Structural and biochemical insights of EFhd1 and EFhd2

Abstract

Ca2+ regulates several cellular functions, including signaling events, energy production, and cell survival. These cellular processes are mediated by Ca2+-binding proteins, such as EF-hand superfamily proteins. Among the EF-hand superfamily proteins, allograft inflammatory factor-1 (AIF-1) and swiprosin-1/EF-hand domain-containing protein 2 (EFhd2) are cytosolic actin-binding proteins. Swiprosin-2/EF-hand domain-containing protein 1 (EFhd1), a mitochondrial fraternal twin of EFhd2, mediates neuronal and pro-/pre-B cell differentiation and mitoflash activation. Although EFhd1 is important for maintaining mitochondrial morphology and energy synthesis, its mechanism of action remains unclear. Here, I report the crystal structure of the EFhd1 core domain comprising a C-terminus of a proline-rich region, two EF-hands, and a ligand mimic helix. Structural comparisons of EFhd1, EFhd2, and AIF-1 revealed similarities in their overall structures. In the structure of the EFhd1 core domain, two Zn2+ ions were observed at the interface of the crystal contact, suggesting the possibility of Zn2+-mediated multimerization. In addition, I found that EFhd1 has Ca2+-independent β-actin-binding and Ca2+-dependent β-actin-bundling activities. These findings suggest that EFhd1 may contribute to the Ca2+-dependent regulation of mitochondrial morphology and energy synthesis. Meanwhile, several proteins occasionally modify their activities by coordinating metals other than Ca2+, including Mg2+, Pb2+ and Zn2+, within their EF-hands. EFhd1 and EFhd2 are EF-hand proteins that modulate F-actin rearrangement through Ca2+-independent actin-binding and Ca2+-dependent actin-bundling activity. Although Ca2+ is known to affect the activities of EFhd1 and EFhd2, it is not known whether their actin-related activities are affected by other metals. Here, I report the crystal structures of the EFhd1 and EFhd2 core domains coordinating Zn2+ ions within their EF-hands. The presence of Zn2+ within EFhd1 and EFhd2 was confirmed by analyzing anomalous signals and the difference between anomalous signals using data collected at the peak position as well as low-energy remote positions at the Zn K-edge. I also found that EFhd1 and EFhd2 exhibit Zn2+-independent actin-binding and Zn2+-dependent actin-bundling activity. This suggests the actin-related activities of EFhd1 and EFhd2 could be regulated by Zn2+ as well as Ca2+.