The role of the M-band myomesin proteins in muscle integrity and cardiac disease

Transversal structural elements in cross-striated muscles, such as the M-band or the Z-disc, anchor and mechanically stabilize the contractile apparatus and its minimal unit—the sarcomere. The ability of proteins to target and interact with these structural sarcomeric elements is an inevitable necessity for the correct assembly and functionality of the myofibrillar apparatus. Specifically, the M-band is a well-recognized mechanical and signaling hub dealing with active forces during contraction, while impairment of its function leads to disease and death. Research on the M-band architecture is focusing on the assembly and interactions of the three major filamentous proteins in the region, mainly the three myomesin proteins including their embryonic heart (EH) isoform, titin and obscurin. These proteins form the basic filamentous network of the M-band, interacting with each other as also with additional proteins in the region that are involved in signaling, energetic or mechanosensitive processes. While myomesin-1, titin and obscurin are found in every muscle, the expression levels of myomesin-2 (also known as M-protein) and myomesin-3 are tissue specific: myomesin-2 is mainly expressed in the cardiac and fast skeletal muscles, while myomesin-3 is mainly expressed in intermediate muscles and specific regions of the cardiac muscle. Furthermore, EH-myomesin apart from its role during embryonic stages, is present in adults with specific cardiac diseases. The current work in structural, molecular, and cellular biology as well as in animal models, provides important details about the assembly of myomesin-1, obscurin and titin, the information however about the myomesin-2 and -3, such as their interactions, localization and structural details remain very limited. Remarkably, an increasing number of reports is linking all three myomesin proteins and particularly myomesin-2 to serious cardiovascular diseases suggesting that this protein family could be more important than originally thought. In this review we will focus on the myomesin protein family, the myomesin interactions and structural differences between isoforms and we will provide the most recent evidence why the structurally and biophysically unexplored myomesin-2 and myomesin-3 are emerging as hot targets for understanding muscle function and disease

Structural basis for DNA strand separation by a hexameric replicative helicase

Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5′ ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted ‘steric exclusion’ model for dsDNA unwinding, the active 3′ ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5′ passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases

Rab regulation by GEFs and GAPs during membrane traffic

Rab GTPases and their regulatory proteins play a crucial role in vesicle-mediated membrane trafficking. During vesicle membrane tethering Rab GTPases are activated by GEFs (guanine nucleotide exchange factors) and then inactivated by GAPs (GTPase activating proteins). Recent evidence shows that in addition to activating and inactivating Rab GTPases, both Rab GEFs and GAPs directly contribute to membrane tethering events during vesicle traffic. Other studies have extended the range of processes, in which Rabs function, and revealed roles for Rabs and their GAPs in the regulation of autophagy. Here, we will discuss these advances and the emerging relationship between the domain architectures of Rab GEFs and vesicle coat protein complexes linked with GTPases of the Sar, ARF and Arl families in animal cells

Characterisation of RB1 derivatives by small-angle X-ray scattering.

<p><b>A</b>. Experimental and calculated scattering patterns of ddRB<b>-</b>NP (1), MBP-ddRB<b>-</b>NP (2), ddRB<b>-</b>NP-MBP (3). Experimental SAXS data as black dots with black error bars. Lines (red) represent the fits from <i>ab initio</i> models shown in <b>C</b> (ddRB-NP), <b>D</b> (MBP-ddRB-NP) and <b>E</b> (ddRB-NP-MBP). The logarithm of the scattering intensity is plotted as a function of momentum transfer, s = 4πsin(θ/2)/λ where θ is the scattering angle and λ is the wavelength of the X-rays (1.5 Å). <b>B.</b> Distance distribution functions for ddRB-NP, MBP-ddRB-NP and ddRB-NP-MBP. <b>C.</b> Averaged <i>ab initio</i> models for ddRB-NP obtained using DAMMIN (grey semi-transparent spheres) and MONSA (RB-N blue spheres, RB-P red spheres) superimposed. The models are shown in two different views rotated by 90°. <b>D., E. </b><i>Ab initio</i> models of MBP-ddRB-NP (<b>D</b>) and ddRB-NP-MBP (<b>E</b>) obtained by MONSA. MBP is shown as green, ddRB-NP as grey spheres. The models are viewed as in <b>C</b>. <b>F.</b> Radius of gyration (<i>R_g</i>) distribution obtained by EOM for ddRB-NP. Distributions correspond to a random pool of 10.000 generated structures (blue) and the EOM optimized ensemble (red).</p

RB1 architecture and study design.

