Herneisen Alice L, 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. 2017 Nov 7;56(44):5955-5963. doi: 10.1021/acs.biochem.7b00924.
Influenza A M2 is a membrane-associated protein with a C-terminal amphipathic helix that plays a cholesterol-dependent role in viral budding. An M2 mutant with alanine substitutions in the C-terminal amphipathic helix is deficient in viral scission. With the goal of providing atomic-level understanding of how the wild-type protein functions, we used a multipronged site-directed spin labeling electron paramagnetic resonance spectroscopy (SDSL-EPR) approach to characterize the conformational properties of the alanine mutant. We spin-labeled sites in the transmembrane (TM) domain and the C-terminal amphipathic helix (AH) of wild-type (WT) and mutant M2, and collected information on line shapes, relaxation rates, membrane topology, and distances within the homotetramer in membranes with and without cholesterol. Our results identify marked differences in the conformation and dynamics between the WT and the alanine mutant. Compared to WT, the dominant population of the mutant AH is more dynamic, shallower in the membrane, and has altered quaternary arrangement of the C-terminal domain. While the AH becomes more dynamic, the dominant population of the TM domain of the mutant is immobilized. The presence of cholesterol changes the conformation and dynamics of the WT protein, while the alanine mutant is insensitive to cholesterol. These findings provide new insight into how M2 may facilitate budding. We propose the AH-membrane interaction modulates the arrangement of the TM helices, effectively stabilizing a conformational state that enables M2 to facilitate viral budding. Antagonizing the properties of the AH that enable interdomain coupling within M2 may therefore present a novel strategy for anti-influenza drug design.
甲型流感病毒M2是一种与膜相关的蛋白质，其C端具有两亲性螺旋结构，在病毒出芽过程中发挥胆固醇依赖性作用。C端两亲性螺旋结构中丙氨酸替代的M2突变体在病毒分裂方面存在缺陷。为了从原子水平理解野生型蛋白的功能，我们采用了多管齐下的定点自旋标记电子顺磁共振光谱法（SDSL-EPR）来表征丙氨酸突变体的构象特性。我们在野生型（WT）和突变型M2的跨膜（TM）结构域和C端两亲性螺旋（AH）中进行自旋标记，并收集了有关线形、弛豫率、膜拓扑结构以及在有胆固醇和无胆固醇的膜中同四聚体内距离的信息。我们的结果表明WT和丙氨酸突变体在构象和动力学方面存在显著差异。与WT相比，突变体AH的主要群体更具动态性，在膜中的深度更浅，并改变了C端结构域的四级排列。虽然AH变得更具动态性，但突变体TM结构域的主要群体却被固定。胆固醇的存在改变了WT蛋白的构象和动力学，而丙氨酸突变体对胆固醇不敏感。这些发现为M2如何促进出芽提供了新的见解。我们提出AH与膜的相互作用调节了TM螺旋的排列，有效地稳定了一种构象状态，使M2能够促进病毒出芽。因此，拮抗M2内能够实现结构域间偶联的AH的特性可能为抗流感药物设计提供一种新策略。
