Grinthal Alison, Guidotti Guido
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Biochemistry. 2007 Jan 9;46(1):279-90. doi: 10.1021/bi061052p.
CD39 can exist in at least two distinct functional states depending on the presence and intact membrane integration of its two transmembrane helices. In native membranes, the transmembrane helices undergo dynamic rotational motions that are required for enzymatic activity and are regulated by substrate binding. In this study, we show that bilayer mechanical properties regulate conversion between the two enzymatic functional states by modulating transmembrane helix dynamics. Alteration of membrane properties by insertion of cone-shaped or inverse cone-shaped amphiphiles or by cholesterol removal switches CD39 to the same enzymatic state that removal or solubilization of the transmembrane domains does. The same membrane alterations increase the propensity of both transmembrane helices to rotate within the packed structure, resulting in a structure with greater mobility but not an altered primary conformation. Membrane alteration also abolishes the ability of the substrate to stabilize the helices in their primary conformation, indicating a loss of coupling between substrate binding and transmembrane helix dynamics. Removal of either transmembrane helix mimics the effect of membrane alteration on the mobility and substrate sensitivity of the remaining helix, suggesting that the ends of the extracellular domain have intrinsic flexibility. We suggest that a mechanical bilayer property, potentially elasticity, regulates CD39 by altering the balance between the stability and flexibility of its transmembrane helices and, in turn, of its active site.
根据其两个跨膜螺旋的存在情况和完整的膜整合情况，CD39可以以至少两种不同的功能状态存在。在天然膜中，跨膜螺旋会进行动态旋转运动，这是酶活性所必需的，并且受底物结合的调节。在本研究中，我们表明双层膜的机械特性通过调节跨膜螺旋动力学来调控两种酶功能状态之间的转换。通过插入锥形或反锥形两亲分子或去除胆固醇来改变膜特性，会将CD39切换到与去除或溶解跨膜结构域相同的酶状态。相同的膜改变会增加两个跨膜螺旋在紧密堆积结构中旋转的倾向，从而形成具有更高流动性但一级构象未改变的结构。膜改变还消除了底物将螺旋稳定在其一级构象的能力，表明底物结合与跨膜螺旋动力学之间的耦合丧失。去除任何一个跨膜螺旋都模拟了膜改变对剩余螺旋的流动性和底物敏感性的影响，这表明细胞外结构域的末端具有内在的灵活性。我们认为，一种潜在为弹性的双层膜机械特性，通过改变CD39跨膜螺旋及其活性位点的稳定性与灵活性之间的平衡来对其进行调节。
