GLUT1是哺乳动物细胞膜上的转运蛋白，能够帮助D-葡萄糖完成沿浓度梯度的跨膜运输。转运蛋白XylE是大肠杆菌中的GLUT1同源蛋白，需要利用质子梯度作为能量来源逆底物梯度转运D-木糖。GLUT1和XylE都是糖转运蛋白家族的成员，序列相似性约为40%，但在功能上前者为协助扩散后者为主动运输。前人对于XylE和GLUT1的研究表明，质子共转运蛋白XylE与单转运蛋白GLUT1的一个保守位点（XylE的质子化位点酸性残基Asp27，GLUT1中对应中性残基Asn29）是决定它们功能差异的关键。在本工作中，我们结合计算与生化实验方法，来研究XylE利用质子偶联的转运机理，并解释GLUT1和XylE功能差异的分子基础。通过使用分子动力学模拟，我们计算了转运蛋白在内向开口和外向开口状态之间发生构象变化的自由能。我们构建了GLUT1，XylE_H（Asp27质子化）和XylE_noH（Asp27去质子化）三个不含底物的系统来研究XylE的质子偶联，以及两个蛋白的功能差异。我们的结果揭示了XylE蛋白的质子化状态与构象偏好性存在联系，并与晶体结构和生化实验吻合。具体而言，GLUT1的自由能表面比较平坦，容易在内向开口与外向开口状态间转换，而质子化的XylE系统存在一个高能量的关闭构象，能够避免在没有底物的情况下泄漏质子。除此之外，我们的模拟结果指出XylE和GLUT1的热力学差异不可能由单个残基的状态变化完全决定。为了理解质子共转运蛋白XylE与单转运蛋白GLUT1热力学差异的分子基础，我们使用了贝叶斯网络模型，来研究构象变化过程中的蛋白氢键网络发生的变化。模型与相应的生化实验表明，XylE和GLUT1的多个不同位点共同导致了它们热力学上和功能上的区别。这些计算和实验的结果首次揭示了质子共转运蛋白和单转运蛋白在机理上的差异。
GLUT1 facilitates the down-gradient translocation of D-glucose across cell membrane in mammals. XylE, an Escherichia coli homolog of GLUT1, utilizes proton gradient as an energy source to drive uphill D-xylose transport. GLUT1 and XylE are members of sugar porter family and share about 40% sequence similarity. However, the former works as a uniporter for facilitated diffusion while the later catalyzes active transport. Previous studies of XylE and GLUT1 suggest that the variation between an acidic residue (Asp27 in XylE) and a neutral one (Asn29 in GLUT1) is a key element for their mechanistic divergence.In this work, we combined computational and biochemical approaches to investigate the mechanism of proton coupling by XylE and the functional divergence between GLUT1 and XylE. Using molecular dynamics simulations, we evaluated the free energy profiles of the transition between inward- and outward-facing conformations. We constructed three systems without substrates: GLUT1, XylE_H (Asp27 protonated), and XylE_noH (Asp27 deprotonated), to study the mechanism of proton coupling and functional distinction. Our results revealed the correlation between the protonation state and conformational preference in XylE, which is supported by the crystal structures. In particular, GLUT1 possesses a rather flat free energy landscape ensuring rapid turnover, while protonated XylE has a high energy transition state at occluded conformation which could presumably prevent proton leak without sugar binding.In addition, our simulations suggested a thermodynamic difference between XylE and GLUT1 that cannot be explained by the single residue variation at the protonation site. To understand the molecular basis, we applied Bayesian network models to analyze the alteration in the architecture of the hydrogen bond networks during conformational transition. The models and subsequent experimental validation suggest that multiple residue substitutions are required to produce the thermodynamic and functional distinction between XylE and GLUT1. These computational and biochemical characterizations provide unprecedented insight into the mechanistic difference between proton symporters and uniporters.
