Molecular basis for defect in Alix-binding by alternatively spliced isoform of ALG-2 (ALG-2DeltaGF122) and structural roles of F122 in target recognition.
Molecular basis for defect in Alix-binding by alternatively spliced isoform of ALG-2 (ALG-2DeltaGF122) and structural roles of F122 in target recognition.
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DOI：
10.1186/1472-6807-10-25
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发表时间：
2010-08-06
影响因子：
--
通讯作者：
Maki M
中科院分区：
文献类型：
--
作者：
Inuzuka T;Suzuki H;Kawasaki M;Shibata H;Wakatsuki S;Maki M
ALG-2（PDCD6 的基因产物）属于 penta-EF-hand (PEF) 蛋白家族，并且 Ca2+ 依赖性地与各种细胞内蛋白相互作用，包括哺乳动物 Alix（ESCRT 系统中的接头蛋白）。我们之前的 X 射线晶体结构分析表明，Ca2+ 与 EF3 的结合使 R125 的侧链能够充分移动，从而使 Alix 的短片段能够接近初级疏水口袋（口袋 1）。 F122 的侧链面向二级疏水口袋（口袋 2），与 Alix 肽相互作用。另一种剪接的较短同工型，称为 ALG-2ΔGF122，缺乏 Gly121Phe122，并且不结合 Alix，但这种缺陷的结构基础仍有待阐明。我们解析了Ca2+结合形式的ALG-2ΔGF122的PEF结构域的X射线晶体结构，并将其与ALG-2的进行了比较。删除这两个残基缩短了 α-螺旋 5 (α5)，并改变了 R125 侧链的构型，使其部分封闭了口袋 1。由 121-GFG-123 主链形成的面向两个口袋的墙被破坏。然而，令人惊讶的是，用 Ala 或 Gly 取代 F122，而不是用 Trp 取代，增加了结合测定中的 Alix 结合能力。 F122 取代对 ALG-2 与其他已知相互作用蛋白（包括 TSG101（肿瘤易感基因 101）和膜联蛋白 A11）的结合表现出不同的影响。 F122A突变体的X射线晶体结构表明，去除庞大的F122侧链不仅在Pocket 2中产生了额外的开放空间，而且还消除了与W95和V98（存在于α4中）的螺旋间相互作用，并且α5倾斜远离α4以扩展Pocket 2，这表明获得了更合适的相互作用残基定位以接受Alix。我们发现，两个残基较短的 ALG-2 亚型无法结合 Alix，并不是因为 F122 缺少庞大的侧链，而是因为面向口袋 1 和 2 的主链壁变形。此外，F122 位置上的残基有助于靶点特异性，较小的侧链有利于 Alix 结合，但不利于结合膜联蛋白 A11。
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-2ΔGF122, lacks Gly121Phe122 and does not bind Alix, but the structural basis of the incompetence has remained to be elucidated. We solved the X-ray crystal structure of the PEF domain of ALG-2ΔGF122 in the Ca2+-bound form and compared it with that of ALG-2. Deletion of the two residues shortened α-helix 5 (α5) 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 α4) and that α5 inclined away from α4 to expand Pocket 2, suggesting acquirement of more appropriate positioning of the interacting residues to accept Alix. 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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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
DOI：
10.1016/j.bbrc.2005.05.074
发表时间：
2005-07-22
影响因子：
3.1
作者：
Chen, CM;Sytkowski, AJ
通讯作者：
Sytkowski, AJ
DOI：
10.1016/j.bbrc.2008.11.021
发表时间：
2009-01-02
影响因子：
3.1
作者：
Hoj, Berit R.;la Cour, Jonas M.;Berchtold, Martin W.
通讯作者：
Berchtold, Martin W.
DOI：
10.1107/s0907444998003254
发表时间：
1998-09-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Brunger, AT;Adams, PD;Warren, GL
通讯作者：
Warren, GL
DOI：
10.1016/s1570-9639(02)00444-2
发表时间：
2002-11-04
影响因子：
3.2
作者：
Maki, M;Kitaura, Y;Shibata, H
通讯作者：
Shibata, H