Taneva Svetla, Johnson Joanne E, Cornell Rosemary B
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada, V5A 1S6.
Biochemistry. 2003 Oct 14;42(40):11768-76. doi: 10.1021/bi035234k.
phosphocholine cytidylyltranferase (CCT) regulates phosphatidylcholine (PC) biosynthesis. Its activity is controlled by reversible interactions with membrane lipids, mediated by an internal segment referred to as domain M. Although domain M peptides adopt an amphipathic alpha-helical structure when membrane bound, the structure of this domain in the context of the whole enzyme in the lipid-free and lipid-bound state is unknown. Here we derive lipid-induced secondary structural changes in CCTalpha using circular dichroism and three deconvolution programs. The analysis of two fragments, CCT236 (CCT1-236, housing the catalytic domain) and a synthetic domain M peptide (CCT237-293) aided the assignment of structural change to specific domains. To carry out this study, we developed a micellar lipid activating system that would avoid generation of CCT-induced lipid vesicle aggregates that interfere with the CD analysis. Lysophosphatidylcholine/phosphatidylglycerol (LPC/PG) mixed micelles supported full activation of CCT and caused an increase in the alpha-helix content of full-length CCT from 25 to 41%, at the expense of all other conformations. LPC/PG also induced an increase in alpha-helix content of the domain M peptide from 7 to 85% at the expense of all other conformers. This lipid system did not significantly affect the secondary structure of CCT236, nor did it affect the proteolytic fragmentation pattern of this region within full-length CCT, suggesting that the region containing the catalytic domain changes very little upon membrane activation of CCT. Our data suggest that lipids trigger a conformational switch in domain M from a mixed structure to an alpha-helix, thus creating a hydrophobic face for membrane insertion. Our results negate the idea that domain M is entirely helical in both the soluble and membrane-bound forms of CCT.
磷酸胆碱胞苷转移酶（CCT）调节磷脂酰胆碱（PC）的生物合成。其活性受与膜脂的可逆相互作用控制，这种相互作用由一个称为结构域M的内部片段介导。尽管结构域M肽在与膜结合时呈现两亲性α-螺旋结构，但在无脂和脂结合状态下，该结构域在整个酶中的结构尚不清楚。在这里，我们使用圆二色性和三种去卷积程序推导了脂质诱导的CCTα二级结构变化。对两个片段CCT236（CCT1 - 236，包含催化结构域）和一个合成的结构域M肽（CCT237 - 293）的分析有助于将结构变化归属于特定结构域。为了进行这项研究，我们开发了一种胶束脂质激活系统，该系统可以避免产生干扰CD分析的CCT诱导的脂质囊泡聚集体。溶血磷脂酰胆碱/磷脂酰甘油（LPC/PG）混合胶束支持CCT的完全激活，并使全长CCT的α-螺旋含量从25%增加到41%，代价是所有其他构象减少。LPC/PG还使结构域M肽的α-螺旋含量从7%增加到85%，代价是所有其他构象异构体减少。这种脂质系统对CCT236的二级结构没有显著影响，也不影响全长CCT中该区域内的蛋白水解片段化模式，这表明包含催化结构域的区域在CCT的膜激活过程中变化很小。我们的数据表明，脂质触发结构域M从混合结构向α-螺旋的构象转变，从而为膜插入创造一个疏水表面。我们的结果否定了结构域M在CCT的可溶性和膜结合形式中均完全呈螺旋状的观点。
