Solution structure of marinostatin, a natural ester-linked protein protease inhibitor
Solution structure of marinostatin, a natural ester-linked protein protease inhibitor
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DOI：
10.1021/bi048034x
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发表时间：
2005-02-22
期刊：
影响因子：
2.9
通讯作者：
Hara, S
中科院分区：
文献类型：
--
作者：
Kanaori, K;Kamei, K;Hara, S
Marinostatin是一种独特的蛋白酶抑制剂，含有两个酯键。我们纯化了一个12-残基的marinostatin [MST（1-12），（1）FATMRYPSDarin（12）]，并通过H-1 NMR光谱和约束分子动力学计算确定了参与酯键形成的残基和溶液结构。MST（1 - 12）的两个酯键在羟基和羧基之间形成，Thr（3）-Asp（9）和Ser（8）-Asp（11），表明MST（1-12）具有在Set（8）和Asp（9）的残基处稠合的两个环状区域。观察到Tyr（6）H-α和Pro（7）H-α之间的强NOE交叉峰，表明Pro 7残基呈顺式构象。MST（1-12）的良好收敛的结构和氢-氘实验表明，P1 '残基Arg（5）的骨架NH质子与Thr（3）和Asp（9）之间的酯键的碳酰氧形成氢键。为了揭示酯键的重要性，还合成了具有两个二硫键Cys（3）-Cys（9）和Cys（8）-Cys（11）的marinostatin类似物MST-2SS（（1）FACMRYPCSCE（12））。MST-2SS的抑制活性与MST（1-12）相当，其脯氨酸7位残基也呈顺式构象。然而，MST-2SS的Arg 5 NH质子交换速率比MST（1-12）快约100倍，并且由于NOE数目少，MST-2SS的结构计算不收敛，表明MST-2SS具有更灵活的结构。由P2位残基Thr（3）形成的酯键的氢可接受性对于抑制反应位点的波动和维持抑制活性至关重要，这使得marinostatin成为自然界中最小的蛋白酶抑制剂之一。
Marinostatin is a unique protein protease inhibitor containing two ester linkages. We have purified a 12-residue marinostatin [MST(1-12), (1)FATMRYPSDSDE(12)] and determined the residues involved in the formation of the ester linkages and the solution structure by H-1 NMR spectroscopy and restrained molecular dynamics calculation. The two ester linkages of MST(1 - 12) are formed between hydroxyl and carboxyl groups, Thr(3)-Asp(9) and Ser(8)-Asp(11), indicating that MST(1-12) has two cyclic regions which are fused at the residues of Set(8) and Asp(9). A strong, NOE cross-peak between Tyr(6) H-alpha and Pro(7) H-alpha was observed, indicating that the Pro7 residue takes a cis-conformation. Well-converged structures and hydrogen-deuterium experiments of MST(1-12) showed that the backbone NH proton of the P1', residue, Arg(5), is hydrogen-bonded to the carboryl oxygen of the ester linkage between Thr(3) and Asp(9). To reveal the significance of the ester linkages, a marinostatin analogue, MST-2SS ((1)FACMRYPCCSCE(12)) with two disulfide bridges of Cys(3)-Cys(9) and Cys(8)-Cys(11), was also synthesized. The inhibitory activity of MST-2SS was as strong as that of MST(1-12), and the Pro 7 residue of MST-2SS also takes a cisconformation. However, the exchange rate of the Arg5 NH proton of MST-2SS was about 100 times faster than that of MST(1-12), and the structure calculation of MST-2SS was not converged on account of the small number of NOEs, indicating that MST-2SS takes a more flexible structure. The hydrogen acceptability of the ester linkage formed by the P2 position residue, Thr(3), is crucial for suppressing the fluctuation of the reactive site and sustaining, the inhibitory activity, which enables marinostatin to be one of the smallest protease inhibitors in nature.