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Structure of the novel C-terminal domain of vacuolar protein sorting 30/autophagy-related protein 6 and its specific role in autophagy.

Noda NN, Kobayashi T, Adachi W, Fujioka Y, Ohsumi Y, Inagaki F - J. Biol. Chem. (2012)

Bottom Line: Thus, the domain is named the β-α repeated, autophagy-specific (BARA) domain.On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar protein sorting.These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this protein family in various biological processes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbial Chemistry, Tokyo, Tokyo 141-0021, Japan. nn@bikaken.or.jp

ABSTRACT
Vacuolar protein sorting 30 (Vps30)/autophagy-related protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this protein family in various biological processes.

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Structure of Vps30 BARA.A, overall structure of Vps30 BARA. The α-helices and β-strands are indicated with red helical ribbons and cyan arrows, respectively. Secondary structures are labeled, and residues adjacent to the disordered regions are numbered. Amino and carboxy termini are denoted as N and C, respectively. B, topology of Vps30 BARA. The α-helices and β-strands are indicated with boxes and arrows, respectively. Repeats 1, 2, and 3 are colored green, red, and cyan, respectively. The disordered regions are indicated with a broken line. C, superimposition of the secondary structural elements of repeats 2 and 3 on those of repeat 1. Coloring is as in B. D, left, ribbon representation of the three-helix bundle of BARA. Side chains involved in the interaction between helices are indicated with a stick model. Coloring is as in B. Right, ribbon representation of secondary structural elements of BARA. The side chains of conserved hydrophobic residues on the three sheets, which are bound to the grooves formed between helices, are indicated with a stick model. Coloring is as in B. All figures representing molecular structures were generated with PyMOL (47).
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Figure 1: Structure of Vps30 BARA.A, overall structure of Vps30 BARA. The α-helices and β-strands are indicated with red helical ribbons and cyan arrows, respectively. Secondary structures are labeled, and residues adjacent to the disordered regions are numbered. Amino and carboxy termini are denoted as N and C, respectively. B, topology of Vps30 BARA. The α-helices and β-strands are indicated with boxes and arrows, respectively. Repeats 1, 2, and 3 are colored green, red, and cyan, respectively. The disordered regions are indicated with a broken line. C, superimposition of the secondary structural elements of repeats 2 and 3 on those of repeat 1. Coloring is as in B. D, left, ribbon representation of the three-helix bundle of BARA. Side chains involved in the interaction between helices are indicated with a stick model. Coloring is as in B. Right, ribbon representation of secondary structural elements of BARA. The side chains of conserved hydrophobic residues on the three sheets, which are bound to the grooves formed between helices, are indicated with a stick model. Coloring is as in B. All figures representing molecular structures were generated with PyMOL (47).

Mentions: S. cerevisiae Vps30 consists of 557 amino acids, and a coiled-coil motif was predicted in its central region (residues 187–319; supplemental Fig. S1). However, structural information on the C-terminal region of the Vps30 family proteins has not yet been reported. The C-terminal region (residues 320–539) of Vps30 was obtained by limited proteolysis, crystallized, and its structure was determined by x-ray crystallography. The structure was refined against 2.3 Å data to an R-factor of 0.209 and a free R-factor of 0.243 (Table 1). The region corresponding to amino acids 320–538 of Vps30 was modeled, but three loop regions (residues 397–411, 422–432, and 461–487) were omitted from the model because they lacked defined electron density. The structure is comprised of three α-helices (H1, H2, and H3) and three β-sheets (S1, S2, and S3) with an approximate 3-fold symmetry (Fig. 1A). S1, S2, and S3 are three-stranded, anti-parallel β-sheets resembling each other. The helices and the sheets are aligned in the order: S1-H1-S2-H2-S3-H3 from the N terminus to the C terminus (Fig. 1B). Three subdomains (S1-H1, S2-H2, and S3-H3) resemble each other and can be superimposed on each other with root mean square differences of 1.5–2.4 Å for main chain atoms (Fig. 1C); therefore, the subdomains are named repeat 1, repeat 2, and repeat 3, respectively. The hydrophobic residues in H1, H2, and H3 interact with each other (Fig. 1D, left) to form an inter-subdomain helix bundle. In addition, the hydrophobic residues in S1, S2, and S3 interact with the hydrophobic grooves formed between H1-H3, H1-H2, and H2-H3, respectively (Fig. 1D, right), stabilizing the helix bundle. These inter-subdomain interactions make the three subdomains into a stable globular fold.


