspond to those of the wild-type reconstructions (sketches) and the better definition of boundaries in the center of the filaments: the actin subunits are well segmented while there is no sub-segmentation of myosin domains as can be obtained for wild-type reconstructions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001123#pone.0001123-Volkmann4" target="_blank">[33]</a>. The sketches show central lines extracted from the density of the wild-type (grey) and the segmentation of the R403Q (black). Only the line segments extracted for the corresponding light-chain regions (LC) are shown, line segments corresponding to the motor domain region (MD) overlap almost completely for all maps.</p

Difference and structural variability maps.

<p>4-nm wide slices perpendicular to the helix axis of several maps are shown on the left. Only peaks significant at a confidence level of 99.99% are shown. A: Wild-type smooth muscle actomyosin in the absence of nucleotide. The motor domain (MD) and light-chain (LC) regions are labeled. A faint ghost image of this map is also overlaid on C and E to aid visualization. B: A difference map generated by subtracting the R403Q mutant smooth muscle actomyosin apo state reconstruction from the R403Q mutant smooth muscle actomyosin ADP state reconstruction. A clear difference peak can be identified in the light-chain region. A faint ghost image of the wild-type ADP state reconstruction is overlaid to aid visualization. This image is also overlaid on D and F. C: Difference map between two independently generated R403Q mutant apo state reconstructions. Only occasional, randomly distributed, isolated pixels can be seen, no coherent difference peaks exist. D: Difference map between two independently generated R403Q mutant ADP state reconstructions. E: Structural variability (AVID map) of R403Q mutant apo state reconstruction. Only randomly distributed peaks can be seen, there is no consistent structural variability in any confined region. F: Structural variability (AVID map) of R403Q mutant ADP state reconstruction. Bar:10 nm. G: Surface representation of the difference map shown in B. The cyan density represents additional density in the R403Q mutant ADP state reconstruction if compared to the R403Q mutant apo state reconstruction. The apo state (pink) and ADP state (blue wireframe) wild-type reconstructions are also shown. The difference between the mutant reconstructions is located in the light-chain region and correlates with the changes observed in wild-type smooth muscle actomyosin.</p

Data collection

a<p>This counts split data (i.e. far and near side) as different data sets (see text for details).</p>b<p>All statistics are calculated over the respective number of data sets.</p>c<p>The phase residual is a measure for the homogeneity and quality of the data. Values below 45° are considered excellent; values below 55° are acceptable.</p