Structural and biochemical insights into Zn²⁺-bound EF-hand proteins, EFhd1 and EFhd2

EF-hand proteins, which contain a Ca²⁺-binding EF-hand motif, are involved in regulating diverse cellular functions. Ca²⁺ binding induces conformational changes that modulate the activities of EF-hand proteins. Moreover, these proteins occasionally modify their activities by coordinating metals other than Ca²⁺, including Mg²⁺, Pb²⁺ and Zn²⁺, within their EF-hands. EFhd1 and EFhd2 are homologous EF-hand proteins with similar structures. Although separately localized within cells, both are actin-binding 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, the crystal structures of the EFhd1 and EFhd2 core domains coordinating Zn²⁺ ions within their EF-hands are reported. 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 positions as well as low-energy remote positions at the Zn K-edge. EFhd1 and EFhd2 were also found to 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⁺.