Sequence and structural alignments reveal insights into ANKLE2 evolution and function.

ANKLE2 is an enigmatic protein with emerging roles in cell division, development, and virus replication. While ANKLE2 orthologs are present in all animals, its domain composition has evolved over time. ANKLE2's two namesake domains, the ankyrin repeat and LEM domains (named for LAP2, Emerin, and MAN1), have clear and defined roles; however nearly all ANKLE2 orthologs have at least three other structured domains with poorly understood purposes. In this study, we performed sequence and structural alignments of ANKLE2 orthologs to improve our understanding of the protein's evolution and function. We identified that ANKLE2's transmembrane domain likely evolved more recently and coincided with loss of VAPA interaction as a membrane anchoring mechanism. We show that despite stark differences in amino acid sequence, the structure of the LEM and ankyrin repeat domains are highly conserved across ANKLE2 orthologs. To investigate ANKLE2's uncharacterized domains, we performed structural alignments to identify similar proteins. This revealed surprising similarities between portions of ANKLE2 and nuclease or nucleic acid-binding proteins. However, ANKLE2 lacks key motifs imparting function in these domains, which was confirmed by experimental interrogation. We further identified that loss of ANKLE2 is correlated with changes in DNA damage response and micronuclei formation. We believe this methodology demonstrates the power of combining structural predictions with classical molecular techniques in exploring poorly understood proteins.