Abramsson Mia L, Corey Robin A, Skerle Jan L, Persson Louise J, Anden Olivia, Oluwole Abraham O, Howard Rebecca J, Lindahl Erik, Robinson Carol V, Strisovsky Kvido, Marklund Erik G, Drew David, Stansfeld Phillip J, Landreh Michael
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Solna, Sweden.
School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom.
Elife. 2025 Apr 30;14:RP104237. doi: 10.7554/eLife.104237.
Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.
整合膜蛋白在细胞中执行重要功能，其活性常常受到膜中特定蛋白质 - 脂质相互作用的调节。在此，我们阐明了作为调节性脂质的心磷脂（CDL）作为膜蛋白及其复合物稳定剂的复杂作用。以计算机设计的模型蛋白TMHC4_R（ROCKET）为支架，我们结合分子动力学模拟和天然质谱来探索促进优先脂质相互作用并介导稳定作用的蛋白质特征。我们发现带正电残基的空间排列以及局部构象灵活性是区分稳定的和不稳定的CDL相互作用的因素。然而，我们还发现，即使在这个可控的人工系统中，也难以实现结合与稳定之间的明确区分，这表明重叠的脂质接触可以部分补偿结合位点突变的影响。将我们的见解扩展到天然存在的蛋白质，我们在菱形膜内蛋白酶GlpG中鉴定出一个稳定的CDL位点，并揭示其对酶底物偏好的调节影响。在这项工作中，我们建立了一个工程化功能性脂质相互作用的框架，为设计具有膜特异性性质或功能的蛋白质铺平了道路。
