Möller Anna-Maria, Brückner Simon, Tilg Lea-Janina, Kutscher Blanka, Nowaczyk Marc M, Narberhaus Franz
Microbial Biology, Ruhr University Bochum, Bochum, Germany.
Plant Biochemistry, Ruhr University Bochum, Bochum, Germany.
Mol Microbiol. 2023 Jan;119(1):29-43. doi: 10.1111/mmi.15005. Epub 2022 Dec 4.
The outer membrane (OM) of Gram-negative bacteria functions as an essential barrier and is characterized by an asymmetric bilayer with lipopolysaccharide (LPS) in the outer leaflet. The enzyme LpxC catalyzes the first committed step in LPS biosynthesis. It plays a critical role in maintaining the balance between LPS and phospholipids (PL), which are both derived from the same biosynthetic precursor. The essential inner membrane proteins YejM (PbgA, LapC), LapB (YciM), and the protease FtsH are known to account for optimal LpxC levels, but the mechanistic details are poorly understood. LapB is thought to be a bi-functional protein serving as an adaptor for FtsH-mediated turnover of LpxC and acting as a scaffold in the coordination of LPS biosynthesis. Here, we provide experimental evidence for the physical interaction of LapB with proteins at the biosynthetic node from where the LPS and PL biosynthesis pathways diverge. By a total of four in vivo and in vitro assays, we demonstrate protein-protein interactions between LapB and the LPS biosynthesis enzymes LpxA, LpxC, and LpxD, between LapB and YejM, the anti-adaptor protein regulating LapB activity, and between LapB and FabZ, the first PL biosynthesis enzyme. Moreover, we uncovered a new adaptor function of LapB in destabilizing not only LpxC but also LpxD. Overall, our study shows that LapB is a multi-functional protein that serves as a protein-protein interaction hub for key enzymes in LPS and PL biogenesis presumably by virtue of multiple tetratricopeptide repeat (TPR) motifs in its cytoplasmic C-terminal region.
革兰氏阴性菌的外膜（OM）起着至关重要的屏障作用，其特征是外小叶含有脂多糖（LPS）的不对称双层膜。LpxC酶催化LPS生物合成的第一步关键反应。它在维持LPS与磷脂（PL）之间的平衡中起关键作用，LPS和PL均来自相同的生物合成前体。已知必需的内膜蛋白YejM（PbgA、LapC）、LapB（YciM）和蛋白酶FtsH可调节LpxC达到最佳水平，但其机制细节尚不清楚。LapB被认为是一种双功能蛋白，作为FtsH介导的LpxC周转的衔接蛋白，并在LPS生物合成的协调中作为支架发挥作用。在这里，我们提供了实验证据，证明LapB与LPS和PL生物合成途径分歧处的生物合成节点上的蛋白质存在物理相互作用。通过总共四项体内和体外试验，我们证明了LapB与LPS生物合成酶LpxA、LpxC和LpxD之间、LapB与调节LapB活性的抗衔接蛋白YejM之间以及LapB与第一个PL生物合成酶FabZ之间存在蛋白质-蛋白质相互作用。此外，我们发现LapB不仅对LpxC，而且对LpxD具有新的衔接蛋白功能，可使其不稳定。总体而言，我们的研究表明，LapB是一种多功能蛋白，可能凭借其细胞质C末端区域的多个四肽重复（TPR）基序，作为LPS和PL生物合成中关键酶的蛋白质-蛋白质相互作用枢纽。
