Abstract:
The nematode Caenorhabditiselegans has two ADF/cofilin isoforms, UNC-60A and UNC-60B, which are expressed from theunc60 gene by alternative splicing.UNC-60A has higher activity to cause net depolymerization, and to inhibit polymerization, than UNC-60B. UNC-60B, on the other hand, shows much stronger severing activity than UNC-60A.To understand the structural basis of their functional differences, we have determined the solution structures of UNC-60A and UNC-60B proteins and characterized backbone dynamics. Both UNC-60A and UNC-60B show conserved ADF/cofilin fold. The G-actin binding regions of the two proteins are structurally and dynamically conserved. Accordingly, UNC-60A and UNC-60B individually bind to rabbit muscle ADP-G-actin with high affinities, with Kd of 32.25 nMand 8.62 nM, respectively. The primary differences between these strong and weak severing proteins were observed in the orientation and dynamics of the F-actin binding loop (F-loop). In the strong severing activity isoform UNC-60B, the orientation of F-loop was towards the recently identified F-loop binding region on F-actin, and the F-loop was relatively more flexible with fourteen residues showing motions on nanosecond-picosecond timescale. In contrast, in the weak severing protein isoform UNC-60A, the orientation of F-loop was away from the F-loop binding region and inclined towards its own C-terminal and strand β6. It was also relatively less flexible with only five residues showing motions on nanosecond-picosecond timescale. These differences in structure and dynamics seem to directly correlate to the differential F-actin site-binding and severing properties of UNC-60A and UNC-60B, and other related ADF/cofilin proteins.
