The divergent domains of the NEFA and nucleobindin proteins are derived from an EF-hand ancestor

The human protein NEFA (DNA binding, EF-hand, Acidic region) has previouslybeen isolated from a KM3 cell line and immunolocalized on the plasmamembrane, in the cytoplasma, and in the culture medium. Sequence analysisof a cDNA clone encoding NEFA identified a hydrophilic domain, twoEF-hands, and a leucine zipper at the C- terminus. These characters areshared with nucleobindin (Nuc). In this paper we have further characterizedNEFA and probed its evolutionary origins. Circular dichroism (CD) spectraof recombinant NEFA indicated a helical content of 51% and showed that theEF-hands are capable of binding Ca2+. Experiments with recombinant NEFA andsynthesized peptides revealed that the leucine zipper cannot form ahomodimer. The leucine zipper may allow heterodimer formation of NEFA andan unknown protein. Phylogenetic analyses suggest that this protein isderived from a four-domain EF-hand ancestor with subsequent duplicationsand fusions. The leucine zipper and putative DNA-binding domains of NEFAhave evolved secondarily from existing EF-hand sequences. These analysesprovide insights into how complex proteins may originate and trace theprecursor of NEFA to the common ancestor of eukaryotes.