Liu Feng, Culham Doreen E, Vernikovska Yaroslava I, Keates Robert A B, Boggs Joan M, Wood Janet M
Department of Structural Biology and Biochemistry, Hospital for Sick Children, Toronto M5G 1X8, ON Canada.
Biochemistry. 2007 May 15;46(19):5647-55. doi: 10.1021/bi062198r. Epub 2007 Apr 19.
Escherichia coli transporter ProP acts as both an osmosensor and an osmoregulator. As medium osmolality rises, ProP is activated and mediates H+-coupled uptake of osmolytes like proline. A homology model of ProP with 12-transmembrane (TM) helices and cytoplasmic termini was created, and the protein's topology was substantiated experimentally. Residues 468-497, at the end of the C-terminal domain and linked to TM XII, form an intermolecular, homodimeric alpha-helical coiled-coil that tunes the transporter's response to osmolality. We aim to further define the structure and function of ProP residues Q415-E440, predicted to include TM XII. Each residue was replaced with cysteine (Cys) in a histidine-tagged, Cys-less ProP variant (ProP*). Cys at positions 415-418 and 438-440 were most reactive with Oregon Green Maleimide (OGM), suggesting that residues 419 through 437 are in the membrane. Except for V429-I433, reactivity of those Cys varied with helical periodicity. Cys predicted to face the interior of ProP were more reactive than Cys predicted to face the lipid. The former may be exposed to hydrated polar residues in the protein interior, particularly on the periplasmic side. Intermolecular cross-links formed when ProP* variants with Cys at positions 419, 420, 422, and 439 were treated with DTME. Thus TM XII can participate, along its entire length, in the dimer interface of ProP. Cys substitution E440C rendered ProP* inactive. All other variants retained more than 30% of the proline uptake activity of ProP* at high osmolality. Most variants with Cys substitutions in the periplasmic half of TM XII activated at lower osmolalities than ProP*. Variants with Cys substitutions on one face of the cytoplasmic half of TM XII required a higher osmolality to activate. They included elements of a GXXXG motif that are predicted to form the interface of TM XII with TM VII. These studies define the position of ProP TM XII within the membrane, further support the predicted structure of ProP, reveal the dimerization interface, and show that the structure of TM XII influences the osmolality at which ProP activates.
大肠杆菌转运蛋白ProP兼具渗透感受器和渗透调节剂的功能。随着培养基渗透压升高，ProP被激活并介导如脯氨酸等渗透溶质的H⁺偶联摄取。构建了具有12个跨膜（TM）螺旋和细胞质末端的ProP同源模型，并通过实验证实了该蛋白的拓扑结构。位于C末端结构域末端并与TM XII相连的468 - 497位残基形成分子间同二聚体α - 螺旋卷曲螺旋，调节转运蛋白对渗透压的响应。我们旨在进一步确定预测包含TM XII的ProP中415 - E440位残基的结构和功能。在一个带有组氨酸标签且无半胱氨酸的ProP变体（ProP*）中，将每个残基替换为半胱氨酸（Cys）。415 - 418位和438 - 440位的Cys与俄勒冈绿马来酰亚胺（OGM）反应性最强，表明419至437位残基位于膜内。除了V429 - I433，这些Cys的反应性随螺旋周期性变化。预测面向ProP内部的Cys比预测面向脂质的Cys反应性更强。前者可能暴露于蛋白质内部的水合极性残基，特别是在周质侧。当用DTME处理在419、420、422和439位带有Cys的ProP变体时形成分子间交联。因此，TM XII可以沿着其全长参与ProP的二聚体界面。将E440替换为Cys使ProP失活。所有其他变体在高渗透压下保留了超过ProP脯氨酸摄取活性的30%。TM XII周质侧一半中带有Cys替代的大多数变体在比ProP更低的渗透压下被激活。TM XII细胞质侧一半一侧带有Cys替代的变体需要更高的渗透压来激活。它们包含预测形成TM XII与TM VII界面的GXXXG基序的元件。这些研究确定了ProP的TM XII在膜内的位置，进一步支持了ProP的预测结构，揭示了二聚化界面，并表明TM XII的结构影响ProP激活时的渗透压。
