Photon Factory Activity Report 2016 #34 (2017)
BL-17A,BL-10C/2014G714,2016G678,2016G680

Crystal structure of RavZ from Legionella pneumophila
Do Hoon Kwon and Hyun Kyu Song*Division of Life Sciences, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, South Korea

1 Introduction
Hosts utilize macroautophagy/autophagy to clear
invading bacteria; however, bacteria have also developed
a specific mechanism to survive by manipulating the hostcell autophagy mechanism. One pathogen, Legionella
pneumophila, can hinder host cell autophagy by using thespecificeffectorproteinRavZ
thatcleaves
phosphatidylethanolamine-conjugated LC3 on thephagophore membrane. [1] However, the detailed
molecular mechanisms associated with the function ofRavZ have hitherto remained unclear. Here, we report on
the biochemical characteristics of the RavZ-LC3interaction, the solution structure of the 1:2 complex
between RavZ and LC3, and crystal structures of RavZ
showing different conformations of the active site loop
without LC3. Based on our biochemical, structural, andcell-based analyses of RavZ and LC3, both distant flexible
N- and C-terminal regions containing LC3-interacting
region (LIR) motifs are important for substrate recognition.
These results suggest a novel mechanism of RavZ action
on the phagophore membrane and lay the groundwork forunderstanding how bacterial pathogens can survive
autophagy. [2]2ExperimentHis-tagged RavZ from Legionella pneumophila wasexpressed and GST-tagged LC3 from Homo sapiens using
modified pGEX4T-1 vector. The overexpressed RavZ
protein was purified by nickel-NTA affinity
chromatography followed by anion exchange and size
exclusion chromatography. And the overexpressed LC3B
protein was purified by GST chromatography and removed
GST tag by TEV protease. For further purification, weperformed cation exchange and size exclusion
chromatography. RavZ was crystallized using the hanging
drop method by vapor-diffusion at 20 ºC. In RavZ crystal
diffracted up to 2.7 Å and the number of molecules inasymmetric unit was one. The space group and unit cellparameters were I422 with a=b=222.0 Å and c=71.3 Å.
Diffraction data were collected using an ADSC quantum
CCD detector at the NW12 beamline of Photon Factory.
180-200 images were collected with 1º oscillation, andeach image was exposed for 1-3 seconds. The diffraction
data were processed and scaled using the HKL2000
software package. Phases were obtained by single
wavelength anomalous dispersion (SAD) using MOLREPin the CCP4 program suite. The initial model was manuallybuilt and refined using COOT and PHENIX.

domains which are catalytic domain and membranetargeting domain. Catalytic domain is similar with humansumo protease and membrane targeting domain has special
protein folding only found bacteria proteins (Fig. 1A and1B). Due to the flexibility, N-terminal 48 residues and Cterminal 71 residues were not visible in the electron density
map.

Fig. 1: Crystal structure of RavZ. (A) Overall structures ofRavZ. (B) Schematic drawing of RavZ overall structure.

We tried to solve the structure of complex between
RavZ and LC3B, but it was not successful in thecrystallization. Therefore, we performed the small-angle
X-ray scattering experiments using a complex between
RavZ and LC3B in solution. According to SAXS model,
we found that N-terminus and C-terminus can interact with
LC3B separately (Fig. 1C and 1D). The binding
stoichiometry between RavZ and LC3B is 1:2.

AFig. 2: Solution structure of RavZ-LC3B complex. (A) SAXS
model of RavZ. (B) SAXS model of RavZ and LC3B complex.

References[1] A. Choy et al., Science 338, 1072 (2012)[2] D.H. Kwon et al., Autophagy 13, 70 (2017)*hksong@korea.ac.kr
3Results
Legionella pneumophilia RavZ consists of two distinct

B