Department of Structural Biology, Genentech, Inc., South San Francisco, CA 94080.
Molecular Biology Institute, University of California, Los Angeles, CA 90095.
Proc Natl Acad Sci U S A. 2017 Jul 25;114(30):E6044-E6053. doi: 10.1073/pnas.1707813114. Epub 2017 Jul 11.
Gram-negative bacteria express a diverse array of lipoproteins that are essential for various aspects of cell growth and virulence, including nutrient uptake, signal transduction, adhesion, conjugation, sporulation, and outer membrane protein folding. Lipoprotein maturation requires the sequential activity of three enzymes that are embedded in the cytoplasmic membrane. First, phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) recognizes a conserved lipobox motif within the prolipoprotein signal sequence and catalyzes the addition of diacylglycerol to an invariant cysteine. The signal sequence is then cleaved by signal peptidase II (LspA) to give an N-terminal S-diacylglyceryl cysteine. Finally, apolipoprotein -acyltransferase (Lnt) catalyzes the transfer of the -1-acyl chain of phosphatidylethanolamine to this N-terminal cysteine, generating a mature, triacylated lipoprotein. Although structural studies of Lgt and LspA have yielded significant mechanistic insights into this essential biosynthetic pathway, the structure of Lnt has remained elusive. Here, we present crystal structures of wild-type and an active-site mutant of Lnt. The structures reveal a monomeric eight-transmembrane helix fold that supports a periplasmic carbon-nitrogen hydrolase domain containing a Cys-Glu-Lys catalytic triad. Two lipids are bound at the active site in the structures, and we propose a putative phosphate recognition site where a chloride ion is coordinated near the active site. Based on these structures and complementary cell-based, biochemical, and molecular dynamics approaches, we propose a mechanism for substrate engagement and catalysis by Lnt.
革兰氏阴性菌表达多种脂蛋白，这些脂蛋白对于细胞生长和毒力的各个方面都是必不可少的，包括营养物质摄取、信号转导、黏附、接合、孢子形成和外膜蛋白折叠。脂蛋白成熟需要三种酶的连续活性，这些酶嵌入细胞质膜中。首先，磷脂酰甘油：原脂蛋白二酰基甘油转移酶（Lgt）识别原脂蛋白信号序列中的保守脂盒基序，并催化二酰基甘油添加到不变半胱氨酸上。然后，信号肽 II（LspA）切割信号序列，得到 N 端 S-二酰基甘油半胱氨酸。最后，载脂蛋白酰基转移酶（Lnt）催化磷脂酰乙醇胺的-1-酰基链转移到这个 N 端半胱氨酸上，生成成熟的三酰基化脂蛋白。尽管 Lgt 和 LspA 的结构研究为这个必需的生物合成途径提供了重要的机制见解，但 Lnt 的结构仍然难以捉摸。在这里，我们展示了野生型和活性位点突变体 Lnt 的晶体结构。这些结构揭示了一个单体八跨膜螺旋折叠，支持含有 Cys-Glu-Lys 催化三联体的周质碳氮水解酶结构域。在结构中结合了两种脂质，我们提出了一个可能的磷酸识别位点，其中一个氯离子在活性位点附近配位。基于这些结构以及互补的基于细胞的生化和分子动力学方法，我们提出了 Lnt 底物结合和催化的机制。
