Structural basis of fast- and slow-severing actin-cofilactin boundaries

Members of the ADF/cofilin family of regulatory proteins bind actin filaments cooperatively, locally change actin subunit conformation and orientation, and sever filaments at 'boundaries' between bare and cofilin-occupied segments. A cluster of bound cofilin introduces two distinct classes of boundaries due to the intrinsic polarity of actin filaments, one at the 'pointed' end-side and the other at the 'barbed' end-side of the cluster; severing occurs more readily at the pointed end side of the cluster ('fast-severing' boundary) than the barbed end side ('slow-severing' boundary). A recent electron-cryomicroscopy (cryo-EM) model of the slow-severing boundary revealed structural 'defects' at the interface that potentially contribute to severing. However, the structure of the fast-severing boundary remains uncertain. Here, we use extensive molecular dynamics simulations to produce atomic resolution models of both severing boundaries. Our equilibrated simulation model of the slow-severing boundary is consistent with the cryo-EM structural model. Our simulations indicate that actin subunits at both boundaries adopt structures intermediate between those of bare and cofilin-bound actin subunits. These 'intermediate' states have compromised intersubunit contacts, but the actin subunit interfaces lacking contacts at the slow-severing boundary are stabilized by cofilin bridging interactions, accounting for its lower fragmentation probability. Simulations where cofilin proteins are removed from cofilactin filaments favor a mechanism in which a cluster of two contiguously bound cofilins is needed to fully stabilize the cofilactin conformation, promote cooperative binding interactions, and accelerate filament severing. Together, these studies provide a molecular-scale foundation for developing coarse-grained and theoretical descriptions of cofilin-mediated actin filament severing.