Prediction of a new ligand-binding site for type 2 motif based on the crystal structure of ALG-2 by dry and wet approaches.
Prediction of a new ligand-binding site for type 2 motif based on the crystal structure of ALG-2 by dry and wet approaches.
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DOI：
10.3390/ijms13067532
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发表时间：
2012
影响因子：
5.6
通讯作者：
Maki M
中科院分区：
文献类型：
--
作者：
Takahashi T;Suzuki H;Inuzuka T;Shibata H;Maki M
ALG-2是一种penta-EF-hand Ca 2+结合蛋白，与多种细胞内蛋白相互作用。已经确定了两种类型的ALG-2结合基序：1型，PXYPXnYP（X，变量; n = 4），在阿利克斯和PLSCR 3中; 2型，PXPGF，在Sec 31 A和PLSCR 3中。先前解析的ALG-2和含有1型基序的阿利克斯肽之间的复合物的X射线晶体结构表明，该肽与口袋1和口袋2结合。ALG-2和含2型基序的肽的共结晶尚未成功。为了深入了解2型基序识别的分子基础，我们使用MetaPocket 2.0基于ALG-2的3D结构通过计算算法搜索新的疏水腔。使用分子对接软件AutoDock维纳，预测的命名为口袋3的疏水口袋与N-乙酰基-ProAlaProGlyPhe-酰胺匹配，N-乙酰基-ProAlaProGlyPhe-酰胺是衍生自PLSCR 3中两种类型的ALG-2结合位点之一的虚拟五肽（2型基序）。我们研究了预测的结合位点的氨基酸取代对结合能力的影响，通过使用谷胱甘肽-S-转移酶融合的ALG-2的野生型和突变体蛋白和细胞裂解物表达绿色荧光蛋白融合的PLSCR 3的野生型和突变体的下拉测定。用ALG-2的Ala取代L52或用Ser取代F148导致对缺乏1型基序的PLSCR 3的结合能力丧失，但保留对缺乏2型基序的PLSCR 3的结合能力，强烈支持口袋3是2型基序的结合位点的假设。
ALG-2 is a penta-EF-hand Ca2+-binding protein and interacts with a variety of intracellular proteins. Two types of ALG-2-binding motifs have been determined: type 1, PXYPXnYP (X, variable; n = 4), in ALIX and PLSCR3; type 2, PXPGF, in Sec31A and PLSCR3. The previously solved X-ray crystal structure of the complex between ALG-2 and an ALIX peptide containing type 1 motif showed that the peptide binds to Pocket 1 and Pocket 2. Co-crystallization of ALG-2 and type 2 motif-containing peptides has not been successful. To gain insights into the molecular basis of type 2 motif recognition, we searched for a new hydrophobic cavity by computational algorithms using MetaPocket 2.0 based on 3D structures of ALG-2. The predicted hydrophobic pocket designated Pocket 3 fits with N-acetyl-ProAlaProGlyPhe-amide, a virtual penta-peptide derived from one of the two types of ALG-2-binding sites in PLSCR3 (type 2 motif), using the molecular docking software AutoDock Vina. We investigated effects of amino acid substitutions of the predicted binding sites on binding abilities by pulldown assays using glutathione-S-transferase -fused ALG-2 of wild-type and mutant proteins and lysates of cells expressing green fluorescent protein -fused PLSCR3 of wild-type and mutants. Substitution of either L52 with Ala or F148 with Ser of ALG-2 caused loss of binding abilities to PLSCR3 lacking type 1 motif but retained those to PLSCR3 lacking type 2 motif, strongly supporting the hypothesis that Pocket 3 is the binding site for type 2 motif.
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DOI：
10.1006/bbrc.2002.6600
发表时间：
2002-03-15
影响因子：
3.1
作者：
Satoh, H;Shibata, H;Maki, M
通讯作者：
Maki, M
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
影响因子：
--
作者：
Huang B;Schroeder M
通讯作者：
Schroeder M
影响因子：
5.8
作者：
Laurie, ATR;Jackson, RM
通讯作者：
Jackson, RM
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