Maynes Jason T, Perreault Kathleen R, Cherney Maia M, Luu Hue Anh, James Michael N G, Holmes Charles F B
Canadian Institutes of Health Research, Group in Protein Structure and Function, Department of Biochemistry, Faculty of Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
J Biol Chem. 2004 Oct 8;279(41):43198-206. doi: 10.1074/jbc.M407184200. Epub 2004 Jul 26.
Protein phosphatase-1 and protein phosphatase-2B (calcineurin) are eukaryotic serine/threonine phosphatases that share 40% sequence identity in their catalytic subunits. Despite the similarities in sequence, these phosphatases are widely divergent when it comes to inhibition by natural product toxins, such as microcystin-LR and okadaic acid. The most prominent region of non-conserved sequence between these phosphatases corresponds to the beta12-beta13 loop of protein phosphatase-1, and the L7 loop of toxin-resistant calcineurin. In the present study, mutagenesis of residues 273-277 of the beta12-beta13 loop of the protein phosphatase-1 catalytic subunit (PP-1c) to the corresponding residues in calcineurin (312-316), resulted in a chimeric mutant that showed a decrease in sensitivity to microcystin-LR, okadaic acid, and the endogenous PP-1c inhibitor protein inhibitor-2. A crystal structure of the chimeric mutant in complex with okadaic acid was determined to 2.0-A resolution. The beta12-beta13 loop region of the mutant superimposes closely with that of wild-type PP-1c bound to okadaic acid. Systematic mutation of each residue in the beta12-beta13 loop of PP-1c showed that a single amino acid change (C273L) was the most influential in mediating sensitivity of PP-1c to toxins. Taken together, these data indicate that it is an individual amino acid residue substitution and not a change in the overall beta12-beta13 loop conformation of protein phosphatase-1 that contributes to disrupting important interactions with inhibitors such as microcystin-LR and okadaic acid.
蛋白磷酸酶-1和蛋白磷酸酶-2B（钙调神经磷酸酶）是真核生物的丝氨酸/苏氨酸磷酸酶，其催化亚基的序列一致性为40%。尽管在序列上有相似性，但在受到微囊藻毒素-LR和冈田酸等天然产物毒素抑制方面，这些磷酸酶差异很大。这些磷酸酶之间最显著的非保守序列区域对应于蛋白磷酸酶-1的β12-β13环，以及抗毒素钙调神经磷酸酶的L7环。在本研究中，将蛋白磷酸酶-1催化亚基（PP-1c）的β12-β13环的273-277位残基突变为钙调神经磷酸酶中的相应残基（312-316），产生了一个嵌合突变体，该突变体对微囊藻毒素-LR、冈田酸和内源性PP-1c抑制剂蛋白抑制剂-2的敏感性降低。测定了与冈田酸结合的嵌合突变体的晶体结构，分辨率为2.0埃。该突变体的β12-β13环区域与结合冈田酸的野生型PP-1c的β12-β13环区域紧密重叠。对PP-1c的β12-β13环中的每个残基进行系统突变表明，单个氨基酸变化（C273L）对介导PP-1c对毒素的敏感性影响最大。综上所述，这些数据表明，是单个氨基酸残基取代而非蛋白磷酸酶-1的整个β12-β13环构象变化导致了与微囊藻毒素-LR和冈田酸等抑制剂的重要相互作用被破坏。
