119.2 - Structural and mechanistic basis for protein glutamylation by the kinase fold

Vincent Tagliabracci (HHMI/UT Southwestern), Adam Osinski (UT Southwestern), Miles Black (UT Southwestern), Krzysztof Pawlowski (UT Southwestern), Zhe Chen (UT Southwestern), Yang Li (UT Southwestern)

The kinase domain transfers phosphate from ATP to substrates.  However, the Legionella effector SidJ adopts a kinase fold yet catalyzes calmodulin (CaM)-dependent glutamylation to inactivate the SidE ubiquitin ligases.  The structural and mechanistic basis in which the kinase domain catalyzes protein glutamylation is unknown.  Here we present cryo-EM reconstructions of SidJ:CaM:SidE reaction intermediate complexes.  We show that the kinase-like active site of SidJ adenylates an active site Glu in SidE resulting in the formation of a stable reaction intermediate complex.  An insertion in the catalytic loop of the kinase domain positions the donor Glu near the acyl-adenylate for peptide bond formation.  Our structural analysis led us to discover that the SidJ paralog SdjA is a glutamylase that differentially regulates the SidE-ligases during Legionella infection.  Our results uncover the structural and mechanistic basis in which the kinase fold catalyzes non-ribosomal amino acid ligations and reveal an unappreciated level of SidE-family regulation.