Structural insight into M-band assembly and mechanics from the titin-obscurin-like-1 complex

Abstract

In the sarcomeric M-band, the giant ruler proteins titin and obscurin, its small homologue obscurin-like-1 (obsl1), and the myosin cross-linking protein myomesin form a ternary complex that is crucial for the function of the M-band as a mechanical link. Mutations in the last titin immunoglobulin (Ig) domain M10, which interacts with the N-terminal Ig-domains of obscurin and obsl1, lead to hereditary muscle diseases. The M10 domain is unusual not only in that it is a frequent target of disease-linked mutations, but also in that it is the only currently known muscle Ig-domain that interacts with two ligands--obscurin and obsl1--in different sarcomeric subregions. Using x-ray crystallography, we show the structural basis for titin M10 interaction with obsl1 in a novel antiparallel Ig-Ig architecture and unravel the molecular basis of titin-M10 linked myopathies. The severity of these pathologies correlates with the disruption of the titin-obsl1/obscurin complex. Conserved signature residues at the interface account for differences in affinity that direct the cellular sorting in cardiomyocytes. By engineering the interface signature residues of obsl1 to obscurin, and vice versa, their affinity for titin can be modulated similar to the native proteins. In single-molecule force-spectroscopy experiments, both complexes yield at forces of around 30 pN, much lower than those observed for the mechanically stable Z-disk complex of titin and telethonin, suggesting why even moderate weakening of the obsl1/obscurin-titin links has severe consequences for normal muscle functions.

Fig. 1.

(A and B) Cartoon representation of the M10-OL1 heterodimer. As indicated, the view in (B) is rotated by 90 degrees around the x axis compared to the view in (A). M10 and OL1 are shown in orange and green, respectively. (C) Enlarged view of the boxed overall representation shown in the top panel highlighting important M10-OL1 interactions. Some structural elements in the overall representation are shown partly transparent to highlight the mixed intermolecular β-sheet. Color coding for carbon atoms is orange and green for M10 and OL1, respectively; nitrogen, blue; oxygen, red; sulfur, yellow. Hydrogen bonds are dotted cyan lines. (D) Sequence and secondary structure of the M10 and OL1 domains. The sequence of the O1 domain is also given. Amino acids are color-coded according to sequence conservation as highlighted by the conservation bar. Colored circles show residues of one domain contacting the other as indicated in the inset. The radius of the circles is proportional to the buried area percentage as calculated by PISA (34). The letter codes H and S indicate residues involved hydrogen bonds and salt bridges, respectively. Figures were generated with Pymol (www.pymol.org) and Adobe Illustrator.

Fig. 2.

(A) Close-up on the OL1 F17 residue with M10 shown as surface representation. The OL1 F17 side chain is buried and stabilized by M10 residues A25, A27 and L61. 2mF_o-DF_c electron density for OL1 F17 is shown in black at the 1.3σ contour level. (B) Closeup of the OL1 R17 residue in the M10-OL1 F17R complex engineered to partly mimic the M10-O1 complex. OL1 R17 points its charged side-chain towards the solvent. 2mF_o-DF_c electron density for OL1 R17 is shown in black at the 1.3σ contour level. The orientation is the same as in A. (C) View of the M10-OL1 complex highlighting the position of the M10 residues involved human cardiomyopathies as a result of four different (Finnish, Belgian, French and Italian) genetic mutations (see main text). Amino acid changes resulting from the different mutations are shown in the inset.

Fig. 3.

(A) Representative example of the competitive effect of overexpressed GFP-fused obscurin/obs|1 (here: GFP-O1 R15F) on endogenous obscurin. The separate channels for GFP, endogenous myomesin and obscurin, and the ratiometric image with overlaid GFP mask for the outline of the transfected cell are shown. The false-color scale range indicator shows a range of 0 (Black) to 3 (White). Scale bar: 10 μm. (B) Average ratio of endogenous obscurin to myomesin in NRC expressing various O1, OL1, and M10 constructs. Whereas the high affinity interactions of O1, O1 R15F and OL1 show no significant differences (p > 0.17), weakening the OL1 interaction leads to a significant loss in M-band affinity (p = 0.003). The weaker competing effect of M10 is completely abrogated in the mutant M10 A9Y. Error bars: SEM.

Fig. 4.

(A) Schematic representation of the chimaeric single-chain titin-obscurin/obsl1 constructs fused to ubiquitin used in AFM. (B and C), representative single-molecule force-extension traces for O1 (B) and OL1 (C) showing the unbinding of the complex at low forces (Boxed) and the subsequent unfolding of the M10 and O1/OL1 and ubiquitin domains. It is important to note that in these traces the unfolding events of M10 and O1/OL1 are indistinguishable in both contour length increase and unfolding force. The orange/green coloring of the unfolding traces is hence merely for illustration. (D) Frequency analysis of the unbinding forces for titin O1 (Black Distribution) and titin-OL1 (Red Distribution) with a force distribution (Dashed Line) calculated with a potential width Δx = 8 Å , an off-rate k_off = 0.7 s^-1 and a loading rate of 2500 pN s^-1. (E) Schematic interpretation of the observed single-molecule experiments where unbinding of the titin-O1/OL1 complex precedes the sequential unfolding of Ig and ubiquitin domains.