<p><b>A.</b> Schematic of RB1 domain structure. RB1 NH2-terminal domain (RB-N, light blue), RB1 pocket-domain (RB-P, raspberry), the position of the twin cyclin folds which form the core of each domain is indicated, RB1 C-terminal region (RB-C, yellow)<b>. B.</b> RB1 constructs used in this study indicating the range of amino-acids covered. In the MBP-RB-NP and MBP-ddRB-NP constructs maltose binding protein (MBP, green) is coupled to the N-terminus of the RB construct, while in ddRB-NP-MBP it is coupled to the C-terminus. In the ddRB-NP, MBP-ddRB-NP and ddRB-NP-MBP constructs two interstitial regions were deleted, corresponding to residues 250–269, the arginine-rich linker (R-linker) of the RB-N domain, and residues 579–643, corresponding to the pocket linker connecting RB-P domain pocket lobes (P-linker). The positions of cyclin-dependent kinase consensus sites in RB-NP are indicated, with sites retained in the ddRB-NP, MBP-ddRB-NP and ddRB-NP-MBP constructs bold and starred. <b>C.</b> Atomic models of the RB-N and RB-P domains, shown in ribbon representations. RB-N left, RB-P right. Cyclin-fold helixes are coloured, RB-N A-fold in cyan, RB-N B-fold in light blue, RB-P A-fold in dark salmon, RB-P B-fold in pink, other helixes and visible loops are shown as grey.</p

Binding surfaces positioning in the active and inactive structure, and predicted molecular movement to yield inactive RB1. A., B.

<p>Relative orientation of the functional surfaces in the model of active, nonphosphorylated (A) and inactive, phosphorylated (B) RB1. Cartoon representation of Rb-NP with overlaid transparent surface with RB-N in light blue, RB-P in light-pink. The residues involved in docking LXCXE are shown in yellow, those forming the FXXXV motif are shown in purple and those for EXXXDLFD in cyan. The residues 346–355 which form a helix in unmodified RB-N but are disordered in inactive RB-NP are represented in dark grey <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058463#pone.0058463-Hassler1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058463#pone.0058463-Burke1" target="_blank">[21]</a>, amino acid groups involved in RB-N:P interphase interaction in the inactive conformation in red ([RB-N K136, D139, T140, T142, D145], [RB-P Q736, E737, K740, K729]) and orange [(RB-N L161, K164, L206-E209, L211-I213, F216, E282, E287, N290, N295] [RB-P Q736, E737, K740, K729]). <b>C., D.</b> Cartoon representation of active, nonphosphorylated, (C) and inactive, phosphorylated (D) RB1. RB-N B-fold is coloured in green and RB-P B-fold in purple (this different colour scheme has not been used elsewhere in the paper and is only used here for clarity). The residues 346–355 which are structured in unmodified RB-N and unstructured in inactive RB-NP are represented in dark grey. <b>E.</b> Predicted molecular movement yielding conformational RB1 inactivation. Note surfaces involved in binding LXCXE motif proteins in RB-P (salmon/pink) and the homologous surface involved in FXXXV binding in RB-N (cyan/blue) are collinear in the active (left) but not inactive form (right).</p

Single particle analysis of electron microscope images of MBP-ddRB-NP. A.-F.

