Domain-domain interactions in Filamin A (16-23) impose a hierarchy of unfolding forces

The quaternary structure of Filamin A (FLNa) 16-23 was recently shown to exhibit multiple domain-domain interactions that lead to a propeller-like construction. Here we present single molecule force spectroscopy experiments to show a wide variety of mechanical responses of this molecule and compare it with its linear counterpart FLNa 1-8. The compact structure of FLNa 16-23 leads to a broad distribution of rupture forces and end-to-end lengths in the force-extension mode and multiple unraveling timescales in the force-clamp mode. Moreover, a subset of force-extension trajectories reveals a mechanical hierarchy in which the rupture of domain-domain interactions at high forces (200 pN) liberates the unfolding of individual domains at low forces (100 pN). This mechanism may also explain the order of magnitude difference in the rates of the biexponential fits to the distribution of unfolding dwell times under force-clamp. Overall, FLNa 16-23 under a force of 100 pN is more compliant than the linear FLNa 1-8. Since a physiological role of FLNa is to crosslink actin filaments, this range of responses allows it to accommodate a broad spectrum of forces exerted by the cell and its environment.

💡 Research Summary

Filamin A (FLNa) is a large actin‑binding protein that cross‑links actin filaments and participates in numerous signaling pathways. Its 24 immunoglobulin‑like (Ig) domains are organized into distinct segments, with the 16‑23 region recently shown by structural studies to adopt a compact, propeller‑like architecture mediated by extensive domain‑domain contacts. This study investigates how such an arrangement influences the mechanical response of FLNa at the single‑molecule level, comparing the 16‑23 segment to the linear 1‑8 segment using atomic force microscopy (AFM) based single‑molecule force spectroscopy (SMFS).

Two experimental modalities were employed. In the force‑extension mode, the AFM tip was retracted at a constant velocity, stretching individual FLNa molecules until rupture events occurred. In the force‑clamp mode, a constant force (primarily 100 pN) was applied, and the time‑dependent extension (unfolding dwell times) was recorded.

Force‑extension traces for FLNa 16‑23 displayed a broad distribution of rupture forces and contour‑length increments. High‑force events (~200 pN) correspond to the rupture of inter‑domain interfaces that hold the compact propeller together. After these high‑force ruptures, a series of lower‑force events (~100 pN) appear, each matching the characteristic unfolding force of an individual Ig domain. By contrast, the linear FLNa 1‑8 segment showed a narrow force distribution centered around 120‑150 pN and relatively uniform contour‑length changes, reflecting the absence of stabilizing domain‑domain contacts.

In force‑clamp experiments, dwell‑time histograms for 16‑23 were best described by a biexponential function, revealing two distinct kinetic components: a fast component (τ₁) associated with the breaking of domain‑domain contacts, and a slower component (τ₂) linked to the unfolding of single Ig domains. The amplitudes of the two components were roughly equal, indicating that both processes contribute significantly to the overall mechanical response. For the 1‑8 segment, the dwell‑time distribution was essentially mono‑exponential, consistent with a single unfolding pathway.

These observations establish a mechanical hierarchy in FLNa 16‑23: high‑force disruption of inter‑domain contacts precedes low‑force unfolding of individual domains. This hierarchy creates a multi‑step buffering system that allows the protein to absorb a wide range of forces. At forces ≤100 pN, the 16‑23 segment is more compliant than the linear 1‑8 segment, suggesting that the compact region can soften under modest loads while still providing structural integrity under larger stresses.

Biologically, cells experience forces ranging from a few piconewtons (e.g., thermal fluctuations) to several hundred piconewtons (e.g., shear stress, contractile tension). FLNa’s ability to respond differently across this spectrum is crucial for its role as an actin cross‑linker. The hierarchical unfolding mechanism enables rapid release of stored elastic energy via domain‑domain rupture, followed by a controlled, stepwise extension of individual domains. This dual‑stage response likely protects cells from abrupt mechanical failure and facilitates adaptation to dynamic mechanical environments.

In summary, the paper provides compelling experimental evidence that the propeller‑like arrangement of FLNa 16‑23 imposes a hierarchy of unfolding forces, generating a broad distribution of rupture events and multi‑timescale kinetics. These findings illuminate how protein architecture can be tuned to achieve sophisticated mechanical functions, offering insights relevant to cellular mechanobiology, disease states involving FLNa mutations, and the design of biomimetic materials that require hierarchical force‑dissipation mechanisms.