Crystallization and preliminary X-ray analysis of Atg3

Yamada, Yuya; Suzuki, Nobuo; Fujioka, Yūko; Ichimura, Yoshinobu; Ohsumi, Yoshinori; Inagaki, Fuyuhiko

doi:10.1107/s1744309106036098

Cited by 7 publications

(7 citation statements)

References 12 publications

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“…The expression and purification of Atg8(K26P) and Atg3 have been described previously (Kumeta et al, 2010;Yamada et al, 2006). Full-length S. cerevisiae Atg7 and its truncates were cloned into a pGEX-6P vector (GE healthcare).…”

Section: Experimental Procedures Protein Expression and Purificationmentioning

confidence: 99%

Structural Basis of Atg8 Activation by a Homodimeric E1, Atg7

et al. 2011

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E1 enzymes activate ubiquitin-like proteins and transfer them to cognate E2 enzymes. Atg7, a noncanonical E1, activates two ubiquitin-like proteins, Atg8 and Atg12, and plays a crucial role in autophagy. Here, we report crystal structures of full-length Atg7 and its C-terminal domain bound to Atg8 and MgATP, as well as a solution structure of Atg8 bound to the extreme C-terminal domain (ECTD) of Atg7. The unique N-terminal domain (NTD) of Atg7 is responsible for Atg3 (E2) binding, whereas its C-terminal domain is comprised of a homodimeric adenylation domain (AD) and ECTD. The structural and biochemical data demonstrate that Atg8 is initially recognized by the C-terminal tail of ECTD and is then transferred to an AD, where the Atg8 C terminus is attacked by the catalytic cysteine to form a thioester bond. Atg8 is then transferred via a trans mechanism to the Atg3 bound to the NTD of the opposite protomer within a dimer.

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Section: Experimental Procedures Protein Expression and Purificationmentioning

confidence: 99%

Structural Basis of Atg8 Activation by a Homodimeric E1, Atg7

et al. 2011

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“…Protein Expression and Purification-Wild-type Atg3 was expressed and purified for crystallization as described previously (15). For in vitro assays, wild-type Atg3 and three Atg3 mutants (Atg3⌬FR, Atg3FR, and Atg3⌬HR) were prepared as follows.…”

Section: Methodsmentioning

confidence: 99%

“…Preparation of Derivative Crystals and Data CollectionCrystallization of Atg3 was carried out as described previously (15). Platinum and two mercury derivatives were prepared by soaking the crystals of Atg3 for 30 min in a solution of 1.8 M lithium sulfate and 0.1 M citrate buffer (pH 6.6) containing 10 mM K 2 PtCl 4 , 1 mM HgCl 2 , or 0.1 mM thimerosal.…”

Section: Methodsmentioning

confidence: 99%

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The Crystal Structure of Atg3, an Autophagy-related Ubiquitin Carrier Protein (E2) Enzyme that Mediates Atg8 Lipidation

Yamada

Suzuki

Hanada

et al. 2007

Journal of Biological Chemistry

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127

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Atg3 is an E2-like enzyme that catalyzes the conjugation of Atg8 and phosphatidylethanolamine (PE). The Atg8-PE conjugate is essential for autophagy, which is the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. We report here the crystal structure of Saccharomyces cerevisiae Atg3 at 2.5-Å resolution. Atg3 has an ␣/␤-fold, and its core region is topologically similar to canonical E2 enzymes. Atg3 has two regions inserted in the core region, one of which consists of ϳ80 residues and has a random coil structure in solution and another with a long ␣-helical structure that protrudes from the core region as far as 30 Å . In vivo and in vitro analyses suggested that the former region is responsible for binding Atg7, an E1-like enzyme, and that the latter is responsible for binding Atg8. A sulfate ion was bound near the catalytic cysteine of Atg3, suggesting a possible binding site for the phosphate moiety of PE. The structure of Atg3 provides a molecular basis for understanding the unique lipidation reaction that Atg3 carries out.

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“…These two Atg3s were subjected to crystallization screening. Intriguingly, crystals were obtained from Atg3 with a Gly-Ser artificial sequence, but not from Atg3 with a Gly-Pro-Leu-Gly-Ser artificial sequence (Yamada et al, 2006). Thus, we used pGEX-4T-Atg3 for further studies, despite the fact that we usually use the pGEX-6P vector rather than the pGEX-4T vector since a PreScission protease has a higher activity than a thrombin protease at low temperature.…”

Section: Atg3mentioning

confidence: 99%