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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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Augmentation of staurosporine-induced cell death by ALG-2F122A. Parental (Prnt) HeLa cells and ALG-2-knockdown (ALG-2KD) HeLa cells were transfected with a vector (pcDNA3) as controls. ALG-2KD cells were transfected with plasmids that express wild-type ALG-2, ALG-2ΔGF122 or ALG-2F122A and cultured for 24 h. The transfected cells were treated with 1 μM staurosporine for 24 h. Cell death was monitored by measuring the amounts of lactate dehydrogenase (LDH) released into the culture medium as described in Materials and Methods. Total amounts of LDH released into the medium and retained in cells were expressed as 100%. Duplicate assays of five independent repetitive experiments were performed and data are presented as means +/- SD (n = 5). Closed column, not treated with staurosporine; open column, treated with staurosporine. Statistically significant differences among staurosporine-treated cells were evaluated by Student's t-test by comparing the measured values of samples with those of control ALG-2KD cells that were transfected with a control vector (*, P < 0.05; **, P < 0.01)
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Figure 5: Augmentation of staurosporine-induced cell death by ALG-2F122A. Parental (Prnt) HeLa cells and ALG-2-knockdown (ALG-2KD) HeLa cells were transfected with a vector (pcDNA3) as controls. ALG-2KD cells were transfected with plasmids that express wild-type ALG-2, ALG-2ΔGF122 or ALG-2F122A and cultured for 24 h. The transfected cells were treated with 1 μM staurosporine for 24 h. Cell death was monitored by measuring the amounts of lactate dehydrogenase (LDH) released into the culture medium as described in Materials and Methods. Total amounts of LDH released into the medium and retained in cells were expressed as 100%. Duplicate assays of five independent repetitive experiments were performed and data are presented as means +/- SD (n = 5). Closed column, not treated with staurosporine; open column, treated with staurosporine. Statistically significant differences among staurosporine-treated cells were evaluated by Student's t-test by comparing the measured values of samples with those of control ALG-2KD cells that were transfected with a control vector (*, P < 0.05; **, P < 0.01)

Mentions: Staurosporine, a microbial alkaloid, acts as a non-selective protein kinase inhibitor with high potency by binding to ATP-binding pockets of kinases [28], and it induces cell death via caspase-dependent and -independent apoptotic pathways [29]. Previously, Vito et al. reported that ALG-2 and Alix (named AIP1 in the article) modulate staurosporine-induced cell death [8]. To investigate whether the enhanced Alix-binding capacity by F122A mutant exerts augmentation of cell death, we employed previously established ALG-2-knockdown (ALG-2KD) HeLa cells whose endogenous ALG-2 level was reduced by the RNA interference (RNAi) method [26]. After transfection with RNAi-resistant expression plasmids of either wild-type or mutant ALG-2 proteins or with a vector as a control, cells were treated with staurosporine for 24 h. The degree of cell death was estimated by measuring the amounts of lactate dehydrogenase (LDH) released into the culture medium. As shown in Figure 5, cells not treated with staurosporine released small amounts of LDH under the conditions used (closed columns, 6-8% of total LDH activities). On the other hand, dramatic increases were observed for the release of LDH from staurosporine-treated cells in all cases tested (open columns, 29-46%), but vector-transfected ALG-2KD HeLa cells (29%) showed lower values than vector-transfected parental HeLa cells (36%). The amounts of released LDH were increased slightly by exogenous expressions of wild-type (WT) ALG-2 (36%) and ALG-2ΔGF122 (33%) in ALG-2KD HeLa cells. The augmentation was statistically significant (P < 0.01) when ALG-2F122A was exogenously expressed (46%) and compared with the vector-transfected ALG-2KD HeLa cells (29%).


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)

Augmentation of staurosporine-induced cell death by ALG-2F122A. Parental (Prnt) HeLa cells and ALG-2-knockdown (ALG-2KD) HeLa cells were transfected with a vector (pcDNA3) as controls. ALG-2KD cells were transfected with plasmids that express wild-type ALG-2, ALG-2ΔGF122 or ALG-2F122A and cultured for 24 h. The transfected cells were treated with 1 μM staurosporine for 24 h. Cell death was monitored by measuring the amounts of lactate dehydrogenase (LDH) released into the culture medium as described in Materials and Methods. Total amounts of LDH released into the medium and retained in cells were expressed as 100%. Duplicate assays of five independent repetitive experiments were performed and data are presented as means +/- SD (n = 5). Closed column, not treated with staurosporine; open column, treated with staurosporine. Statistically significant differences among staurosporine-treated cells were evaluated by Student's t-test by comparing the measured values of samples with those of control ALG-2KD cells that were transfected with a control vector (*, P < 0.05; **, P < 0.01)
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Figure 5: Augmentation of staurosporine-induced cell death by ALG-2F122A. Parental (Prnt) HeLa cells and ALG-2-knockdown (ALG-2KD) HeLa cells were transfected with a vector (pcDNA3) as controls. ALG-2KD cells were transfected with plasmids that express wild-type ALG-2, ALG-2ΔGF122 or ALG-2F122A and cultured for 24 h. The transfected cells were treated with 1 μM staurosporine for 24 h. Cell death was monitored by measuring the amounts of lactate dehydrogenase (LDH) released into the culture medium as described in Materials and Methods. Total amounts of LDH released into the medium and retained in cells were expressed as 100%. Duplicate assays of five independent repetitive experiments were performed and data are presented as means +/- SD (n = 5). Closed column, not treated with staurosporine; open column, treated with staurosporine. Statistically significant differences among staurosporine-treated cells were evaluated by Student's t-test by comparing the measured values of samples with those of control ALG-2KD cells that were transfected with a control vector (*, P < 0.05; **, P < 0.01)
Mentions: Staurosporine, a microbial alkaloid, acts as a non-selective protein kinase inhibitor with high potency by binding to ATP-binding pockets of kinases [28], and it induces cell death via caspase-dependent and -independent apoptotic pathways [29]. Previously, Vito et al. reported that ALG-2 and Alix (named AIP1 in the article) modulate staurosporine-induced cell death [8]. To investigate whether the enhanced Alix-binding capacity by F122A mutant exerts augmentation of cell death, we employed previously established ALG-2-knockdown (ALG-2KD) HeLa cells whose endogenous ALG-2 level was reduced by the RNA interference (RNAi) method [26]. After transfection with RNAi-resistant expression plasmids of either wild-type or mutant ALG-2 proteins or with a vector as a control, cells were treated with staurosporine for 24 h. The degree of cell death was estimated by measuring the amounts of lactate dehydrogenase (LDH) released into the culture medium. As shown in Figure 5, cells not treated with staurosporine released small amounts of LDH under the conditions used (closed columns, 6-8% of total LDH activities). On the other hand, dramatic increases were observed for the release of LDH from staurosporine-treated cells in all cases tested (open columns, 29-46%), but vector-transfected ALG-2KD HeLa cells (29%) showed lower values than vector-transfected parental HeLa cells (36%). The amounts of released LDH were increased slightly by exogenous expressions of wild-type (WT) ALG-2 (36%) and ALG-2ΔGF122 (33%) in ALG-2KD HeLa cells. The augmentation was statistically significant (P < 0.01) when ALG-2F122A was exogenously expressed (46%) and compared with the vector-transfected ALG-2KD HeLa cells (29%).

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