Apel Sabrina I, Schaffter Emily, Melisi Nicholas, Gage Matthew J
Chemistry Department, University of Massachusetts Lowell, Lowell, MA, USA.
UMass Movement Center, University of Massachusetts Lowell , Lowell, MA, USA.
J Gen Physiol. 2025 Jan 6;157(1). doi: 10.1085/jgp.202313472. Epub 2024 Dec 31.
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.
肌联蛋白是肌节中的第三种收缩性细丝，在肌节完整性以及被动和主动张力方面发挥着关键作用。与分别由肌球蛋白和肌动蛋白聚合而成的粗肌丝和细肌丝不同，肌联蛋白是一种从Z盘延伸至M线的单一蛋白质。肌联蛋白中的N2A区域已被确定为肌肉的信号枢纽，并显示参与多种相互作用。N2A区域内的插入序列（UN2A）被预测为钙结合蛋白S100A1的潜在结合位点。我们通过尺寸排阻色谱、表面等离子体共振和荧光共振能量转移相结合的方法证明，S100A1可以与UN2A区域结合。我们进一步证明，这种相互作用发生在钙与S100A1结合的条件下，这表明钙结合时S100A1的构象变化很重要。我们还观察到pH值变化引起的UN2A构象变化，这表明UN2A的构象灵活性在与S100A1的相互作用中起关键作用。这些结果使我们提出，S100A1与UN2A的相互作用可能作为一种传感器，根据肌肉中的生理变化来调节肌联蛋白的功能。