Structure of the novel C-terminal domain of vacuolar protein sorting 30/autophagy-related protein 6 and its specific role in autophagy.

Noda NN, Kobayashi T, Adachi W, Fujioka Y, Ohsumi Y, Inagaki F - J. Biol. Chem. (2012)

Structure of Vps30 BARA.A, overall structure of Vps30 BARA. The α-helices and β-strands are indicated with red helical ribbons and cyan arrows, respectively. Secondary structures are labeled, and residues adjacent to the disordered regions are numbered. Amino and carboxy termini are denoted as N and C, respectively. B, topology of Vps30 BARA. The α-helices and β-strands are indicated with boxes and arrows, respectively. Repeats 1, 2, and 3 are colored green, red, and cyan, respectively. The disordered regions are indicated with a broken line. C, superimposition of the secondary structural elements of repeats 2 and 3 on those of repeat 1. Coloring is as in B. D, left, ribbon representation of the three-helix bundle of BARA. Side chains involved in the interaction between helices are indicated with a stick model. Coloring is as in B. Right, ribbon representation of secondary structural elements of BARA. The side chains of conserved hydrophobic residues on the three sheets, which are bound to the grooves formed between helices, are indicated with a stick model. Coloring is as in B. All figures representing molecular structures were generated with PyMOL (47).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351336&req=5

Figure 1: Structure of Vps30 BARA.A, overall structure of Vps30 BARA. The α-helices and β-strands are indicated with red helical ribbons and cyan arrows, respectively. Secondary structures are labeled, and residues adjacent to the disordered regions are numbered. Amino and carboxy termini are denoted as N and C, respectively. B, topology of Vps30 BARA. The α-helices and β-strands are indicated with boxes and arrows, respectively. Repeats 1, 2, and 3 are colored green, red, and cyan, respectively. The disordered regions are indicated with a broken line. C, superimposition of the secondary structural elements of repeats 2 and 3 on those of repeat 1. Coloring is as in B. D, left, ribbon representation of the three-helix bundle of BARA. Side chains involved in the interaction between helices are indicated with a stick model. Coloring is as in B. Right, ribbon representation of secondary structural elements of BARA. The side chains of conserved hydrophobic residues on the three sheets, which are bound to the grooves formed between helices, are indicated with a stick model. Coloring is as in B. All figures representing molecular structures were generated with PyMOL (47).
Mentions: S. cerevisiae Vps30 consists of 557 amino acids, and a coiled-coil motif was predicted in its central region (residues 187–319; supplemental Fig. S1). However, structural information on the C-terminal region of the Vps30 family proteins has not yet been reported. The C-terminal region (residues 320–539) of Vps30 was obtained by limited proteolysis, crystallized, and its structure was determined by x-ray crystallography. The structure was refined against 2.3 Å data to an R-factor of 0.209 and a free R-factor of 0.243 (Table 1). The region corresponding to amino acids 320–538 of Vps30 was modeled, but three loop regions (residues 397–411, 422–432, and 461–487) were omitted from the model because they lacked defined electron density. The structure is comprised of three α-helices (H1, H2, and H3) and three β-sheets (S1, S2, and S3) with an approximate 3-fold symmetry (Fig. 1A). S1, S2, and S3 are three-stranded, anti-parallel β-sheets resembling each other. The helices and the sheets are aligned in the order: S1-H1-S2-H2-S3-H3 from the N terminus to the C terminus (Fig. 1B). Three subdomains (S1-H1, S2-H2, and S3-H3) resemble each other and can be superimposed on each other with root mean square differences of 1.5–2.4 Å for main chain atoms (Fig. 1C); therefore, the subdomains are named repeat 1, repeat 2, and repeat 3, respectively. The hydrophobic residues in H1, H2, and H3 interact with each other (Fig. 1D, left) to form an inter-subdomain helix bundle. In addition, the hydrophobic residues in S1, S2, and S3 interact with the hydrophobic grooves formed between H1-H3, H1-H2, and H2-H3, respectively (Fig. 1D, right), stabilizing the helix bundle. These inter-subdomain interactions make the three subdomains into a stable globular fold.

Bottom Line: Thus, the domain is named the β-α repeated, autophagy-specific (BARA) domain.On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar protein sorting.These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this protein family in various biological processes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbial Chemistry, Tokyo, Tokyo 141-0021, Japan. nn@bikaken.or.jp

ABSTRACT
Vacuolar protein sorting 30 (Vps30)/autophagy-related protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this protein family in various biological processes.

Show MeSH