Binding of Myomesin to Obscurin-Like-1 at the Muscle M-Band Provides a Strategy for Isoform-Specific Mechanical Protection

Figure 1. Schematic of the M-Band Network (A) Principal sarcomere regions are marked by the letters Z, I, A, and M. (B) Modular myomesin, titin, and obscurin/obscurin-like-1 proteins form an intricate M-band network with C-terminal myomesin dimers crosslinking myosin filaments. The inset highlights the interaction between myomesin and obscurin/obscurin-like-1, which has been mapped to linker sequence (L) located between the myomesin fibronectin (Fn-III) domains My4 and My5 and the third immunoglobulin (Ig) domain of obscurin/obscurin-like-1 (O3/OL3, respectively) (Fukuzawa et al., 2008).

Figure 2. The (OL3:My4L)2 Complex Dimer (A) In the crystal, two OL3:My4L heterodimers are arranged around a non-crystallographic two-fold axis (vertical in this view) forming a W-shaped (OL3:My4L)2 dimeric assembly. My4L and OL3 are in slate blue and green, respectively, with one heterodimer shown as a solid surface and the other in cartoon mode with a transparent surface. The dotted line at the C-terminus of L indicates the flexible region leading to My5 not visible in the structure. (B) Superdex S75 10/300 SEC traces for the OL3:My4LMy5 complex injected in the column at 3 mg/mL (thin continuous line) and 8.2 mg/mL (thick continuous line). The OL3:My4LMy5 complex forms a mixture of monomers and dimers in a concentration-dependent manner. The elution profile of calibration markers is shown with a gray broken line. Molecular weights are indicated.

Figure 3. OL3:My4L Heterodimer (A and B) Cartoon representation of the OL3:My4L heterodimer. The view in (B) is rotated by 90° around the x axis compared with (A). My4L and OL3 are shown in slate blue and green, respectively. In (A), the three main regions contributing to the complex are highlighted: the My4 Fn-III domain, its C-terminal helical spacer LH, and the OL3A (A for augmented) domain in which the Ig OL3 domain is stabilized by myomesin fold complementation in-trans. (C) Topology diagram of the complex. (D and E) Sequence alignment of the linker region for the different myomesin isoforms (D) and of the OL3 and O3 domains (E). Identical amino acids are highlighted. Colored circles above the secondary structure cartoons show residues of one domain contacting the other as indicated in the inset. Their radius is proportional to the buried area. The letter codes H and S indicate residues involved hydrogen bonds and salt bridges, respectively. (F) Enlarged stick representation of the boxed region in the top left-hand corner (view rotated approximately 90° around the x axis compared with B) highlighting important myomesin:obscurin-like-1 interactions. Nitrogen, oxygen, and sulfur atoms…

Figure 4. Cellular Validation and Myomesin Isoform Specificity (A) Close up of the OL3:My4L interface. OL3 is shown as surface representation and colored according to its electrostatic potential. The myomesin L linker interacting with OL3 is shown as stick representation. (B) Quantification of endogenous obscurin displaced in neonatal rat cardiomyocytes expressing GFP-fused wild-type My4LMy5 (n = 11) and its T622V (n = 10), T622K (n = 9), I625T (n = 10), V627Y (n = 13) variants. Amino acid replacements in the L linker inspired by myomesin-2 (T622K and V627Y) and myomesin-3 (I625T) sequences abrogate competition. Error bars are SEM values. ∗∗∗p ≤ 0.001. (C) Example of the competitive effect of overexpressed GFP-fused My4LMy5 (WT) on endogenous obscurin in NRCs. The separate channels for endogenous myomesin, GFP, endogenous obscurin, as well as the combined and ratiometric images with overlaid GFP mask for the outline of the transfected cell are shown. The false-color scale range indicator shows an increased obscurin/myomesin ratio. The scale bar represents 10 μm. (D–G) Similar to (C) for overexpressed GFP-fused My4LMy5 T622V (D), T622K (E), I625T (F), and V627Y (G). The scale bar represents…

Figure 5. Small-Angle X-Ray Scattering Analysis of the Myomesin:Obscurin-Like-1 Complex (A) Molecular parameters calculated from SAXS data. MM, Rg, Dmax are the molecular mass, radius of gyration, and maximum size, respectively. The superscript exp denotes experimental values while xt, ai, and tC refer to crystal, ab initio, and tCONCOORD fitted models, respectively. MMcalc is the theoretical MM computed from the protein sequence. χ is the discrepancy between experimental data and those computed from models. (B) Experimental scattering intensities for OL3:My4L (upper curve) and OL3:My4LMy5 (lower curve) are displayed as dots with error bars. Curves computed from the crystallographic model and the best tC models are shown as solid blue and red lines, respectively, while the curve computed from the ab initio models is shown as a dotted green line. (C) Cartoon-tube representation of a selection of 20 OL3:My4L tCONCOORD models (out of >30,000) aligned with respect to their My4 domain. My4L is in slate blue and OL3 is in green. (D) Cartoon-tube representation of the ten OL3:My4L tCONCOORD models providing the best fit to the SAXS data (0.78 < χ < 0.81). (E and F) OL3:My4L SAXS molecular…

Figure 6. Fold Complementation by the Myomesin L Linker Protects the Mechanically Labile OL3 Domain (A) Schematics of the ubiquitin-L-(connector)-OL3-ubiquitin polyprotein used in the single-molecule mechanical experiment. The 43 amino acid flexible connector covalently joining L to OL3 is depicted as a dashed line. The color code for L and OL3 is as in previous images. Ubiquitin (Ub) molecules bracketing the complex are in gray while the gold substrate and the atomic force microscopy (AFM) cantilever tip are shown in yellow and black, respectively. (B) Typical force-extension trace for the ubiquitin-L-(connector)43-OL3-ubiquitin polyprotein exhibiting a saw-tooth pattern of unfolding events that does not follow a hierarchy in the mechanical stability: the first unfolding event occurs at higher forces than the second unfolding event. The unfolding of the ubiquitin modules, occurring at forces of ∼200 pN and characterized by an increment of contour length of ΔLUb ∼24.5 nm, ensures the single-molecule nature of the experiment. (C) Histogram of unfolding forces. The first two events can be readily identified in light of their different mechanical stability and increment in contour length.…

Figure 7. Possible Model for the Organization of the Titin:Obscurin(-like-1):Myomesin Ternary Complex at the M-Band Experimental complexes are shown as surface representation: OL3:My4L dimer (this work), M10:OL1 (Pernigo et al., 2010, Pernigo et al., 2015, Sauer et al., 2010), My9-M13 (Pinotsis et al., 2008, Pinotsis et al., 2012).