Purification, characterization and functional site prediction of the vaccinia-related kinase 2A small transmembrane domain

Vaccinia-related kinases (VRK) are serine-threonine kinases that regulate several signaling pathways. The isoform-VRK2A of one such kinase VRK2 controls cell stress response by interacting with TAK1, a mitogen-activated protein 3 kinase (MAP3K), via its partly cytosolic C-terminal transmembrane domain (VTMD). To establish the driving force and identify the key residues of the VRK2A-TAK1 interaction, we expressed and purified the standalone 3.6 kDa VTMD in the bacterial system using a unique and atypical two-step approach, when the effort to obtain full-length VRK2A remained unsuccessful. Characterization of biophysical properties demonstrated that VTMD domain maintains its structural integrity. Furthermore, dissecting the VRK2A-TAK1 binding interface using in silico • VRK2A is a highly disordered transmembrane (TM) kinase, whose TM domain interacts with TAK1 (transforming growth factor-β-activated kinase). • The standalone VRK2A-TM domain (VTMD) was purified using affinity chromatography followed by two-step centricon based approach. • Biophysical and in silico analyses confirmed structural integrity of the domain.