Study On Crystallization Of Human MST4and Its Interaction With MO25α

MST4(mammalian Ste20-like kinases, MST) is a member of GCKⅢ subfamily which belongs to the Ste-20Ser/Thr protein kinase family, and is widely involved in cell polarity, cell growth and apoptosis. MST4contains a highly conserved kinase domain at the N-terminal region, and C-teminal regulatory domain, which mediates its dimerization and regulates its kinase activity. In this study, we focus on crystal analysis of MST4kinase domain and its interaction analysis with MO25α. With clone, expression and purification of MO25α, MST4full length (1-416), MST4(1-326), MST4(325-416), MST4kinase domain (1-300), kinase dead mutant MST4(1-300)-D162A and A-loop mimic phosphorylation mutant MST4(1-300)-D162A-T172/178E, we’ve got stable and homogeneous protein with high purity. By crystallization of MST4(1-300)-D162A-T172/178E, we got a crystal which diffracts to2.9A, preliminary data shows its crystal belongs to space group P212121with unit cell parameters a=69.63A, b=73.78A,c=127.10A, α=β=γ=90.0°. To investigate the interaction mechanism of MST4and MO25α, we processed pull-down assay and KD determination, and found that MST4kinase domain is sufficient to bind MO25α in a kinase activity independent way. By co-purified through size exclusive chromatography we also found that MST4kinase domain and MO25α can form a heterodimer while MST4full length forms a tetramer to bind with a tetramer MO25α. These data provides a solid foundation for revealing the crystal structure of MST4-MO25complex.