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Structure-based analyses reveal distinct binding sites for Atg2 and phosphoinositides in Atg18.

ABSTRACT: Autophagy is an intracellular degradation system by which cytoplasmic materials are enclosed by an autophagosome and delivered to a lysosome/vacuole. Atg18 plays a critical role in autophagosome formation as a complex with Atg2 and phosphatidylinositol 3-phosphate (PtdIns(3)P). However, little is known about the structure of Atg18 and its recognition mode of Atg2 or PtdIns(3)P. Here, we report the crystal structure of Kluyveromyces marxianus Hsv2, an Atg18 paralog, at 2.6 Å resolution. The structure reveals a seven-bladed ?-propeller without circular permutation. Mutational analyses of Atg18 based on the K. marxianus Hsv2 structure suggested that Atg18 has two phosphoinositide-binding sites at blades 5 and 6, whereas the Atg2-binding region is located at blade 2. Point mutations in the loops of blade 2 specifically abrogated autophagy without affecting another Atg18 function, the regulation of vacuolar morphology at the vacuolar membrane. This architecture enables Atg18 to form a complex with Atg2 and PtdIns(3)P in parallel, thereby functioning in the formation of autophagosomes at autophagic membranes.

SUBMITTER: Watanabe Y

PROVIDER: S-EPMC3442503 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Structure-based analyses reveal distinct binding sites for Atg2 and phosphoinositides in Atg18.

Watanabe Yasunori Y Kobayashi Takafumi T Yamamoto Hayashi H Hoshida Hisashi H Akada Rinji R Inagaki Fuyuhiko F Ohsumi Yoshinori Y Noda Nobuo N NN

The Journal of biological chemistry 20120731 38

Autophagy is an intracellular degradation system by which cytoplasmic materials are enclosed by an autophagosome and delivered to a lysosome/vacuole. Atg18 plays a critical role in autophagosome formation as a complex with Atg2 and phosphatidylinositol 3-phosphate (PtdIns(3)P). However, little is known about the structure of Atg18 and its recognition mode of Atg2 or PtdIns(3)P. Here, we report the crystal structure of Kluyveromyces marxianus Hsv2, an Atg18 paralog, at 2.6 Å resolution. The struc ...[more]

PMID: 22851171

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Project description:The reversible phosphorolysis of purine and pyrimidine nucleosides is an important biochemical reaction in the salvage pathway, which provides an alternative to the de novo purine and pyrimidine biosynthetic pathways. Structural studies in our laboratory and by others have revealed that only two folds exist that catalyse the phosphorolysis of all nucleosides, and provide the basis for defining two families of nucleoside phosphorylases. The first family (nucleoside phosphorylase-I) includes enzymes that share a common single-domain subunit, with either a trimeric or a hexameric quaternary structure, and accept a range of both purine and pyrimidine nucleoside substrates. Despite differences in substrate specificity, amino acid sequence and quaternary structure, all members of this family share a characteristic subunit topology. We have also carried out a sequence motif study that identified regions of the common subunit fold that are functionally significant in differentiating the various members of the nucleoside phosphorylase-I family. Although the substrate-binding sites are arranged similarly for all members of the nucleoside phosphorylase-I family, a comparison of the active sites from the known structures of this family indicates significant differences between the trimeric and hexameric family members. Sequence comparisons also suggest structural identity between the nucleoside phosphorylase-I family and both 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase and AMP nucleosidase. Members of the second family of nucleoside phosphorylases (nucleoside phosphorylase-II) share a common two-domain subunit fold and a dimeric quaternary structure, share a significant level of sequence identity (>30%) and are specific for pyrimidine nucleosides. Members of this second family accept both thymidine and uridine substrates in lower organisms, but are specific for thymidine in mammals and other higher organisms. A possible relationship between nucleoside phosphorylase-II and anthranilate phosphoribosyltransferase has been identified through sequence comparisons. Initial studies in our laboratory suggested that members of the nucleoside phosphorylase-II family require significant domain movements in order for catalysis to proceed. A series of recent structures has confirmed our hypothesis and provided details of these conformational changes. Structural studies of the nucleoside phosphorylases have resulted in a wealth of information that begins to address fundamental biological questions, such as how Nature makes use of the intricate relationships between structure and function, and how biological processes have evolved over time. In addition, the therapeutic potential of suppressing the nucleoside phosphorylase activity in either family of enzymes has motivated efforts to design potent inhibitors. Several research groups have synthesized a variety of nucleoside phosphorylase inhibitors that are at various stages of preclinical and clinical evaluation.

Dataset Information

Structure-based analyses reveal distinct binding sites for Atg2 and phosphoinositides in Atg18.

Publications

Structure-based analyses reveal distinct binding sites for Atg2 and phosphoinositides in Atg18.

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