Molecular Dynamics Simulations of the Bacterial UraA H+-Uracil Symporter in Lipid Bilayers Reveal a Closed State and a Selective Interaction with Cardiolipin

The Escherichia coli UraA H+-uracil symporter is a member of the nucleobase/ascorbate transporter (NAT) family of proteins, and is responsible for the proton-driven uptake of uracil. Multiscale molecular dynamics simulations of the UraA symporter in phospholipid bilayers consisting of: 1) 1-palmitoyl 2-oleoyl-phosphatidylcholine (POPC); 2) 1-palmitoyl 2-oleoyl-phosphatidylethanolamine (POPE); and 3) a mixture of 75% POPE, 20% 1-palmitoyl 2-oleoyl-phosphatidylglycerol (POPG); and 5% 1-palmitoyl 2-oleoyl-diphosphatidylglycerol/cardiolipin (CL) to mimic the lipid composition of the bacterial inner membrane, were performed using the MARTINI coarse-grained force field to self-assemble lipids around the crystal structure of this membrane transport protein, followed by atomistic simulations. The overall fold of the protein in lipid bilayers remained similar to the crystal structure in detergent on the timescale of our simulations. Simulations were performed in the absence of uracil, and resulted in a closed state of the transporter, due to relative movement of the gate and core domains. Anionic lipids, including POPG and especially CL, were found to associate with UraA, involving interactions between specific basic residues in loop regions and phosphate oxygens of the CL head group. In particular, three CL binding sites were identified on UraA: two in the inner leaflet and a single site in the outer leaflet. Mutation of basic residues in the binding sites resulted in the loss of CL binding in the simulations. CL may play a role as a “proton trap” that channels protons to and from this transporter within CL-enriched areas of the inner bacterial membrane.大肠杆菌UraA H+尿嘧啶同向转运蛋白（Escherichia coli UraA H+-uracil symporter）属于碱基/抗坏血酸转运蛋白（nucleobase/ascorbate transporter, NAT）家族，负责质子驱动的尿嘧啶摄取。本数据集针对该同向转运蛋白开展了多尺度分子动力学模拟，模拟体系的磷脂双分子层包含三类组分：1）1-棕榈酰-2-油酰磷脂酰胆碱（1-palmitoyl 2-oleoyl-phosphatidylcholine, POPC）；2）1-棕榈酰-2-油酰磷脂酰乙醇胺（1-palmitoyl 2-oleoyl-phosphatidylethanolamine, POPE）；3）75% POPE、20% 1-棕榈酰-2-油酰磷脂酰甘油（1-palmitoyl 2-oleoyl-phosphatidylglycerol, POPG）与5% 1-棕榈酰-2-油酰二磷脂酰甘油/心磷脂（1-palmitoyl 2-oleoyl-diphosphatidylglycerol/cardiolipin, CL）的混合体系，以模拟细菌内膜的脂质组成。模拟采用MARTINI粗粒化力场（MARTINI coarse-grained force field），先完成脂质在该膜转运蛋白晶体结构周围的自组装过程，随后开展全原子模拟。在本次模拟的时间尺度内，磷脂双分子层中该蛋白的整体折叠与去垢剂环境下的晶体结构保持一致。模拟在无尿嘧啶的条件下进行，由于门域与核心结构域发生相对位移，转运蛋白最终呈现闭合状态。研究发现包括POPG尤其是CL在内的阴离子脂质会与UraA结合，相互作用涉及环区特定碱性残基与CL头部基团磷酸氧原子之间的结合。具体而言，UraA上共鉴定出3个CL结合位点：2个位于内膜小叶，1个位于外膜小叶。结合位点的碱性残基发生突变后，模拟中CL的结合行为消失。CL可能作为"质子陷阱"发挥作用，在细菌内膜的CL富集区域介导质子向该转运蛋白的传递与反向传递。