Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine
Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine
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DOI：
10.1073/pnas.1511167112
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发表时间：
2015-08-25
影响因子：
11.1
通讯作者：
Moser, Juergen
中科院分区：
文献类型：
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作者：
Hebecker, Stefanie;Krausze, Joern;Moser, Juergen
细胞质膜可能是微生物与周围环境之间最重要的物理屏障。由Ala-tRNAAla依赖性丙氨酰磷脂酰甘油合酶（A-PGS）或Lys-tRNA（Lys）依赖性赖氨酰磷脂酰甘油合酶（L-PGS）催化的磷脂磷脂酰甘油（PG）的极性头部基团的氨酰化使得细菌能够科普对细胞膜的完整性有害的阳离子肽。因此，这些酶也被命名为多肽抗性因子（MprF）。它们由可分离的C-末端催化结构域和N-末端跨膜翻转酶结构域组成。在这里，我们提出了从人类机会致病菌铜绿假单胞菌的A-PGS的催化结构域的X-射线晶体结构。与此同时，与底物类似物L-赖氨酸酰胺复合的地衣芽孢杆菌相关赖氨酰-磷脂酰甘油特异性L-PGS结构域的结构被呈现。这两种蛋白质揭示了一个连续的隧道，允许疏水性脂质底物PG和极性氨酰-tRNA底物从相反的方向进入催化位点。使用错酰化tRNA变体研究A-PGS与L-PGS的底物识别。这里提出的结构工作结合使用人工tRNA或人工脂质底物的生物化学实验揭示了tRNA受体茎，氨基酰基部分，和PG的极性头基作为底物识别的主要决定因素。诱变方法产生了tRNA相互作用的互补氨基酸决定簇。这些结果对于L-PGS和A-PGS抑制剂的设计具有广泛的意义，这些抑制剂可以使微生物病原体对抗菌化合物更敏感。
The cytoplasmic membrane is probably the most important physical barrier between microbes and the surrounding habitat. Aminoacylation of the polar head group of the phospholipid phosphatidylglycerol (PG) catalyzed by Ala-tRNAAla-dependent alanyl-phosphatidylglycerol synthase (A-PGS) or by Lys-tRNA(Lys)-dependent lysyl-phosphatidylglycerol synthase (L-PGS) enables bacteria to cope with cationic peptides that are harmful to the integrity of the cell membrane. Accordingly, these synthases also have been designated as multiple peptide resistance factors (MprF). They consist of a separable C-terminal catalytic domain and an N-terminal transmembrane flippase domain. Here we present the X-ray crystallographic structure of the catalytic domain of A-PGS from the opportunistic human pathogen Pseudomonas aeruginosa. In parallel, the structure of the related lysyl-phosphatidylglycerol-specific L-PGS domain from Bacillus licheniformis in complex with the substrate analog L-lysine amide is presented. Both proteins reveal a continuous tunnel that allows the hydrophobic lipid substrate PG and the polar aminoacyl-tRNA substrate to access the catalytic site from opposite directions. Substrate recognition of A-PGS versus L-PGS was investigated using misacylated tRNA variants. The structural work presented here in combination with biochemical experiments using artificial tRNA or artificial lipid substrates reveals the tRNA acceptor stem, the aminoacyl moiety, and the polar head group of PG as the main determinants for substrate recognition. A mutagenesis approach yielded the complementary amino acid determinants of tRNA interaction. These results have broad implications for the design of L-PGS and A-PGS inhibitors that could render microbial pathogens more susceptible to antimicrobial compounds.