Kim Sangwoo S, Upshur Mary Alice, Saotome Kei, Sahu Indra D, McCarrick Robert M, Feix Jimmy B, Lorigan Gary A, Howard Kathleen P
Department of Chemistry and Biochemistry, Swarthmore College , Swarthmore, Pennsylvania 19081, United States.
Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States.
Biochemistry. 2015 Dec 15;54(49):7157-67. doi: 10.1021/acs.biochem.5b01065. Epub 2015 Nov 30.
The C-terminal amphipathic helix of the influenza A M2 protein plays a critical cholesterol-dependent role in viral budding. To provide atomic-level detail on the impact cholesterol has on the conformation of M2 protein, we spin-labeled sites right before and within the C-terminal amphipathic helix of the M2 protein. We studied the spin-labeled M2 proteins in membranes both with and without cholesterol. We used a multipronged site-directed spin-label electron paramagnetic resonance (SDSL-EPR) approach and collected data on line shapes, relaxation rates, accessibility of sites to the membrane, and distances between symmetry-related sites within the tetrameric protein. We demonstrate that the C-terminal amphipathic helix of M2 populates at least two conformations in POPC/POPG 4:1 bilayers. Furthermore, we show that the conformational state that becomes more populated in the presence of cholesterol is less dynamic, less membrane buried, and more tightly packed than the other state. Cholesterol-dependent changes in M2 could be attributed to the changes cholesterol induces in bilayer properties and/or direct binding of cholesterol to the protein. We propose a model consistent with all of our experimental data that suggests that the predominant conformation we observe in the presence of cholesterol is relevant for the understanding of viral budding.
甲型流感病毒M2蛋白的C末端两亲性螺旋在病毒出芽过程中发挥着关键的胆固醇依赖性作用。为了提供关于胆固醇对M2蛋白构象影响的原子水平细节，我们在M2蛋白C末端两亲性螺旋之前及其中间位置进行了自旋标记。我们研究了在有胆固醇和无胆固醇的膜中的自旋标记M2蛋白。我们采用了多管齐下的定点自旋标记电子顺磁共振（SDSL-EPR）方法，并收集了关于线形、弛豫速率、位点对膜的可及性以及四聚体蛋白中对称相关位点之间距离的数据。我们证明，M2蛋白的C末端两亲性螺旋在POPC/POPG 4:1双层膜中至少呈现两种构象。此外，我们表明，在有胆固醇存在时占比增加的构象状态比另一种状态的动态性更低、膜埋藏程度更低且堆积更紧密。M2蛋白中胆固醇依赖性的变化可能归因于胆固醇诱导的双层膜性质变化和/或胆固醇与蛋白的直接结合。我们提出了一个与我们所有实验数据一致的模型，该模型表明我们在有胆固醇存在时观察到的主要构象与理解病毒出芽相关。
