Legionella Translocates an E3 Ubiquitin Ligase That Has Multiple U-boxes with Distinct Functions. the Crystal Structure of Plant-specific Calcium-binding Protein Atcbl2 in Complex with the Regulatory Domain of Atcipk14

Legionella pneumophila has a Dot/Icm type IV secretion system (T4SS) used to translocate a number of ‘effector proteins’ which subvert host cell functions. In this study we identified 19 novel Dot/Icm substrate proteins using a systematic screening technique. A BLAST analysis revealed that one of substrates, which we named LubX, contains two domains that have a remarkable similarity to the U-box, a domain found in eukaryotic E3 ubiquitin ligases. The expression of LubX is induc ed upon infection, and most of the LubX produced was translocated into the host cells. LubX has ubiquitin ligase activity in conjunction with UbcH5a or UbcH5c E2 enzymes and mediates polyubiquitination of host Clk1 (Cdc2-like kinase 1). We demonstrate that one of the U-boxes (U-box 1) is critical to the ubiquitin ligation and the other U-box (U-box 2) mediates interaction with Clk1. Thus the two U-boxes of LubX have distinct functions, and U-box 2 plays a non-canonical role in substrate binding. Although we demonstrate that inhibition of Clk kinase results in a marked reduction of Legionella growth within mouse macrophages, the consequence of Clk1 ubiquitination is still being elucidated,. Together these data suggest that Clk1 is the target host molecule which Legionella modulates during infection.