A covalent linker allows for membrane targeting of an oxylipin biosynthetic complex
A covalent linker allows for membrane targeting of an oxylipin biosynthetic complex
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DOI：
10.1021/bi800751p
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发表时间：
2008-10-07
期刊：
影响因子：
2.9
通讯作者：
Newcomer, Marcia E.
中科院分区：
文献类型：
--
作者：
Gilbert, Nathaniel C.;Niebuhr, Marc;Newcomer, Marcia E.
一种来自Plexaura homomalla的自然产生的双功能蛋白将氧脂生物合成途径中的顺序催化活性联系在一起。分子的C-末端脂肪氧合酶(LOX)部分催化花生四烯酸(AA)转化为相应的SR-氢过氧化氢，N-末端丙二烯氧化物合成酶(AOS)结构域促进氢过氧化氢转化为产物氧化丙二烯(AO)。小角X射线散射数据表明，在没有共价键的情况下，两个催化结构域将AA转化为AO，形成一个复合体，概括了双功能蛋白的结构。SAXS数据还支持从低分辨率晶体结构推断融合蛋白中LOX和AOS结构域定向的模型。然而，膜结合实验的结果表明，尽管存在非共价域的结合，但依赖于钙离子的膜靶向序列活性需要结构域的共价键。此外，膜靶向伴随着构象的变化，通过连接AOS和LOX结构域的连接体的特定蛋白分解来监测。我们的数据与依赖于钙离子的膜结合解除了AOS和LOX结构域之间的非共价相互作用的模型一致，并表明LOX的C2样结构域既介导蛋白质-蛋白质相互作用，也介导蛋白质-膜相互作用。
A naturally occurring bifunctional protein from Plexaura homomalla links sequential catalytic activities in an oxylipin biosynthetic pathway. The C-terminal lipoxygenase (LOX) portion of the molecule catalyzes the transformation of arachidonic acid (AA) to the corresponding SR-hydroperoxide, and the N-terminal allene oxide synthase (AOS) domain promotes the conversion of the hydroperoxide intermediate to the product allene oxide (AO). Small-angle X-ray scattering data indicate that in the absence of a covalent linkage the two catalytic domains that transform AA to AO associate to form a complex that recapitulates the structure of the bifunctional protein. The SAXS data also support a model for LOX and AOS domain orientation in the fusion protein inferred from a low-resolution crystal structure. However, results of membrane binding experiments indicate that covalent linkage of the domains is required for Ca2+-dependent membrane targeting of the sequential activities, despite the noncovalent domain association. Furthermore, membrane targeting is accompanied by a conformational change as monitored by specific proteolysis of the linker that joins the AOS and LOX domains. Our data are consistent with a model in which Ca2+-dependent membrane binding relieves the noncovalent interactions between the AOS and LOX domains and suggests that the C2-like domain of LOX mediates both protein-protein and protein-membrane interactions.