Solution structures and dynamics of ADF/cofilins UNC-60A and UNC-60B fromCaenorhabditis elegans

Abstract

The nematode Caenorhabditis elegans has two ADF/cofilin isoforms, UNC-60A and UNC-60B, which are expressed from the unc60 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 nM and 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.

Improved resolution crystal structure of Acanthamoeba actophorin reveals structural plasticity not induced by microgravity

“…However, superposition of PDB entry 7rtx onto PDB entry 5yu8 shows that the -turn of actophorin is in the vicinity of the ADP-bound actin and this may be related to the observation that actophorin has a tenfold higher binding affinity for ADP-F-actin over ATP-F-actin (Blanchoin & Pollard, 1999), and is consistent with molecular-dynamics simulations and other computations of the effect of ADF/C binding to actin filaments (Pfaendtner et al, 2010;Schramm et al, 2017). In addition, NMR studies of two isoforms of cofilin from Caenorhabditis elegans, UNC-60A and UNC-60B, revealed nanosecond-to-picosecond dynamics localized to their F-loop; these dynamics were used to explain the differential activity toward actin depolymerization of these homologs in this invertebrate species (Shukla et al, 2015). The severing action of actophorin (Maciver et al, 1991) is similar to that of other members of the ADF/C family, notably cofilin (De La Cruz, 2009;Ono, 2007).…”

Phosphomimetic S3D cofilin binds but only weakly severs actin filaments

“…7 and Ref. 45). This may contribute to the observed differences in binding affinity, cooperativity, and severing activity.…”

Cofilin: A Promising Protein Implicated in Cancer Metastasis and Apoptosis

“…The F-site locates in the N-terminus, which is responsible for binding to F-actin and severing actin filaments. The G/F-site, locates in the C-terminus, binds to both G-actin and F-actin in the same ratio (Nishida et al, 1984 ; Lappalainen et al, 1998 ; Shukla et al, 2015 ). The sequence schematic and ribbon diagrams are shown in Figure 1 .…”