The role of L1 loop in the mechanism of rhomboid intramembrane protease GlpG.
The role of L1 loop in the mechanism of rhomboid intramembrane protease GlpG.
复制标题
L1环在菱形膜内蛋白酶GlpG机制中的作用。
DOI：
10.1016/j.jmb.2007.10.014
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发表时间：
2007
影响因子：
5.6
通讯作者：
Ha,Ya
中科院分区：
文献类型：
--
作者：
Wang,Yongcheng;Maegawa,Saki;Akiyama,Yoshinori;Ha,Ya
膜内蛋白水解酶是生物学中的重要酶。最近解决的菱形蛋白水解酶GlpG的晶体结构为这些膜蛋白的机制提供了有用的见解。除了揭示了含有Ser-His催化二聚体的内部充满水的空腔外，晶体结构还确定了一个位于跨膜螺旋一侧的新结构域(L1环)。在这里，利用定点突变，我们证实了L1环部分嵌入到膜中，并表明在L1环靠近脂：水界面的末端，丙氨酸取代了高度优先的色氨酸(Trp136)，降低了GlpG的蛋白分解活性。结晶学分析表明，W136A突变没有改变该酶的结构。相反，突变部位的一个小的、暴露在脂肪中的蛋白质表面的极性已经改变。晶体结构，现在细化到1.7奥分辨率，还清楚地定义了一条20奥宽的疏水带环绕着蛋白酶，这很可能对应于蛋白质周围压缩的膜双层的厚度。这个改进的结构模型预测，催化的所有关键元件，包括催化丝氨酸和L5帽，需要位于膜表面的几埃内，并可能解释为什么蛋白酶活性对蛋白质的变化敏感：脂相互作用。基于这些发现，我们提出了一个模型，即底物跨膜螺旋的末端首先从膜的疏水核心区分离出来，然后弯曲到蛋白酶活性部位进行切割。
Intramembrane proteases are important enzymes in biology. The recently solved crystal structures of rhomboid protease GlpG have provided useful insights into the mechanism of these membrane proteins. Besides revealing an internal water-filled cavity that harbored the Ser–His catalytic dyad, the crystal structure identified a novel structural domain (L1 loop) that lies on the side of the transmembrane helices. Here, using site-directed mutagenesis, we confirmed that the L1 loop is partially embedded in the membrane, and showed that alanine substitution of a highly preferred tryptophan (Trp136) at the distal tip of the L1 loop near the lipid:water interface reduced GlpG proteolytic activity. Crystallographic analysis showed that W136A mutation did not modify the structure of the protease. Instead, the polarity for a small and lipid-exposed protein surface at the site of the mutation has changed. The crystal structure, now refined at 1.7 Å resolution, also clearly defined a 20-Å-wide hydrophobic belt around the protease, which likely corresponded to the thickness of the compressed membrane bilayer around the protein. This improved structural model predicts that all critical elements of the catalysis, including the catalytic serine and the L5 cap, need to be positioned within a few angstroms of the membrane surface, and may explain why the protease activity is sensitive to changes in the protein:lipid interaction. Based on these findings, we propose a model where the end of the substrate transmembrane helix first partitions out of the hydrophobic core region of the membrane before it bends into the protease active site for cleavage.