EFhd2 is a novel protein conserved from C. elegans to H. sapiens. This novel protein was originally identified
in cells of the immune and central nervous systems. However, it is most abundant in the central nervous system, where it
has been found associated with pathological forms of the microtubule-associated protein tau. The physiological or pathological
roles of EFhd2 are poorly understood. In this study, a functional and structural analysis was carried to characterize
the molecular requirements for EFhd2’s calcium binding activity. The results showed that mutations of a conserved aspartate
on either EF-hand motif disrupted the calcium binding activity, indicating that these motifs work in pair as a functional
calcium binding domain. Furthermore, characterization of an identified single-nucleotide polymorphisms (SNP) that
introduced a missense mutation indicates the importance of a conserved phenylalanine on EFhd2 calcium binding activity.
Structural analysis revealed that EFhd2 is predominantly composed of alpha helix and random coil structures and that this
novel protein is thermostable. EFhd2’s thermo stability depends on its N-terminus. In the absence of the N-terminus, calcium
binding restored EFhd2’s thermal stability. Overall, these studies contribute to our understanding on EFhd2 functional
and structural properties, and introduce it into the family of canonical EF-hand domain containing proteins.