Smyd2 Interacts With Titin At The Ig80 Domain Within N2a

The integrity of muscle cells are dependent on their functional unit known as the sarcomere and myofibril. During stress, the sarcomere or myofibril loses its regularity implying degradation of its structural components. Sarcomere bears accessory proteins with roles in maintaining their phenotype and homeostasis. One of such proteins is SMYD2, a protein methyltransferase that associates with titin via its N2A region. Studies have suggested that titin is cleaved by proteases within its N2A domain, and its treatment with SMYD2 protects it from protease digestion, but SMYD2's mode of interaction is not well understood. Using molecular cloning techniques, we made various sub-domain constructs of the N2A domain and quantitatively studied their binding with SMYD2 by bio-layer interferometry (BLI). We have identified Ig80 as the N2A domain facilitating SMYD2's binding to N2A. We also mapped the amino acid sequences in Ig80 enabling SMYD2's binding to a PAVAP motif in proximity of its N-terminus. Furthermore, we identified an additional motif within Ig80 that interacts with SMYD2. Our subsequent analysis revealed a 13 sequence residue within Ig80 that may be exposed to bind to SMYD2 during unfolding of Ig80. As Ig80 unfolding may occur during muscle contraction and relaxation or pathologic conditions, our data may find a novel functional role of SMYD2 associated with an unfolded state of Ig80. Finally, we found that SMYD2 was able to bind and protect Ig80 that is susceptible to digestion by calpain-1. This indicates that Ig80 may be the segment of N2A predisposing its vulnerability to proteases. Putting together, our findings presents new understanding about SMYD2's interaction in titin.