The calcium-binding protein S100A1 binds to titin’s N2A insertion sequence in a pH-dependent manner

Titin is the third contractile filament in the sarcomere, and it plays a critical role in sarcomere integrity and both passive and active tension. Unlike the thick and thin filaments, which are polymers of myosin and actin, respectively, titin is a single protein that spans from Z-disk to M-line. The N2A region within titin has been identified as a signaling hub for the muscle and is shown to be involved in multiple interactions. The insertion sequence (UN2A) within the N2A region was predicted as a potential binding site for the Ca2+-binding protein, S100A1. We demonstrate using a combination of size exclusion chromatography, surface plasmon resonance, and fluorescence resonance energy transfer that S100A1 can bind to the UN2A region. We further demonstrate that this interaction occurs under conditions where calcium is bound to S100A1, suggesting that the conformational shift in S100A1 when calcium binds is important. We also observed a conformational change in UN2A induced by shifts in pH, suggesting that conformational flexibility in UN2A plays a critical role in the interaction with S100A1. These results lead us to propose that the interaction of S100A1 and UN2A might act as a sensor to regulate titin's function in response to physiological changes in the muscle.