Structural and Biochemical Characterization of EFhd1/Swiprosin-2, an Actin-Binding Protein in Mitochondria.

Sang A Mun,Jongseo Park,Chang-Duk Jun,Youngjin Lee,Taein Park,Jihyeong Yang,Kyoung Ryoung Park,Soo Hyun Eom,Jung Youn Kang,Minwoo Jin

doi:10.3389/fcell.2020.628222

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. AIF-1 modulates the cytoskeleton and increases the migration of immune cells. EFhd2 is also a cytoskeletal protein implicated in immune cell activation and brain cell functions. 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, we report the crystal structure of the EFhd1 core domain comprising a C-terminus of a proline-rich region, two EF-hand domains, 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, we found that EFhd1 has Ca2+-independent β-actin-binding and Ca2+-dependent β-actin-bundling activities. These findings suggest that EFhd1, an actin-binding and -bundling protein in the mitochondria, may contribute to the Ca2+-dependent regulation of mitochondrial morphology and energy synthesis.

Highlights

Regulation of the cytoskeleton is essential for cell dynamics, such as the maintenance of cell shape or motility (Egelman, 2004; Wu et al, 2016)
This study demonstrated the crystal structure of the EF-hand domaincontaining protein 1 (EFhd1) core domain, whose overall structure was similar to that of EF-hand domain-containing protein 2 (EFhd2) and allograft inflammatory factor-1 (AIF-1)
We found two Zn2+ ions in the crystal packing interface, providing new insights into the Zn2+-mediated multimerization of EFhd1

Summary

Introduction

Regulation of the cytoskeleton is essential for cell dynamics, such as the maintenance of cell shape or motility (Egelman, 2004; Wu et al, 2016). As the major cytoskeletal protein is actin, its regulation is responsible for several cellular functions, Crystal Structure of EFhd1/Swiprosin-2 including maintenance of cellular morphology and formation of lamellipodia or filopodia (Lee and Dominguez, 2010). Actin monomers form actin filaments, and the actin filament networks are modulated by several actinbinding proteins (ABPs), including profilin and cofilin, which regulate the polymerization of actin and actinin, fascin, allograft inflammatory factor-1 (AIF-1), and EF-hand domain-containing protein 2 (EFhd2), which facilitate actin-bundling or crosslinking (Dubernard et al, 1997; Autieri et al, 2003; Aratyn et al, 2007; Lee and Dominguez, 2010; Kwon et al, 2013; Ali et al, 2016). The maintenance of morphology and function requires the mitochondrial actin, βactin (Xie et al, 2018). Since the gene encoding EFhd is not present in the mitochondrial DNA, following its translation in cytoplasm, EFhd translocates from the cytoplasm to the mitochondria (Anderson et al, 1981)

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Conclusion