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Molecular basis for defect in Alix-binding by alternatively spliced isoform of ALG-2 (ALG-2DeltaGF122) and structural roles of F122 in target recognition.

Inuzuka T, Suzuki H, Kawasaki M, Shibata H, Wakatsuki S, Maki M - BMC Struct. Biol. (2010)

Bottom Line: The F122 substitutions exhibited different effects on binding of ALG-2 to other known interacting proteins, including TSG101 (Tumor susceptibility gene 101) and annexin A11.We found that the inability of the two-residue shorter ALG-2 isoform to bind Alix is not due to the absence of bulky side chain of F122 but due to deformation of a main-chain wall facing pockets 1 and 2.Moreover, a residue at the position of F122 contributes to target specificity and a smaller side chain is preferable for Alix binding but not favored to bind annexin A11.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.

ABSTRACT

Background: ALG-2 (a gene product of PDCD6) belongs to the penta-EF-hand (PEF) protein family and Ca2+-dependently interacts with various intracellular proteins including mammalian Alix, an adaptor protein in the ESCRT system. Our previous X-ray crystal structural analyses revealed that binding of Ca2+ to EF3 enables the side chain of R125 to move enough to make a primary hydrophobic pocket (Pocket 1) accessible to a short fragment of Alix. The side chain of F122, facing a secondary hydrophobic pocket (Pocket 2), interacts with the Alix peptide. An alternatively spliced shorter isoform, designated ALG-2DeltaGF122, lacks Gly121Phe122 and does not bind Alix, but the structural basis of the incompetence has remained to be elucidated.

Results: We solved the X-ray crystal structure of the PEF domain of ALG-2DeltaGF122 in the Ca2+-bound form and compared it with that of ALG-2. Deletion of the two residues shortened alpha-helix 5 (alpha5) and changed the configuration of the R125 side chain so that it partially blocked Pocket 1. A wall created by the main chain of 121-GFG-123 and facing the two pockets was destroyed. Surprisingly, however, substitution of F122 with Ala or Gly, but not with Trp, increased the Alix-binding capacity in binding assays. The F122 substitutions exhibited different effects on binding of ALG-2 to other known interacting proteins, including TSG101 (Tumor susceptibility gene 101) and annexin A11. The X-ray crystal structure of the F122A mutant revealed that removal of the bulky F122 side chain not only created an additional open space in Pocket 2 but also abolished inter-helix interactions with W95 and V98 (present in alpha4) and that alpha5 inclined away from alpha4 to expand Pocket 2, suggesting acquirement of more appropriate positioning of the interacting residues to accept Alix.

Conclusions: We found that the inability of the two-residue shorter ALG-2 isoform to bind Alix is not due to the absence of bulky side chain of F122 but due to deformation of a main-chain wall facing pockets 1 and 2. Moreover, a residue at the position of F122 contributes to target specificity and a smaller side chain is preferable for Alix binding but not favored to bind annexin A11.

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Loss of a wall surrounding hydrophobic pockets in ALG-2ΔGF122. Surface structures of (A) Ca2+-bound des3-20ALG-2, (B) Ca2+-bound des3-23ALG-2ΔGF122, (C) Zn2+-bound ALG-2, and (D) Zn2+-bound des3-20ALG-2F122A are presented in gray except for indicated residues of Gly121Phe122 (green), G123 (or G121 in des3-23ALG-2ΔGF122) (magenta), A122 in the F122A mutant (orange), R125 (or R123 in des3-23ALG-2ΔGF122) (cyan), Y180 (or Y178 of des3-23ALG-2ΔGF122) from a dimerized counterpart molecule (yellow).
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Figure 3: Loss of a wall surrounding hydrophobic pockets in ALG-2ΔGF122. Surface structures of (A) Ca2+-bound des3-20ALG-2, (B) Ca2+-bound des3-23ALG-2ΔGF122, (C) Zn2+-bound ALG-2, and (D) Zn2+-bound des3-20ALG-2F122A are presented in gray except for indicated residues of Gly121Phe122 (green), G123 (or G121 in des3-23ALG-2ΔGF122) (magenta), A122 in the F122A mutant (orange), R125 (or R123 in des3-23ALG-2ΔGF122) (cyan), Y180 (or Y178 of des3-23ALG-2ΔGF122) from a dimerized counterpart molecule (yellow).

Mentions: As shown in Figure 3, the deletion of two residues caused a noticeable change in the surface structure surrounding hydrophobic pockets (designated Pocket 1 and Pocket 2), which were shown to accommodate the Alix ABS peptide in our previous study [21]. The bottom of Pocket 1 was supported by Y180 derived from a dimerizing counterpart molecule of ALG-2 (Figure 3, yellow). A wall formed by Gly121Phe122 in des3-20ALG-2/Ca2+-bound (Figure 3A, green) disappeared in des3-23ALG-2ΔGF122/Ca2+-bound, and a surface structure represented by G123 was also changed (Figure 3B, magenta) The continuous wall formed by 121-GFG-123 was maintained in Zn2+-bound des3-20ALG-2F122A (Figure 3D, orange: Gly121Ala122; magenta, G123). The R125 side chain (Figure 3, cyan) was oriented away from Pocket 1 in the metal-bound ALG-2 proteins (Figure 3, A, C and 3D). On the other hand, the side chain directed toward Pocket 1 in Ca2+-bound des3-23ALG-2ΔGF122 (Figure 3B), but it did not fully block the entry path of Pocket 1 as in the case of metal-free ALG-2 (Additional file 1, Figure S2B).


