质子偶联的寡肽转运蛋白(POT)由于其混杂的底物结合位点而具有很大的药学意义,该位点与几类药物的口服生物利用度的提高有关。POT家族的成员在所有系统发育王国中都是保守的,并通过将肽吸收与质子电化学梯度偶联而发挥作用。Cryo-EM结构和α折叠模型最近为两种哺乳动物POT的不同构象状态提供了新的见解,SLC15A1和SLC15A2。然而,这些研究留下了关于质子和底物耦合机制的悬而未决的重要问题,同时提供了使用分子动力学(MD)模拟研究这些过程的独特机会。这里,我们采用广泛的无偏和增强采样MD来绘制完整的SLC15A2构象循环及其热力学驱动力。通过计算不同质子化状态下和不存在或存在肽底物的构象自由能景观,我们确定了可能的中间质子化步骤序列,这些步骤驱动了向内的交替进入。这些模拟确定了哺乳动物和细菌POT之间细胞外门的关键差异,我们在基于细胞的转运试验中进行了实验验证。我们来自恒定PHMD和绝对结合自由能(ABFE)计算的结果也建立了质子结合和肽识别之间的机械联系,揭示了POTs二次主动运输的关键细节。这项研究为理解哺乳动物中的质子偶联肽和药物转运提供了重要的一步,并为整合溶质载体结构生物学知识和增强的药物设计以靶向组织和器官生物利用度铺平了道路。
我们体内的细胞被周围的膜密封,使它们能够控制哪些分子可以进入或离开。所需的分子通常通过需要能量来源的转运蛋白输入。转运蛋白实现这一目标的一种方法是通过同时移动称为质子的带正电荷的粒子穿过膜。称为POTs的蛋白质(质子偶联的寡肽转运蛋白的缩写)使用这种机制将小肽和药物素导入肾脏和小肠的细胞。这些转运蛋白的中心是一个与进口肽结合的口袋,它的两侧都有一个门:一个朝向细胞外部打开的外部门,和一个通向牢房内部的内门。质子从外门到内门的运动被认为将运输装置的形状从向外转移到面向内的状态。然而,这种高能偶联的分子细节还没有很好的理解。为了探索这个,Lichtinger等人。使用计算机模拟来确定质子在POT上的结合位置,以触发栅极打开。模拟建议两个地点一起组成朝外的大门,它在质子结合时打开。Lichtinger等人。然后在产生突变POT的培养的人体细胞中实验验证了这些位点。在所需的肽/药物附着到结合袋之后,然后质子移动到运输器下方的另外两个位置。这触发了内门打开,最终允许小分子进入细胞。这些发现代表了了解POT如何运输货物的重要一步。由于POTs可以将一系列药物从消化道运输到体内,这些结果可以帮助研究人员设计更好吸收的分子。这可能会导致更多的口服药物,使患者更容易坚持他们的治疗方案。
Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1, and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different
protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate
protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability.
The cells in our body are sealed by a surrounding membrane that allows them to control which molecules can enter or leave. Desired molecules are often imported via transport proteins that require a source of energy. One way that transporter proteins achieve this is by simultaneously moving positively charged particles called protons across the membrane. Proteins called POTs (short for proton-coupled oligopeptide transporters) use this mechanism to import small peptides and drugsin to the cells of the kidney and small intestine. Sitting in the centre of these transporters is a pocket that binds to the imported peptide which has a gate on either side: an outer gate that opens towards the outside of the cell, and an inner gate that opens towards the cell’s interior. The movement of protons from the outer to the inner gate is thought to shift the shape of the transporter from an outwards to an inwards-facing state. However, the molecular details of this energetic coupling are not well understood. To explore this, Lichtinger et al. used computer simulations to pinpoint where protons bind on POTs to trigger the gates to open. The simulations proposed that two sites together make up the outward-facing gate, which opens upon proton binding. Lichtinger et al. then validated these sites experimentally in cultured human cells that produce mutant POTs. After the desired peptide/drug has attached to the binding pocket, the protons then move to two more sites further down the transporter. This triggers the inner gate to open, which ultimately allows the small molecule to move into the cell. These findings represent a significant step towards understanding how POTs transport their cargo. Since POTs can transport a range of drugs from the digestive tract into the body, these results could help researchers design molecules that are better absorbed. This could lead to more orally available medications, making it easier for patients to adhere to their treatment regimen.