Titin N2A Domain and Its Interactions at the Sarcomere

Figures

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  Figure 1. Structural organization of sarcomere, titin, and the N2A insertion of titin. (A) General depiction of the sarcomere in striated muscle showing the Z-disk demarcation of each sarcomere and the M-band in the middle of the sarcomere. The I-band and A-band sections of the sarcomere are indicated. The myofilament proteins, myosin (thick filament, green), actin (thin filament, blue), and titin (red), are shown. (B) Schematic representation of titin in striated cardiac muscle with a depiction of the N2B and N2BA isoforms in the half sarcomere. The N2BA isoform has additional segments, namely the middle immunoglobulins (Ig) and the N2A insertion. (C) Structural domains of the N2A insertion of titin. The N2A insertion has five sub-domains, among which four are immunoglobulin-like (Ig80, Ig81, Ig82, and Ig83) and one is a unique sequence (uN2A). (D) Crystal structure of the Ig81 sub-domain of N2A. The β-strands are aligned in an antiparallel orientation to form two groups of β-sheets, ABED and A’GFC. The β-sheets lie over each other in parallel to form a β-sandwich structure. (A,B) were adapted and redrawn from Koser et al.’s publication [4], and (D) was adapted and redrawn from Meyer and…

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  Figure 2. Calcium ion’s interactions with the N2A domain of titin during contractions and stretching. (A) Alignment of the sarcomere and sarcoplasmic reticulum (SR). The SR stores and releases calcium ions toward the sarcomere. The triad structure made of the SR and T-tubules overlaps with the sarcomere’s A-I junction close to the N2A region of titin. (B) The potential role of calcium ion in the N2A–actin-filament interactions. The influx of Ca2+ may enhance the N2A’s binding with actin filaments, which needs additional investigation due to conflicting data. (C) The role of calcium ion in stabilizing the N2A’s sub-domain. The Ig83 domain is stabilized and keeps a more folded structure in the presence of calcium ion, whereas it may be subjected to unfolding during stretches of titin. (A) was adapted and redrawn from Rossi et al.’s publication [51].

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  Figure 3. N2A’s interacting partners and potential roles of N2A–protein interactions. (A) N2A’s interactions with SMYD2, methyl-Hsp90, and CARP. SMYD2–methyl-Hsp90 and CARP play chaperone roles in protecting or stabilizing their interacting N2A sub-domains against protease activity or unfolding events. (B) N2A’s interactions with calpain-3. Calpain-3 binds at least two regions that encompass the N2A sub-domains. N2A’s interaction with calpain-3 makes the calpain-3 inactive, thus regulating the calpain-3 protease activity. (C) N2A–F-actin complex formation in the presence of CARP. CARP locks titin’s N2A domain to the actin filament, thus increasing titin’s passive force or stiffness during skeletal myofibril stretch. (D) N2A’s interactions with αB-crystallin. N2A is prone to aggregation in an acidic environment upon unfolding that exposes its buried hydrophobic cores. αB-crystallin binds and stabilizes the N2A region to prevent its aggregation. (D) was adapted and redrawn from Kotter et al.’s publication [14].