Structural Study Of The Legionella Pneumophila Type â…£ Effector RidL

Serious infection of Legionella pneumophila causes a human syndrome named Legionnaires’disease. Once uptaken into the human macrophage, the bacteria replicate within a Legnella-containing vacuoles (LCV). RidL, an effector protein secreted by the Icm/Dot type IV secretion system of Legionella, localizes on the LCVs and hijacks the host retrograde trafficking pathway to benefit the bacterial intracellular growth. Previous studies have suggested that RidL specifically interacts with the Vps29subunit of the retromer complex to modulate the host retrograde trafficking pathway. However, the detailed interactions and modulation mechanism are unclear.To uncover the molecular mechanism of RidL, we expressed a truncated RidL (residues183-734) in E. coli. After mutipule steps of purification, we obtained the highly soluble and stable RidL recombinant protein. The purified protein was finally concentrated to20mg/ml for our crystal screening. Fortunately, we obtained the needle-shaped crystals of RidL in initial screening. After crystal optimization, we successfully obtained the diffraction-qualified crystals of RidL. The native crystals of RidL diffracted to a2.7A resolution on the beamline BLU171at Shanghai SSRF. We also expressed the selenomethionine-derivatized protein of RidL and got the SeMet-labeled crystals. The crystals of SeMet-labeled RidL diffracted to the3.5A resolution at the Shanghai synchrotron. The space group of native and SeMet Crystals of RidL has been determined to be C2, with unit-cell parameters a, b, c (A):259.64,78.394,70.07; a,β, γ (°): 90.00,91.49,90.00.In summary, I successfully expressed and crystallized the Legionella type Ⅳ effector RidL. After optimization, the native and SeMet-labeled crystals of RidL diffracted to2.7A and3.5A, respectively. My studies provide the basis for further structural determination of RidL and will be very important for uncovering its molecular mechanism.