Structural Study Of The Legionella Pneumophila Effector Lem27

Ubiquitin(Ub)chains regulate many cellular processes,including NF-?B immune signalling.Legionella pneumophila have evolved to secrete effector proteins that harbour deubiquitinase(DUB)activity into host cells to disrupt host ubiquitination signaling.Serious infection of Legionella pneumophila causes a human syndrome named Legionnaires?disease.We performed extensive screening of the effectors from Legionella pneumophila and found that Legionella pneumophila encodes an effector protein,Lem27,which harbours deubiquitinase activity for Ub chains.We have used crystallography and biochemistry experiments to study the mechanism of Lem27 binding the Ub chains,especially the Lys48-and 63-linked-Ub chains.We have amplified Lem27 from Legionella pneumophila genomic DNA.After expression,purification and crystallization,crystals of the complex was obtained.After crystal optimization,we successfully obtained the diffraction-qualified crystals of the complex.The native crystals diffracted to the 3.0 ? resolution.We also expressed the selenomethionine-derivatized protein of Lem27 and got the SeMet-labeled crystals.The crystals of SeMet-labeled diffracted to the 2.7 ? resolution at the Shanghai synchrotron.We have successfully solved the complex structure of Lem27(1-429)C24A with monoUb,which contains the catalytic assembled as dimer in structure.Other experiments revealed that the dimer conformation is reliable.Analysis of representative residues around the cleavage pocket revealed that His304 may cooperate with Cys24 to exert DUB activity.The results reported here provide a basis for further studies of the mechanism of Lem27 binding the Ub chains by mutants construction.