Differential Mechanical Stability of Filamin a Rod Segments and Domain Pair Interaction

Filamin A (FLNa), an actin cross-linking protein, consists of two subunits that dimerize through C-terminal self-association domain. Each subunit contains an N-terminal spectrin-related actin-binding domain followed by 24 immunoglobulin-like (Ig) repeats. Two flexible hinges separate the 24 Ig repeats into rod 1 (repeats 1-15), rod 2 (repeats 16-23), and self-association domain 24. Rod 1 is like a linear array of Ig repeats, whereas rod 2 is more compact due to inter-domain interactions. FLNa not only support the tension of actin network but also interact with many transmembrane and signaling proteins mostly in the rod 2 segment.Prompted by recent reports suggesting that interaction of FLNa with its binding partners is regulated by mechanical force, we examined mechanical properties of FLNa domains by magnetic tweezers. The three segments of Ig 1-8, Ig 9-15, Ig 16-23 are unfolded at different forces under the same loading rate. Remarkably, we found that repeats 16-23 are susceptible to ∼10 pN force, while the repeats in the rod 1 segment can withstand significantly higher forces. In rod 2, nearest neighboring domains 16-17, 18-19, and 20-21 form domain pairs. Cryptic binding sites in rod 2 can be blocked by inter-domain interactions. For example, A strand of domain 20 blocks the binding site of β-integrin tail on domain 21. If force can unpeel strand A of domain 20 from domain 21, the binding site of β-integrin tail will be exposed, and the binding will be facilitated by the force. This is just one kind of proposed mechanism of force sensor. The specific domain pair interaction between 20 and 21 was studied by magnetic tweezers. Preliminary data shows that the disruption of the domain pair interaction between domain 20 and 21 occurs at ∼15 pN.