<p>3D reconstruction of unmodified MBP-ddRB-NP. <b>A.</b>, <b>B</b>. Single particle reconstruction for unmodified MBP-ddRB-NP. Calculated density map of MBP-ddRB-NP, shown as surface representations in grey related by a 90^o rotation. <b>C.</b> 3D reconstruction in mesh representation oriented as in <b>B</b> with the docked structures of the RB-N and RB-P domains (PDB codes 2QDJ and 3POM) shown as cartoons colour-coded as follows: RB-N domain lobe A -cyan, lobe B -light blue; RB-P domain lobe A -dark salmon and lobe B – pink. <b>D., E.</b> Segmented densities shown as solid surface representation with overlaid surface representation of the unmodified RB-NP 3D reconstruction in mesh. The density attributed to the MBP tag is shown in light green, that attributed to RB-N in light blue and to RB-P in light pink. <b>F.</b> Docked structures of the RB-N and RB-P domains (PDB codes 2QDJ and 3POM) without density mesh, shown as cartoons and colour-coded as in C. <b>G.–L.</b> 3D reconstruction of phosphorylated MBP-ddRB-NP. <b>G., H</b>. 3D reconstruction shown as a grey surface in two orthogonal views. <b>I.</b> 3D reconstruction in mesh representation oriented as in <b>H</b> with the docked structures of inactive RB-NP (PDB code 4ELJ) shown as cartoons colour-coded as follows:-. RB-N domain lobe A -cyan, lobe B -light blue; RB-P domain lobe A -dark salmon and lobe B – pink. <b>J., K.</b> Segmented densities shown as solid surface representation with overlaid surface representation of the 3D reconstruction in mesh<b>.</b> Same colour coding as in <b>D</b> and <b>E. L.</b> Docked structures of inactive RB-NP (PDB code 4ELJ) without density mesh, shown as cartoons colour-coded as in I.</p

Low resolution structural models of the basic helix-loop-helix leucine zipper domain of upstream stimulatory factor 1 and its complexes with DNA from small angle X-ray scattering data

The upstream stimulatory factor 1 (USF1) belongs to the basic helix-loop-helix leucine zipper (b/HLH/Z) transcription factor family, recognizing the CACGTG DNA motive as a dimer and playing an important role in the regulation of transcription in a variety of cellular and viral promoters. In this study we investigate the USF1 b/HLH/Z domain and its complexes with DNA by small angle x-ray scattering. We present low resolution structural models of monomeric b/HLH/Z USF1 in the absence of DNA and USF1 dimeric (b/HLH/Z)(2)-DNA and tetrameric (b/HLH/Z)(4)-DNA(2) complexes. The data reveal a concentration-dependent USF1 dimer (b/HLH/Z)(2)-DNA-tetramer (b/HLH/Z)(4)-DNA(2) equilibrium. The ability of b/HLH/Z USF1 to form a tetrameric assembly on two distant DNA binding sites as a consequence of increased protein concentration suggest a USF1 concentration-dependant mechanism of transcription activation involving DNA loop formation

Regulation of the transcription factor Ets-1 by DNA-mediated homo-dimerization

The function of the Ets-1 transcription factor is regulated by two regions that flank its DNA-binding domain. A previously established mechanism for auto-inhibition of monomeric Ets-1 on DNA response elements with a single ETS-binding site, however, has not been observed for the stromelysin-1 promoter containing two palindromic ETS-binding sites. We present the structure of Ets-1 on this promoter element, revealing a ternary complex in which protein homo-dimerization is mediated by the specific arrangement of the two ETS-binding sites. In this complex, the N-terminal-flanking region is required for ternary protein–DNA assembly. Ets-1 variants, in which residues from this region are mutated, loose the ability for DNA-mediated dimerization and stromelysin-1 promoter transactivation. Thus, our data unravel the molecular basis for relief of auto-inhibition and the ability of Ets-1 to function as a facultative dimeric transcription factor on this site. Our findings may also explain previous data of Ets-1 function in the context of heterologous transcription factors, thus providing a molecular model that could also be valid for Ets-1 regulation by hetero-oligomeric assembly