Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada; Centre for Research in Molecular Modeling, Concordia University, Montréal, Québec, Canada.
Biophys J. 2018 Dec 4;115(11):2152-2166. doi: 10.1016/j.bpj.2018.10.023. Epub 2018 Nov 2.
ProP is a member of the major facilitator superfamily, a proton-osmolyte symporter, and an osmosensing transporter. ProP proteins share extended cytoplasmic carboxyl terminal domains (CTDs) implicated in osmosensing. The CTDs of the best characterized, group A ProP orthologs, terminate in sequences that form intermolecular, antiparallel α-helical coiled coils (e.g., ProPEc, from Escherichia coli). Group B orthologs lack that feature (e.g., ProPXc, from Xanthomonas campestris). ProPXc was expressed and characterized in E. coli to further elucidate the role of the coiled coil in osmosensing. The activity of ProPXc was a sigmoid function of the osmolality in cells and proteoliposomes. ProPEc and ProPXc attained similar activities at the same expression level in E. coli. ProPEc transports proline and glycine betaine with comparable high affinities at low osmolality. In contrast, proline weakly inhibited high-affinity glycine-betaine uptake via ProPXc. The K for proline uptake via ProPEc increases dramatically with the osmolality. The K for glycine-betaine uptake via ProPXc did not. Thus, ProPXc is an osmosensing transporter, and the C-terminal coiled coil is not essential for osmosensing. The role of CTD-membrane interaction in osmosensing was examined further. As for ProPEc, the ProPXc CTD co-sedimented with liposomes comprising E. coli phospholipid. Molecular dynamics simulations illustrated association of the monomeric ProPEc CTD with the membrane surface. Comparison with the available NMR structure for the homodimeric coiled coil formed by the ProPEc-CTD suggested that membrane association and homodimeric coiled-coil formation by that peptide are mutually exclusive. The membrane fluidity in liposomes comprising E. coli phospholipid decreased with increasing osmolality in the range relevant for ProP activation. These data support the proposal that ProP activates as cellular dehydration increases cytoplasmic cation concentration, releasing the CTD from the membrane surface. For group A orthologs, this also favors α-helical coiled-coil formation that stabilizes the transporter in an active form.
ProP 是主要易化剂超家族的成员，是质子渗透物协同转运蛋白，也是一种渗透感应转运蛋白。ProP 蛋白共享涉及渗透感应的延长细胞质羧基末端结构域 (CTD)。最佳表征的 A 组 ProP 同源物的 CTD 以形成分子间反平行α-螺旋卷曲螺旋的序列为末端（例如，来自大肠杆菌的 ProPEc）。B 组同源物缺乏该特征（例如，来自野油菜黄单胞菌的 ProPXc）。在大肠杆菌中表达和表征 ProPXc 以进一步阐明卷曲螺旋在渗透感应中的作用。ProPXc 的活性是细胞和质体囊泡渗透压的 S 形函数。在大肠杆菌中，ProPEc 和 ProPXc 在相同的表达水平下达到相似的活性。ProPEc 在低渗透压下以类似的高亲和力转运脯氨酸和甘氨酸甜菜碱。相比之下，脯氨酸通过 ProPXc 弱抑制高亲和力甘氨酸甜菜碱摄取。通过 ProPEc 摄取脯氨酸的 K 随渗透压急剧增加。通过 ProPXc 摄取甘氨酸甜菜碱的 K 没有。因此，ProPXc 是一种渗透感应转运蛋白，而 C 末端卷曲螺旋对于渗透感应不是必需的。进一步研究了 CTD-膜相互作用在渗透感应中的作用。对于 ProPEc，ProPXc CTD 与包含大肠杆菌磷脂的脂质体共沉淀。分子动力学模拟说明了单体 ProPEc CTD 与膜表面的结合。与可用的用于形成 ProPEc-CTD 的同二聚卷曲螺旋的 NMR 结构的比较表明，该肽的膜结合和同二聚卷曲螺旋形成是相互排斥的。包含大肠杆菌磷脂的脂质体中的膜流动性随着渗透压的增加而降低，渗透压范围与 ProP 激活相关。这些数据支持这样的假设，即随着细胞脱水导致细胞质阳离子浓度增加，ProP 激活，从而将 CTD 从膜表面释放出来。对于 A 组同源物，这也有利于 α-螺旋卷曲螺旋的形成，从而使转运蛋白以活性形式稳定。
