Intermolecular Interactions between a Membrane Protein and a Glycolipid Essential for Membrane Protein Integration
Intermolecular Interactions between a Membrane Protein and a Glycolipid Essential for Membrane Protein Integration
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DOI：
10.1021/acschembio.1c00882
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发表时间：
2022-03-18
影响因子：
4
通讯作者：
Shimamoto, Keiko
中科院分区：
文献类型：
--
作者：
Mori, Shoko;Nomura, Kaoru;Shimamoto, Keiko
诱导新合成的蛋白质到适当的位置是每个生物体不可或缺的生物学功能。在大肠杆菌中，蛋白质整合到生物膜上是由蛋白质因子介导的，如SEC转录子和插入酶YidC。此外，一种名为MPIase(膜蛋白整合酶)的糖脂被证明是膜蛋白整合所必需的，它由长糖链和焦磷脂组成。我们报道了一个人工合成的最小单位的MPIase，只有一个三糖，即mini-MPIase-3，涉及整合活性所必需的结构。在这里，为了阐明MPIase的整合机制，我们使用物理化学方法分析了MPIase或其合成类似物与模型底物Pf3外壳蛋白的分子间相互作用。表面等离子体共振(SPR)分析揭示了焦磷酸对Pf3外壳蛋白亲和力的重要性。与迷你MPIase-3相比，尽管亲和力略有不同，但天然MPIase由于糖链较长，结合和解离速度更快。为了研究更详细的MPIase亚结构，我们进行了对接模拟和饱和转移差分-核磁共振。这些实验表明氨基葡萄糖上的6-O-乙酰基和MPIase的磷酸在与Pf3外壳蛋白的相互作用中起着重要的作用。对接模拟表明MPIase对蛋白质的疏水区和碱性氨基酸残基具有很高的亲和力，并通过使用缺失靶区的蛋白质突变体的SPR实验证明了这一点。这些结果证明了MPIase与底物蛋白的直接相互作用，并揭示了膜蛋白整合的详细机制。
Inducing newly synthesized proteins to appropriate locations is an indispensable biological function in every organism. Integration of proteins into biomembranes in Escherichia coli is mediated by proteinaceous factors, such as Sec translocons and an insertase YidC. Additionally, a glycolipid named MPIase (membrane protein integrase), composed of a long sugar chain and pyrophospholipid, was proven essential for membrane protein integration. We reported that a synthesized minimal unit of MPIase possessing only one trisaccharide, mini-MPIase-3, involves an essential structure for the integration activity. Here, to elucidate integration mechanisms using MPIase, we analyzed intermolecular interactions of MPIase or its synthetic analogs with a model substrate, the Pf3 coat protein, using physicochemical methods. Surface plasmon resonance (SPR) analyses revealed the importance of a pyrophosphate for affinity to the Pf3 coat protein. Compared with mini-MPIase-3, natural MPIase showed faster association and dissociation due to its long sugar chain despite the slight difference in affinity. To focus on more detailed MPIase substructures, we performed docking simulations and saturation transfer difference-nuclear magnetic resonance. These experiments yielded that the 6-O-acetyl group on glucosamine and the phosphate of MPIase play important roles leading to interactions with the Pf3 coat protein. The high affinity of MPIase to the hydrophobic region and the basic amino acid residues of the protein was suggested by docking simulations and proven experimentally by SPR using protein mutants devoid of target regions. These results demonstrated the direct interactions of MPIase with a substrate protein and revealed detailed mechanisms of membrane protein integration.