The Membrane-Binding Domain of an Amphitropic Enzyme Suppresses Catalysis by Contact with an Amphipathic Helix Flanking Its Active Site
The Membrane-Binding Domain of an Amphitropic Enzyme Suppresses Catalysis by Contact with an Amphipathic Helix Flanking Its Active Site
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DOI：
10.1016/j.jmb.2012.12.003
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发表时间：
2013-05-13
影响因子：
5.6
通讯作者：
Cornell, Rosemary B.
中科院分区：
文献类型：
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作者：
Huang, Harris K-H.;Taneva, Svetla G.;Cornell, Rosemary B.
CTP：磷酸胆碱胞苷酰转移酶（CCT），是磷脂酰胆碱合成中的调节酶，通过使用脂质诱导的两亲性螺旋（结构域M）结合膜来激活。结构域M的功能是在CCT不与膜接合时使催化沉默。沉默机制尚不清楚。我们使用光交联和质谱法来鉴定结构域M与其可溶形式的其他CCT结构域之间的接触。结构域M中的四个位点中的每一个都与活性位点侧翼的同一组肽交联，并在活性位点的基础上在螺旋α E处重叠。这些交联在活化脂质囊泡的存在下被破坏。结构域M的突变揭示了推定的自抑制（AI）基序内的多个疏水残基有助于与螺旋α E接触和沉默。通过氘交换分析证实HLA-alpha E是结构域M的对接位点。我们通过定点荧光各向异性和尿素变性比较了CCT结构域的动力学和折叠稳定性。结果表明，结构域M的二分结构：一个无序的N-末端部分和一个有序的C-末端AI基序，其展开过渡与螺旋α E相同。AI基序疏水性的降低降低了其顺序和折叠稳定性，催化结构域的缺失也是如此。这些结果支持了一种模型，其中催化沉默是由两亲性AI基序对接到两亲性螺旋α E上介导的。连接α E与AI基序的非结构化皮带可能有助于沉默接触及其膜触发的破坏。(C)2012爱思唯尔有限公司保留所有权利。
CTP:phosphocholine cytidylyltransferase (CCT), the regulatory enzyme in the synthesis of phosphatidylcholine, is activated by binding membranes using a lipid-induced amphipathic helix (domain M). Domain M functions to silence catalysis when CCT is not membrane engaged. The silencing mechanism is unknown. We used photo-cross-linking and mass spectrometry to identify contacts between domain M and other CCT domains in its soluble form. Each of four sites in domain M forged cross-links to the same set of peptides that flank the active site and overlap at helix alpha E at the base of the active site. These cross-links were broken in the presence of activating lipid vesicles. Mutagenesis of domain M revealed that multiple hydrophobic residues within a putative auto-inhibitory (AI) motif contribute to the contact with helix alpha E and silencing. Helix alpha E was confirmed as the docking site for domain M by deuterium exchange analysis. We compared the dynamics and fold stability of CCT domains by site-directed fluorescence anisotropy and urea denaturation. The results suggest a bipartite structure for domain M: a disordered N-terminal portion and an ordered C-terminal AI motif with an unfolding transition identical with that of helix alpha E. Reduction in hydrophobicity of the AI motif decreased its order and fold stability, as did deletion of the catalytic domain. These results support a model in which catalytic silencing is mediated by the docking of an amphipathic AI motif onto the amphipathic helices alpha E. An unstructured leash linking alpha E with the AI motif may facilitate both the silencing contact and its membrane-triggered disruption. (C) 2012 Elsevier Ltd. All rights reserved.