Requirements for peptidyl-prolyl isomerization activity: A comprehensive mutational analysis of the substrate-binding cavity of FK506-binding protein 12
Requirements for peptidyl-prolyl isomerization activity: A comprehensive mutational analysis of the substrate-binding cavity of FK506-binding protein 12
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DOI：
10.1110/ps.073203707
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发表时间：
2007-12-01
期刊：
影响因子：
8
通讯作者：
Ito, Nobutoshi
中科院分区：
文献类型：
--
作者：
Ikura, Teikichi;Ito, Nobutoshi
属于 PPIase 家族的许多蛋白质均表现出肽基脯氨酰异构酶 (PPIase) 活性。然而，这种活性的催化机制仍有待完全阐明。在这里，我们选择人 FK506 结合蛋白 12 (FKBP12) 作为模型 PPIase，并研究了该活性所必需的氨基酸残基的性质。 PPIase 与短肽的几种复合物的晶体结构表明，FKBP12 底物结合腔中的残基 Asp37、Arg42、Phe46、Val55、Trp59 和 Tyr82 似乎在 PPIase 活性中发挥关键作用。这 6 个残基中的每一个都被 20 个常见氨基酸残基取代。通过胰凝乳蛋白酶消化测定，使用肽类似物测量每种突变蛋白的活性，然后与野生型 FKBP12 进行比较。研究发现，构成底物结合腔的氨基酸残基侧链的位点特异性相互作用对于 PPIase 活性并不是必需的，尽管第 37、55 和 82 个氨基酸残基对该活性有显着贡献。这表明 PPIase 活性仅需要捕获含 Pro 肽的疏水空腔。
Peptidyl-prolyl isomerase (PPIase) activity is exhibited by many proteins belonging to the PPIase family. However, the catalytic mechanism of this activity remains to be completely elucidated. Here, we selected human FK506-binding protein 12 (FKBP12) as the model PPIase and investigated the nature of amino acid residues essential for the activity. The crystal structures of several complexes of PPIase with short peptides revealed that the residues Asp37, Arg42, Phe46, Val55, Trp59, and Tyr82 in the substrate-binding cavity of FKBP12 appear to play key roles in the PPIase activity. Each of these six residues was substituted by 20 common amino acid residues. The activity of each mutant protein was measured using a peptide analog by the chymotrypsin digestion assay and then compared with wild-type FKBP12. It was found that site-specific interactions by the side chains of amino acid residues constituting the substrate-binding cavity were not essential for the PPIase activity, although the 37th, 55th, and 82nd amino acid residues significantly contributed to the activity. This suggests that the PPIase activity requires only the hydrophobic cavity that captures the Pro-containing peptide.