Regulation of Shigella Effector Kinase OspG through Modulation of Its Dynamic Properties

Gram-negative pathogens secrete effector proteins into human cells to modulate normal cellular processes and establish a bacterial replication niche. Shigella and pathogenic Escherichia coli possess homologous effector kinases, OspG and NleH1/2, respectively. Upon translocation, OspG but not NleH binds to ubiquitin and a subset of E2 ~ Ub conjugates, which was shown to activate its kinase activity. Here we show that OspG, having a minimal kinase fold, acquired a novel mechanism of regulation of its activity. Binding of the E2 ~ Ub conjugate to OspG not only stimulates its kinase activity but also increases its optimal temperature for activity to match the human body temperature and stabilizes its labile C-terminal domain. The melting temperature (T_m) of OspG alone is only 31?°C, as compared to 41?°C to NleH1/2 homologs. In the presence of E2 ~ Ub, the T_m of OspG increases to ~ 42?°C, while Ub by itself increases the T_m to 39?°C. Moreover, OspG alone displays maximal activity at 26?°C, while in the presence of E2 ~ Ub, maximal activity occurs at ~ 42?°C. Using NMR and molecular dynamics calculations, we have identified the C-terminal lobe and, in particular, the C-terminal helix, as the key elements responsible for lower thermal stability of OspG as compared to homologous effector kinases.