The crystal structure of an oxidatively stable subtilisin-like alkaline serine protease, KP-43, with a C-terminal β-barrel domain
The crystal structure of an oxidatively stable subtilisin-like alkaline serine protease, KP-43, with a C-terminal β-barrel domain
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DOI：
10.1074/jbc.m409089200
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发表时间：
2004-11-05
影响因子：
4.8
通讯作者：
Miki, K
中科院分区：
文献类型：
--
作者：
Nonaka, T;Fujihashi, M;Miki, K
采用反常散射的多重同构替换法，研究了Bacillus sp. KSM-KP43中具有c端延伸结构域的氧化稳定性枯草杆菌样碱性丝氨酸蛋白酶KP-43的晶体结构。在1.30埃分辨率下，原生形态的晶体学R因子为0.134（无R因子为0.169）。KP-43由两个结构域组成，一个是枯草杆菌样α / β结构域，一个是c端果冻卷β桶结构域。分子的拓扑结构与类枯草菌素蛋白转化酶的kexin和furin相似，而c端β -桶结构域的氨基酸序列和结合方向在每种情况下都不同。由于枯草杆菌样蛋白转化酶的c端结构域对于自身折叠是必不可少的，因此KP-43结构域也被认为起着这样的作用。已知KP-43是一般枯草杆菌样蛋白酶中的一种抗氧化蛋白酶。为了研究KP-43的抗氧化性，在1.73埃的分辨率下，测定了氧化后的KP-43的结构，并将其细化为0.142（无R为0.212）的晶体学R因子。结构分析表明，邻近催化Ser-255的Met-256与枯草菌素BPN'中的等效残基类似。虽然KP-43以及蛋白酶K和嘉士伯枯草菌素在氧化处理后失去了对合成肽的水解活性，但它们对蛋白质底物的活性都保持了70 - 80%。这些结果以及β -酪蛋白消化模式分析表明，邻近催化丝氨酸的蛋氨酸氧化不是主要修饰，但可能改变底物特异性。
The crystal structure of an oxidatively stable subtilisin-like alkaline serine protease, KP-43 from Bacillus sp. KSM-KP43, with a C-terminal extension domain, was determined by the multiple isomorphous replacements method with anomalous scattering. The native form was refined to a crystallographic R factor of 0.134 (R-free of 0.169) at 1.30-Angstrom resolution. KP-43 consists of two domains, a subtilisin-like alpha/beta domain and a C-terminal jelly roll beta-barrel domain. The topological architecture of the molecule is similar to that of kexin and furin, which belong to the subtilisin-like proprotein convertases, whereas the amino acid sequence and the binding orientation of the C-terminal beta-barrel domain both differ in each case. Since the C-terminal domains of subtilisin-like proprotein convertases are essential for folding themselves, the domain of KP-43 is also thought to play such a role. KP-43 is known to be an oxidation-resistant protease among the general subtilisin-like proteases. To investigate how KP-43 resists oxidizing reagents, the structure of oxidized KP-43 was also determined and refined to a crystallographic R factor of 0.142 (R-free of 0.212) at 1.73-Angstrom resolution. The structure analysis revealed that Met-256, adjacent to catalytic Ser-255, was oxidized similarly to an equivalent residue in subtilisin BPN'. Although KP-43, as well as proteinase K and subtilisin Carlsberg, lose their hydrolyzing activity against synthetic peptides after oxidation treatment, all of them retain 70 - 80% activity against proteinaceous substrates. These results, as well as the beta-casein digestion pattern analysis, have indicated that the oxidation of the methionine adjacent to the catalytic serine is not a dominant modification but might alter the substrate specificities.