Biochemical and structural characterization of the interaction of memapsin 2 (β-secretase) cytosolic domain with the VHS domain of GGA proteins
Biochemical and structural characterization of the interaction of memapsin 2 (β-secretase) cytosolic domain with the VHS domain of GGA proteins
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DOI：
10.1021/bi035199h
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发表时间：
2003-10-28
期刊：
影响因子：
2.9
通讯作者：
Tang, J
中科院分区：
文献类型：
--
作者：
He, XY;Zhu, GY;Tang, J
膜蛋白酶2（β-分泌酶）是一种膜相关天冬氨酸蛋白酶，其启动β-淀粉样前体蛋白（APP）的水解，导致淀粉样蛋白-P的产生和阿尔茨海默病（AD）的发作。膜蛋白酶2和APP都从细胞表面转运到内体，在那里APP水解发生。因此，memapsin 2的细胞内转运机制对于理解AD的发病机制是重要的。我们先前已经表明，膜蛋白酶2的胞质结构域含有结合GGA蛋白的VHS结构域的酸簇双亮氨酸（ACDL）基序（He等人（2002）FEBS Lett. 524，183-187）。该机制是推测的膜蛋白酶2运输到内体的囊泡包装的识别步骤。已报道ACDL基序内丝氨酸残基的磷酸化调节膜蛋白酶2从早期内体回到细胞表面的再循环。在这里，我们报告的memapsin 2/VHS结构域的相互作用的研究。使用等温滴定量热法，来自GGA 1、GGA 2和GGA 3的VHS结构域的解离常数K-d值分别为4.0 x 10（-4）、4.1 x 10（-4）和3.1 x 10（-4）M。随着丝氨酸残基被磷酸丝氨酸取代，相同的三个VHS结构域的Kd降低约10倍、4倍和14倍。在2.6 A分辨率下解析了膜蛋白酶2磷酸丝氨酸肽和GGA 1 VHS之间的复合物的晶体结构。磷酸丝氨酸基团的侧链不与VHS结构域相互作用，但与配体肽的C-末端赖氨酸的侧链形成离子相互作用。天然和磷酸化肽与VHS结构域结合的能量计算表明，溶液中的这种肽内离子键可能会降低结合熵的变化，从而增加结合亲和力。
Memapsin 2 (beta-secretase) is a membrane-associated aspartic protease that initiates the hydrolysis of beta-amyloid precursor protein (APP) leading to the production of amyloid-P and the onset of Alzheimer's disease (AD). Both memapsin 2 and APP are transported from the cell surface to endosomes where APP hydrolysis takes place. Thus, the intracellular transport mechanism of memapsin 2 is important for understanding the pathogenesis of AD. We have previously shown that the cytosolic domain of memapsin 2 contains an acid-cluster-dileucine (ACDL) motif that binds the VHS domain of GGA proteins (He et al. (2002) FEBS Lett. 524, 183-187). This mechanism is the presumed recognition step for the vesicular packaging of memapsin 2 for its transport to endosomes. The phosphorylation of a serine residue within the ACDL motif has been reported to regulate the recycling of memapsin 2 from early endosomes back to the cell surface. Here, we report a study on the memapsin 2/VHS domain interaction. Using isothermal titration calorimetry, the dissociation constant, K-d, values are 4.0 x 10(-4), 4.1 x 10(-4), and 3.1 x 10(-4) M for VHS domains from GGA1, GGA2, and GGA3, respectively. With the serine residue replaced by phosphoserine, the Kd decreased about 10-, 4-, and 14-fold for the same three VHS domains. A crystal structure of the complex between memapsin 2 phosphoserine peptide and GGA1 VHS was solved at 2.6 A resolution. The side chain of the phosphoserine group does not interact with the VHS domain but forms an ionic interaction with the side chain of the C-terminal lysine of the ligand peptide. Energy calculation of the binding of native and phosphorylated peptides to VHS domains suggests that this intrapeptide ionic bond in solution may reduce the change in binding entropy and thus increase binding affinity.