Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, Sweden; Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles, Belgium; Medical Inflammation Research, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
FEBS J. 2014 Aug;281(16):3667-84. doi: 10.1111/febs.12889. Epub 2014 Jul 21.
Intracellular vesicles are abundant in eukaryotic cells but absent in the Gram-negative bacterium Escherichia coli. However, strong overexpression of a monotopic glycolipid-synthesizing enzyme, monoglucosyldiacylglycerol synthase from Acholeplasma laidlawii (alMGS), leads to massive formation of vesicles in the cytoplasm of E. coli. More importantly, alMGS provides a model system for the regulation of membrane properties by membrane-bound enzymes, which is critical for maintaining cellular integrity. Both phenomena depend on how alMGS binds to cell membranes, which is not well understood. Here, we carry out a comprehensive investigation of the membrane binding of alMGS by combining bioinformatics methods with extensive biochemical studies, structural modeling and molecular dynamics simulations. We find that alMGS binds to the membrane in a fairly upright manner, mainly by residues in the N-terminal domain, and in a way that induces local enrichment of anionic lipids and a local curvature deformation. Furthermore, several alMGS variants resulting from substitution of residues in the membrane anchoring segment are still able to generate vesicles, regardless of enzymatic activity. These results clarify earlier theories about the driving forces for vesicle formation, and shed new light on the membrane binding properties and enzymatic mechanism of alMGS and related monotopic GT-B fold glycosyltransferases.
真核细胞中富含细胞内囊泡，但革兰氏阴性细菌大肠杆菌中却不存在。然而，强烈过表达来自粘质支原体的单糖基二酰基甘油合成酶（alMGS）这种单萜糖脂合成酶，会导致大肠杆菌细胞质中大量囊泡的形成。更重要的是，alMGS 为通过膜结合酶调节膜性质提供了一个模型系统，这对于维持细胞完整性至关重要。这两种现象都取决于 alMGS 与细胞膜结合的方式，但目前对此了解甚少。在这里，我们通过将生物信息学方法与广泛的生化研究、结构建模和分子动力学模拟相结合，对 alMGS 的膜结合进行了全面研究。我们发现，alMGS 以相当垂直的方式与膜结合，主要通过 N 端结构域中的残基结合，并且以诱导阴离子脂质局部富集和局部曲率变形的方式结合。此外，来自膜锚定片段中残基取代的几种 alMGS 变体仍能够产生囊泡，而与酶活性无关。这些结果阐明了早期关于囊泡形成驱动力的理论，并为 alMGS 及其相关的单萜 GT-B 折叠糖基转移酶的膜结合特性和酶机制提供了新的认识。
