Photon Factory Activity Report 2019 #37 (2020)

BL-1A, AR-NE3A/2019RP-19

Structural study on Atg8-receptor complexes that mediate ER-phagy
1

Akinori YAMASAKI1 and Nobuo N. NODA1,*
Institute of Microbial Chemistry, Microbial Chemistry Research Foundation,
3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan

1 Introduction
Autophagy is an intracellular degradation system andplays a critical role in the degradation of various organelles[1]. Selective degradation of endoplasmic reticulum (ER)by autophagy is named ER-phagy, which is mediated by anER-phagy receptor Atg40 in budding yeast and several ERphagy receptors that include Sec62 in mammals [2, 3].
These ER-phagy receptors possess a transmembraneregion and is embedded in the ER membrane. Upon ERphagy induction, ER-phagy receptors interact with Atg8 on
the newly generated isolation membrane (IM), a precursorof autophagosomes, using Atg8-family interacting motif(AIM) [4], thereby tethering a portion of the ER to the IM
and promoting ER-phagy. In this study, we studied theinteraction of Atg8 with two distinct ER-phagy receptors,Atg40 and Sec62, by X-ray crystallography.
2 Experiment
Plasmids for expression of fusion proteins forcrystallization were constructed by inserting the genes
encoding Atg40 (237-252) (Atg40AIM) and human SEC62(361-376) with the T367D mutation (Sec62AIM) intoupstream of the sequence encoding Atg8 K26P of pGEX6PAtg8K26P (the K26P mutation was introduced for stabilizing
Atg8 [5]) or upstream of the sequence encoding
GABARAP of pGEX6P-GABARAP (with the F3S V4T
mutations for enhancing crystallization). Proteins wereexpressed in E. coli BL21 (DE3). Bacteria were cultured at
37°C until OD600 became 0.8-1.0 and further cultured at16°C with 100 M IPTG overnight. After centrifugation,
the pellets were resuspended with 20 ml PBS with 0.5 mM
EDTA and lysed by sonication for 10 min. Aftercentrifugation, the supernatants were incubated with GSTaccept resin (Nacalai tesque). After 3 times wash by PBS,
the proteins were eluted with glutathione buffer (10 mM
glutathione and 50 mM Tris pH 8.0). The buffer of theeluates was exchanged with PBS by a Bio-Scale Mini
BioGel P-6 desalting column (Bio-Rad). After incubation
with GST-fused HRV 3C protease at 4°C overnight, thesamples were subjected to a GST-accept resin column toremove excised GST and the protease.
All crystallization trials were performed by the sittingdrop vapor-diffusion method at 20°C. For crystallization ofAtg40AIM-Atg8 fusion, 41 mg/ml protein was incubatedwith 10% PEG 8000, 0.1 M HEPES pH 7.5, 8% ethylene
glycol as a reservoir and C6 well of silver bullets bio(0.04% Cortisone, 0.04% (±)-Epinephrine, 0.04%
Protoporphyrin disodium salt, 0.04% Pyridoxine, 0.04%Thymidine 5’-monophosphate disodium salt hydrate, and0.02 M HEPES buffer pH 6.8) (Hampton Research) as anadditive. The volume ratio of protein, reservoir, andadditive was 2:1:1. For crystallization of SEC62AIMGABARAP fusion, 40 mg/ml protein was mixed with areservoir solution consisting of 1.5 M ammonium sulfate
and 0.1 M Tris pH 8.5. The mixing volume ratio of protein
and reservoir solution was 1:1 and mixed solution wasequilibrated against the reservoir solution. All crystals
were obtained within 4 days.
Crystals were soaked in cryoprotectant and frozen inliquid nitrogen. The cryoprotectant for Atg40AIM-Atg8
fusion was prepared by adding 25% ethylene glycol to thereservoir solution. The cryoprotectants for SEC62AIMGABARAP fusions were prepared by adding 33%
glycerol to each reservoir solution. The flash-cooled
crystals were kept in a stream of nitrogen gas at -178°
C during data collection. Diffraction data collections ofthe crystals for Atg40 and Sec62 were performed at thebeamlines BL-1A and NE3A at KEK, Japan, with thewavelength of 1.1000 and 1.0000 Å, respectively. The
diffraction data were indexed, integrated, and scaled
using XDS [6]. The structures of Atg40AIM-Atg8 andSEC62AIM-GABARAP weredetermined bythemolecular replacement method using Phenix [7]. Atg8
(PDBID: 2ZPN) and GABARAP (PDBID: 1GNU)
structures were used as a search model. Crystallographic
refinement was done by using Phenix and COOT
programs [7, 8]. All structural images in themanuscript were prepared by PyMOL (The PyMOL
Molecular Graphics System, Version 2.0 Schrödinger,LLC.). Parameters of diffraction data collection andcrystallographic refinement are summarized in Table 1.
3 Results and Discussion
The core of the AIM is composed of four amino-acidresidues [W/F/Y]-X-X-[L/I/V]. The first aromatic residue
and the fourth aliphatic residue bind to two hydrophobic
pockets in Atg8 (the W- and L-sites, respectively), and themain chain of the motif forms an intermolecular parallel βsheet with β2 of Atg8 [4]. The crystal structure ofAtg40AIM-Atg8 revealed that as with canonical AIMs, thefirst and fourth residues Y242 and M245 of Atg40AIM
bound to the W- and L-sites of Atg8, respectively. In
addition, the second residue D243 formed salt bridges with
the R67 residue of Atg8. Moreover, the Atg40 region Cterminal to M245 assumed a helical conformation, andD247 in this helix formed salt bridges with R67 of Atg8.
Residue F238 on the N-terminal side of the core motif alsointeracted with I21 and the aliphatic portion of R20 ofAtg8.
We also determined the crystal structure of GABARAP(a mammalian Atg8 homologue) bound to SEC62AIM.
Remarkably, the AIM containing region of Sec62 isstructurally similar to that of Atg40, consisting of the core
AIM and its C-terminal short helix. The core AIM residues

