Photon Factory Activity Report 2014 #32 (2015) B

AR-NW12A/2014G714

Structure of complexes between ATG5 and its binding partners for autophagosome
maturation
Jun Hoe Kim and Hyun Kyu Song*Division of Life Sciences, Korea University
145 Anam-ro, Seongbuk-gu, Seoul 136-713, South Korea
1Introduction
Autophagy is a tightly regulated lysosome-mediatedcatabolic process in eukaryotes that maintains cellular
homeostasis. A distinguishable feature of autophagy is
the formation of double-membrane structures,
autophagosome, which envelopes the intracellularcargoes and finally degrades them by fusion withlysosomes. So far, many structures of Atg proteinsworking on the autophagosome formation have beenreported, however those involved in autophagosome
maturation, a fusion with lysosome, are relatively
unknown. One of the molecules in autophagosome
maturation, TECPR1, has been identified and recently,
structural studies on both ATG5-TECPR1 and ATG5ATG16L1 complexes revealed that TECPR1 andATG16L1 share the same binding site on ATG5. We
reported these results in combination with supporting
biochemical and cellular biological data providing aninsight into a model for swapping ATG5 partners forautophagosome maturation [1,2].

Experiment
Human His-tagged ATG5 was co-expressed with Nterminal 69 residues of ATG16L1 (ATG16N69) or AIR of
TECPR1 (TECAIR) using pETDuet-1 vector. The overexpressed proteins were co-purified by nickel-NTA
affinity chromatography followed by anion exchange andsize exclusion chromatography. ATG5-ATG16N69 was3Results
Human ATG5 consists of 2 ubiquitin-fold domains
which are the equivalent of the ubiquitin-like domains ofyeast Atg5 and has a similar binding mode on
ATG16N69 and TECAIR (Fig. 1A). According to asequence alignment, it was revealed that several residues
on the helix of ATG16L1 and TECPR1 are conserved
among species. We identified the motif as W-X3-I-X2-LX3-Q/E and named as ATG5-interacting motif (AFIM).
Biochemical and cell biological data supported thesignificance of the motif [2] and finally we suggested amodel of autophagosome maturation mechanism by
swapping of ATG5 interaction partners (Fig. 1B).

A2Bcrystallized using the hanging drop method byvapor-diffusion at 20 ºC. In case of ATG5-TECAIR,initial twinned crystals were improved by the microseeding technique and growth at 4 ºC. ATG5-ATG16N69
crystal diffracted up to 2.7 Å and the number ofmolecules in asymmetric unit was 3. The spacegroup and unit cell parameters were P41212 with
a=b=93.09 Å and c=245.6 Å . ATG5-TECAIR crystal
diffracted up to 1.8 Å with 1 molecule per asymmetricunit and belongs to P212121 space group with a=43.62
Å , b=71.92 Å and c=96.35 Å . Diffraction data werecollected using an ADSC quantum CCD detector at theNW12 beamline of Photon Factory. 180-200 images werecollected with 1º oscillation, and each image was exposedfor 3-5 seconds. The diffraction data were processed andscaled using the HKL2000 software package. Phases
were obtained by molecular replacement with previously
determined yeast Atg5 structure [3] using MOLREP inthe CCP4 program suite. The initial model was manuallybuilt and refined using COOT and PHENIX.

Fig. 1 (A) Overall structures of ATG5-ATG16N69 (left) andATG5-TECAIR (right). (B) A simplified model for
autophagosome maturation mechanism by swapping ATG5
binding partners.

References[1] Kim, J.H. et al. BMB Reports 48(3), 129-130 (2015)[2] Kim, J.H. et al. Autophagy 11:1, 75-87 (2015)[3] Matsushita, M. et al. JBC 282: 6763-6772 (2007)*hksong@korea.ac.kr