Abstract
EFhd2/Swiprosin-1 is a cytoskeletal Ca2+-binding protein implicated in Ca2+-dependent cell spreading and migration in epithelial cells. EFhd2 domain architecture includes an N-terminal disordered region, a PxxP motif, two EF-hands, a ligand mimic helix and a C-terminal coiled-coil domain. We reported previously that EFhd2 displays F-actin bundling activity in the presence of Ca2+ and this activity depends on the coiled-coil domain and direct interaction of the EFhd2 core region. However, the molecular mechanism for the regulation of F-actin binding and bundling by EFhd2 is unknown. Here, the Ca2+-bound crystal structure of the EFhd2 core region is presented and structures of mutants defective for Ca2+-binding are also described. These structures and biochemical analyses reveal that the F-actin bundling activity of EFhd2 depends on the structural rigidity of F-actin binding sites conferred by binding of the EF-hands to Ca2+. In the absence of Ca2+, the EFhd2 core region exhibits local conformational flexibility around the EF-hand domain and C-terminal linker, which retains F-actin binding activity but loses the ability to bundle F-actin. In addition, we establish that dimerisation of EFhd2 via the C-terminal coiled-coil domain, which is necessary for F-actin bundling, occurs through the parallel coiled-coil interaction.
Highlights
More than 100 actin-related proteins exist in eukaryotic cells, and these proteins regulate the transition of actin polymerisation and depolymerisation to form highly complex structures[1,2,3]
We reported previously that the EF-hands of EFhd[2] are involved directly in F-actin bundling in a Ca2+-dependent manner and the coiled-coil domain is essential to the F-actin bundling activity by homodimerisation[26]
We found that Ca2+ or ethylene glycoltetraacetic acid (EGTA) had little effect on EFhd[2] binding to F-actin; the F-actin bundling activity was significantly reduced in the Ca2+-free state and these results were visualised by electron microscopy[26]
Summary
More than 100 actin-related proteins exist in eukaryotic cells, and these proteins regulate the transition of actin polymerisation and depolymerisation to form highly complex structures[1,2,3]. Fimbrin and non-muscle α-actinin contain multiple calponin-homology (CH) domains and EF-hands These proteins associate with actin through CH domains, and F-actin binding or bundling activity is inhibited by Ca2+ 11. The structure of Ca2+-free EF-hands of non-muscle α-actinin-1 revealed a flexible conformation around the connecting linker between the N-lobe and C-lobe, and binding of Ca2+ to EF-hands induced structural rigidification, which affected the orientation of adjacent CH domains resulting in inhibition of F-actin crosslinking activity[15]. In some cases, such as gelsolin, villin, fragmin and severin, Ca2+ directly affects actin-related functions through binding to multiple actin-binding sites. We established that dimerisation of EFhd[2] via the C-terminal coiled-coil domain, which is necessary for F-actin bundling, occurs through the parallel coiled-coil interaction
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