Miller Silas T, Henzler-Wildman Katherine A, Raman Srivatsan
Cellular and Molecular Biology Graduate Program, University of Wisconsin-Madison, Madison, WI 53706, USA.
DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706, USA.
bioRxiv. 2025 Apr 10:2025.04.09.647630. doi: 10.1101/2025.04.09.647630.
Multidrug efflux pumps are dynamic molecular machines that drive antibiotic resistance by harnessing ion gradients to export chemically diverse substrates. Despite their clinical importance, the molecular principles underlying multidrug promiscuity and energy efficiency remain poorly understood. Using multiparametric deep mutational scanning across eight substrates and two energy conditions, we deconvolute the contributions of substrate recognition, energetic coupling, and protein stability, providing an integrated, high-resolution view of multidrug transport. We find that substrate specificity arises from a distributed network of residues extending beyond the binding site, with mutations that reshape binding, coupling, conformational flexibility, and membrane interactions. Further, we apply a pH-based selection scheme to measure the effect of mutation on pH-dependent transport efficiency. By integrating these data, we reveal a fundamental relationship between efficiency and promiscuity: highly efficient variants exhibit broad substrate profiles, while inefficient variants are narrower. These findings establish a direct link between energy coupling and polyspecificity, uncovering the biochemical logic underlying multidrug transport.
多药外排泵是一种动态分子机器，它通过利用离子梯度输出化学性质多样的底物来驱动抗生素耐药性。尽管它们在临床上很重要，但多药滥交和能量效率背后的分子原理仍知之甚少。通过对八种底物和两种能量条件进行多参数深度突变扫描，我们解析了底物识别、能量偶联和蛋白质稳定性的贡献，提供了多药转运的综合高分辨率视图。我们发现底物特异性源于延伸到结合位点之外的分布式残基网络，突变会重塑结合、偶联、构象灵活性和膜相互作用。此外，我们应用基于pH的选择方案来测量突变对pH依赖性转运效率的影响。通过整合这些数据，我们揭示了效率和滥交之间的基本关系：高效变体表现出广泛的底物谱，而低效变体则较窄。这些发现建立了能量偶联和多特异性之间的直接联系，揭示了多药转运背后的生化逻辑。