Photon Factory Activity Report 2019 #37 (2020)of SEC62 (F363 and I366) interacted with GABARAP ina similar manner. In addition, as with Atg40, the second
AIM residues (E364 of SEC62) and acidic residues in theshort helices (E370 of SEC62) interacted with R67 ofGABARAP. Thus, the C-terminal helix-assisted AIM is aunique structural basis commonly seen in the interactionsof ER-phagy receptors with Atg8-family proteins [9].
Table 1: Data collection and refinement statistics
Atg40AIM-Atg8
Sec62AIM(PDB 7BRN)GABARAP(PDB 7BRT)
Data collection
Space group
P3221
P212121
Cell dimensions
a, b, c (Å)
74.7, 74.7, 57.1
42.9, 42.9, 144.0
90,90,
120
90, 90, 90
   ()Resolution (Å)a37.4-2.23
41.1-2.00(2.31-2.23)a(2.07-2.00)
Rmerge0.141 (0.595)
0.164 (0.920)
13.78 (2.44)
9.27 (2.02)
I/(I)
Completeness (%) 99.58 (97.91)
99.73 (98.34)
Redundancy
9.6 (7.3)
6.5 (5.8)
Refinement
Resolution (Å)
37.4-2.23
41.1-2.00
No. reflections
9227
18676
Rwork / Rfree
0.2031/0.2187
0.1656 / 0.1836
No. atoms
Protein
1076
2172
Water
44291
B factors
Protein
45.99
30.61
Water
45.06
34.55
R.m.s deviations
Bond lengths (Å) 0.008
0.007
0.91
Bond angles () 1.29
aValues in parentheses are for highest-resolution shell.

Fig. 1: Structure of Atg40AIM-Atg8 complex (left) andSec62AIM-GABARAP complex (right).
Acknowledgement
We thank Y. Ishii for her help with protein purificationand crystallization. This work was supported by Japan
Science and Technology Agency (JST) CREST(JPMJCR13M7) (to N.N.N.), the Japan Society for thePromotion of Science (JSPS) KAKENHI (19H05707 toN.N.N., 17K18339 to A.Y.) and the Takeda Science
Foundation (to A.Y. and N.N.N.).
References[1] N. N. Noda and F. Inagaki. Annu. Rev. Biophys. 44, 101(2015).
[2] K. Mochida et al. Nature 522, 359 (2015).
[3] F. Fumagalli et al. Nat. Cell Biol. 18, 1173 (2016).
[4] N. N. Noda et al. FEBS Lett. 584, 1379 (2010).
[5] H. Kumeta et al. J. Biomol. NMR 47, 237 (2010).
[6] W. Kabsch. Acta Crystallogr. D Biol. Crystallogr. 66,125 (2010).
[7] D. Liebschner et al. Acta Crystallogr. D Struct. Biol.
75, 861 (2019).
[8] P. Emsley et al. Acta Crystallogr. D Biol. Crystallogr.
66, 486 (2010).
[9] K. Mochida et al. Nat. Commun., in press, (2020).
* nn@bikaken.or.jp