Integrative HDX-MS enables quantification of the conformational landscape of the sugar transporter XylE

A yet unresolved challenge in structural biology is to quantify conformational states of proteins underpinning function. This challenge is particularly acute for membrane proteins owing to the difficulties in stabilising them for in vitro studies. To address this challenge, we present here an integrative strategy that combines hydrogen-deuterium exchange mass spectrometry (HDX-MS) with ensemble modelling. We benchmark our strategy on wild type and mutant conformers of XylE, a prototypical member of the ubiquitous Major Facilitator Superfamily (MFS) of transporters. Next, we apply our strategy to quantify conformational ensembles of XylE embedded in different lipid environments and identify key lipid contacts that modulate protein conformations. Further application of our integrative strategy to substrate-bound and inhibitor-bound ensembles, allowed us to unravel protein-ligand interactions contributing to the alternating access mechanism of secondary transport in atomistic detail. Overall, our study highlights the potential of integrative HDX-MS modelling to capture, accurately quantify and subsequently visualise co-populated states of membrane proteins in association with mutations and diverse substrates and inhibitors. For Table of Content Only

结构生物学中一个尚未解决的挑战是量化支撑蛋白质功能的构象状态。由于在体外研究中稳定膜蛋白存在困难，这一挑战对于膜蛋白来说尤为严峻。为了应对这一挑战，我们在此提出一种综合策略，将氢 - 氘交换质谱（HDX - MS）与集合建模相结合。我们用XylE（一种普遍存在的主要易化子超家族（MFS）转运蛋白的原型成员）的野生型和突变构象体对我们的策略进行了基准测试。接下来，我们应用我们的策略来量化嵌入不同脂质环境中的XylE的构象集合，并确定调节蛋白质构象的关键脂质接触。我们的综合策略进一步应用于底物结合和抑制剂结合的集合，使我们能够在原子细节上揭示有助于二次转运交替通路机制的蛋白质 - 配体相互作用。总体而言，我们的研究强调了综合HDX - MS建模在捕捉、准确定量以及随后可视化与突变以及不同底物和抑制剂相关的膜蛋白共分布状态方面的潜力。（仅供目录使用）