The structure of apo protein-tyrosine phosphatase 1B C215S mutant:: More than just an S → O change
The structure of apo protein-tyrosine phosphatase 1B C215S mutant:: More than just an S → O change
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DOI：
10.1110/ps.11001
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发表时间：
2001-08-01
期刊：
影响因子：
8
通讯作者：
Asante-Appiah, E
中科院分区：
文献类型：
--
作者：
Scapin, G;Patel, S;Asante-Appiah, E
蛋白质酪氨酸磷酸酶通过涉及共价磷酸酶中间体的两步机制催化磷酸单酯的水解。生物化学和定点诱变实验表明，存在于PTB特征基序（H/V）CX 5 R（S/T）中的不变Cys残基（即，PTP 1B中的C215）是活性绝对必需的。不变的Cys突变为Set导致催化失活的酶，其仍然能够结合底物和抑制剂。尽管人们通常认为底物捕获突变体（如C215 S）在溶液中保留了野生型PTPases的结构和结合动力，但在解决这一问题的少数研究中发现了显著差异，表明突变可能导致PTP 1B结合位点或附近的结构/构象改变。ape-WT PTP 1B和WT-和C215 S-突变型PTP 1B与不同配体的复合物的几种晶体结构是可用的，但从未报道过apo-PTP 1B C215 S的结构。在所有先前报道的结构中，PTR标签基序的残基具有相同的构象，而WPD环（包括催化性Asp的表面环）的残基在存在或不存在配体的情况下呈现不同的构象。这些观察结果导致的假设，不同的光谱和热力学性质的突变蛋白质可能是一个不同的构象的WPD环的结果。我们在这里报告的apo-PTP 1B C215 S突变体的结构，这表明，虽然WPD环是在apo WT酶晶体结构中观察到的开放构象，PTPases签名基序的残基是在一个显着不同的构象。这些结果提供了一个结构的基础上观察到的野生型和突变酶的抑制剂结合的光谱特性和热力学参数的差异。
Protein-tyrosine phosphatases catalyze the hydrolysis of phosphate monoesters via a two-step mechanism involving a covalent phospho-enzyme intermediate. Biochemical and site-directed mutagenesis experiments show that the invariant Cys residue present in the PTPase signature motif (H/V)CX5R(S/T) (i.e., C215 in PTP1B) is absolutely required for activity. Mutation of the invariant Cys to Set results in a catalytically inactive enzyme, which still is capable of binding substrates and inhibitors. Although it often is assumed that substrate-trapping mutants such as the C215S retain, in solution, the structural and binding propel ties of wild-type PTPases, significant differences have been found in the few studies that have addressed this issue, suggesting that the mutation may lead to structural/conformational alterations in or near the PTP1B binding site. Several crystal structures of ape-WT PTP1B, and of WT- and C215S-mutant PTP1B in complex with different ligands are available, but no structure of the apo-PTP1B C215S has ever been reported. In all previously reported structures, residues of the PTPase signature motif have an identical conformation, while residues of the WPD loop (a surface loop which includes the catalytic Asp) assume a different conformation in the presence or absence of ligand. These observations led to the hypothesis that the different spectroscopic and thermodynamic properties of the mutant protein may be the result of a different conformation for the WPD loop. We report here the structure of the apo-PTP1B C215S mutant, which reveals that, while the WPD loop is in the open conformation observed in the apo WT enzyme crystal structure, the residues of the PTPases signature motif are in a dramatically different conformation. These results provide a structural basis for the differences in spectroscopic properties and thermodynamic parameters in inhibitor binding observed for the wild-type and mutant enzymes.