Jayasekera Hiruni S, Mohona Farhana Afrin, De Jesus Madison J, Miller Katherine M, Marty Michael T
Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona, 85721, USA.
bioRxiv. 2025 Feb 3:2024.10.24.620105. doi: 10.1101/2024.10.24.620105.
Lipids surrounding membrane proteins interact with different sites on the protein at varying specificities, ranging from highly specific to weak interactions. These interactions can modulate the structure, function, and stability of membrane proteins. Thus, to better understand membrane protein structure and function, it is important to identify the locations of lipid binding and the relative specificities of lipid binding at these sites. In our previous native mass spectrometry (MS) study, we developed a single and double mutant analysis approach to profile the contribution of specific residues toward lipid binding. Here, we extend this method by screening a broad range of mutants of AqpZ to identify specific lipid binding sites and by measuring binding of different lipid types to measure the selectivity of different lipids at selected binding sites. We complemented these native MS studies with molecular dynamics (MD) simulations to visualize lipid interactions at selected sites. We discovered that AqpZ is selective towards cardiolipins (CL) but only at specific sites. Specifically, CL orients with its headgroup facing the cytoplasmic side, and its acyl chains interact with a hydrophobic pocket located at the monomeric interface within the lipid bilayer. Overall, this integrative approach provides unique insights into lipid binding sites and the selectivity of various lipids towards AqpZ, enabling us to map the AqpZ protein structure based on the lipid affinity.
围绕膜蛋白的脂质以不同的特异性与蛋白上的不同位点相互作用，范围从高度特异性到弱相互作用。这些相互作用可以调节膜蛋白的结构、功能和稳定性。因此，为了更好地理解膜蛋白的结构和功能，确定脂质结合的位置以及这些位点处脂质结合的相对特异性非常重要。在我们之前的天然质谱（MS）研究中，我们开发了一种单突变和双突变分析方法来描述特定残基对脂质结合的贡献。在这里，我们通过筛选广泛的水通道蛋白Z（AqpZ）突变体来识别特定的脂质结合位点，并通过测量不同脂质类型的结合来测定所选结合位点处不同脂质的选择性，从而扩展了该方法。我们用分子动力学（MD）模拟对这些天然质谱研究进行补充，以可视化所选位点处的脂质相互作用。我们发现AqpZ对心磷脂（CL）具有选择性，但仅在特定位点。具体而言，CL以其头部基团朝向细胞质侧的方式定向，其酰基链与位于脂质双分子层内单体界面处的疏水口袋相互作用。总体而言，这种综合方法为脂质结合位点以及各种脂质对AqpZ的选择性提供了独特的见解，使我们能够基于脂质亲和力绘制AqpZ蛋白结构。