Molecular basis for defect in Alix-binding by alternatively spliced isoform of ALG-2 (ALG-2DeltaGF122) and structural roles of F122 in target recognition.

Inuzuka T, Suzuki H, Kawasaki M, Shibata H, Wakatsuki S, Maki M - BMC Struct. Biol. (2010)

Loss of a wall surrounding hydrophobic pockets in ALG-2ΔGF122. Surface structures of (A) Ca2+-bound des3-20ALG-2, (B) Ca2+-bound des3-23ALG-2ΔGF122, (C) Zn2+-bound ALG-2, and (D) Zn2+-bound des3-20ALG-2F122A are presented in gray except for indicated residues of Gly121Phe122 (green), G123 (or G121 in des3-23ALG-2ΔGF122) (magenta), A122 in the F122A mutant (orange), R125 (or R123 in des3-23ALG-2ΔGF122) (cyan), Y180 (or Y178 of des3-23ALG-2ΔGF122) from a dimerized counterpart molecule (yellow).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2927601&req=5

Figure 3: Loss of a wall surrounding hydrophobic pockets in ALG-2ΔGF122. Surface structures of (A) Ca2+-bound des3-20ALG-2, (B) Ca2+-bound des3-23ALG-2ΔGF122, (C) Zn2+-bound ALG-2, and (D) Zn2+-bound des3-20ALG-2F122A are presented in gray except for indicated residues of Gly121Phe122 (green), G123 (or G121 in des3-23ALG-2ΔGF122) (magenta), A122 in the F122A mutant (orange), R125 (or R123 in des3-23ALG-2ΔGF122) (cyan), Y180 (or Y178 of des3-23ALG-2ΔGF122) from a dimerized counterpart molecule (yellow).
Mentions: As shown in Figure 3, the deletion of two residues caused a noticeable change in the surface structure surrounding hydrophobic pockets (designated Pocket 1 and Pocket 2), which were shown to accommodate the Alix ABS peptide in our previous study [21]. The bottom of Pocket 1 was supported by Y180 derived from a dimerizing counterpart molecule of ALG-2 (Figure 3, yellow). A wall formed by Gly121Phe122 in des3-20ALG-2/Ca2+-bound (Figure 3A, green) disappeared in des3-23ALG-2ΔGF122/Ca2+-bound, and a surface structure represented by G123 was also changed (Figure 3B, magenta) The continuous wall formed by 121-GFG-123 was maintained in Zn2+-bound des3-20ALG-2F122A (Figure 3D, orange: Gly121Ala122; magenta, G123). The R125 side chain (Figure 3, cyan) was oriented away from Pocket 1 in the metal-bound ALG-2 proteins (Figure 3, A, C and 3D). On the other hand, the side chain directed toward Pocket 1 in Ca2+-bound des3-23ALG-2ΔGF122 (Figure 3B), but it did not fully block the entry path of Pocket 1 as in the case of metal-free ALG-2 (Additional file 1, Figure S2B).

Bottom Line: The F122 substitutions exhibited different effects on binding of ALG-2 to other known interacting proteins, including TSG101 (Tumor susceptibility gene 101) and annexin A11.We found that the inability of the two-residue shorter ALG-2 isoform to bind Alix is not due to the absence of bulky side chain of F122 but due to deformation of a main-chain wall facing pockets 1 and 2.Moreover, a residue at the position of F122 contributes to target specificity and a smaller side chain is preferable for Alix binding but not favored to bind annexin A11.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.

ABSTRACT

Background: ALG-2 (a gene product of PDCD6) belongs to the penta-EF-hand (PEF) protein family and Ca2+-dependently interacts with various intracellular proteins including mammalian Alix, an adaptor protein in the ESCRT system. Our previous X-ray crystal structural analyses revealed that binding of Ca2+ to EF3 enables the side chain of R125 to move enough to make a primary hydrophobic pocket (Pocket 1) accessible to a short fragment of Alix. The side chain of F122, facing a secondary hydrophobic pocket (Pocket 2), interacts with the Alix peptide. An alternatively spliced shorter isoform, designated ALG-2DeltaGF122, lacks Gly121Phe122 and does not bind Alix, but the structural basis of the incompetence has remained to be elucidated.

Results: We solved the X-ray crystal structure of the PEF domain of ALG-2DeltaGF122 in the Ca2+-bound form and compared it with that of ALG-2. Deletion of the two residues shortened alpha-helix 5 (alpha5) and changed the configuration of the R125 side chain so that it partially blocked Pocket 1. A wall created by the main chain of 121-GFG-123 and facing the two pockets was destroyed. Surprisingly, however, substitution of F122 with Ala or Gly, but not with Trp, increased the Alix-binding capacity in binding assays. The F122 substitutions exhibited different effects on binding of ALG-2 to other known interacting proteins, including TSG101 (Tumor susceptibility gene 101) and annexin A11. The X-ray crystal structure of the F122A mutant revealed that removal of the bulky F122 side chain not only created an additional open space in Pocket 2 but also abolished inter-helix interactions with W95 and V98 (present in alpha4) and that alpha5 inclined away from alpha4 to expand Pocket 2, suggesting acquirement of more appropriate positioning of the interacting residues to accept Alix.

Conclusions: We found that the inability of the two-residue shorter ALG-2 isoform to bind Alix is not due to the absence of bulky side chain of F122 but due to deformation of a main-chain wall facing pockets 1 and 2. Moreover, a residue at the position of F122 contributes to target specificity and a smaller side chain is preferable for Alix binding but not favored to bind annexin A11.

Show MeSH
Related in: MedlinePlus