Dual Role of the C-Terminal Domain in Osmosensing by Bacterial Osmolyte Transporter ProP
Dual Role of the C-Terminal Domain in Osmosensing by Bacterial Osmolyte Transporter ProP
复制标题
DOI：
10.1016/j.bpj.2018.10.023
复制
发表时间：
2018-12-04
影响因子：
3.4
通讯作者：
Wood, Janet M.
中科院分区：
文献类型：
--
作者：
Culham, Doreen E.;Marom, David;Wood, Janet M.
ProP是主要易化剂超家族的成员，是质子-渗压剂同向转运体，也是质子传感转运体。ProP蛋白质共享延伸的细胞质羧基末端结构域（CTD），其与细胞内信号传导有关。最佳表征的A组ProP直系同源物的CTD终止于形成分子间反平行α-螺旋卷曲螺旋的序列（例如，ProPEc，来自大肠杆菌）。组B直向同源物缺乏该特征（例如，ProPXc，来自野油菜黄单胞菌）。ProPXc在E.进一步阐明卷曲的大肠杆菌在荧光传感中的作用。ProPXc的活性是细胞和脂蛋白体中渗透压的S形函数。ProPEc和ProPXc在E.杆菌ProPEc在低渗透压下以相当高的亲和力转运脯氨酸和甘氨酸甜菜碱。与此相反，脯氨酸弱抑制高亲和力甘氨酸甜菜碱的摄取通过ProPXc。通过ProPEc摄取脯氨酸的K-M随着渗透压摩尔浓度显著增加。通过ProPXc的甘氨酸-甜菜碱摄取的K-M没有。因此，ProPXc是一种β-敏感转运蛋白，C-末端卷曲的大肠杆菌对β-敏感不是必需的。进一步研究了CTD-膜相互作用在生物传感中的作用。对于ProPEc，ProPXc CTD与包含E.大肠杆菌磷脂。分子动力学模拟说明关联的单体ProPEc CTD与膜表面。与由ProPEc-CTD形成的同源二聚体卷曲大肠杆菌的可用NMR结构的比较表明，该肽的膜缔合和同源二聚体卷曲大肠杆菌的形成是相互排斥的。脂质体的膜流动性随E.大肠杆菌磷脂在与ProP活化相关的范围内随着渗透压的增加而降低。这些数据支持ProP在细胞脱水增加细胞质阳离子浓度时激活，从膜表面释放CTD的提议。对于A组直系同源物，这也有利于α-螺旋卷曲大肠杆菌的形成，使转运蛋白稳定在活性形式。
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 alpha-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 coli 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-M for proline uptake via ProPEc increases dramatically with the osmolality. The K-M for glycine-betaine uptake via ProPXc did not. Thus, ProPXc is an osmosensing transporter, and the C-terminal coiled coli 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 coli formed by the ProPEc-CTD suggested that membrane association and homodimeric coiled-coli 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 alpha-helical coiled-coli formation that stabilizes the transporter in an active form.