Conformational flexibility of helix VI is essential for substrate permeation of the human apical sodium-dependent bile acid transporter
Conformational flexibility of helix VI is essential for substrate permeation of the human apical sodium-dependent bile acid transporter
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DOI：
10.1124/mol.107.041640
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发表时间：
2008-02-01
影响因子：
3.6
通讯作者：
Swaan, Peter W.
中科院分区：
文献类型：
--
作者：
Hussainzada, Naissan;Khandewal, Akash;Swaan, Peter W.
本研究表征了26个连续的半胱氨酸取代突变体的甲乙硫代磺酸（MTS）抑制谱，这些突变体包括人类顶端Na+依赖性胆囊酸转运体（SLC10A2）的跨膜（TM）螺旋6。TM6通过细胞外环3与TM7表面相连。TM7先前被确定为底物渗透路径的衬里部分（Mol Pharmacol 70: 1565, 2006）。大多数TM6半胱氨酸替代物耐受性良好，除了5个残基的活性严重受阻（1229C, G249C）或消失（P234C, G237C, G241C）。蛋白质合成或折叠和稳定性的破坏可能是突变体P234C缺乏活性的原因。随后的Pro234氨基酸替换揭示了它在运输循环的结构和功能方面的参与。极性MTS试剂（1 mM）的应用显著抑制了六个突变体（V235C, S239C, F242C, R246C， A248C和Y253C）的活性，在包含胆汁酸底物或从MTS预孵育培养基中去除Na+后，其修饰率几乎完全逆转（Y253C除外）。平衡[Na+]的活性评估显示了许多Na+敏感残基，表明它们靠近Na+相互作用位点或周围。硅模型揭示了mts可接近的TM6残基对功能重要的TM7氨基酸的密切和潜在的合作取向，证实了TM6在运输周期中的参与。我们得出结论，Pro234， Gly237和Gly241赋予螺旋柔韧性的功能要求，可能形成底物周转所需的“构象开关”；与此同时，与该开关在空间上不同的螺旋面排列的MTSaccessible残基可能参与底物渗透。
The present study characterizes the methanethiosulfonate (MTS) inhibition profiles of 26 consecutive cysteine-substituted mutants comprising transmembrane (TM) helix 6 of the human apical Na+-dependent bile acid transporter (SLC10A2). TM6 is linked exofacially to TM7 via extracellular loop 3. TM7 was identified previously as lining part of the substrate permeation path (Mol Pharmacol 70: 1565, 2006). Most TM6 cysteine replacements were well tolerated, except for five residues with either severely hampered (1229C, G249C) or abolished (P234C, G237C, G241C) activity. Disruption of protein synthesis or folding and stability may account for lack of activity for mutant P234C. Subsequent Pro234 amino acid replacement reveals its participation in both structural and functional aspects of the transport cycle. Application of polar MTS reagents (1 mM) significantly inhibited the activity of six mutants (V235C, S239C, F242C, R246C, A248C, and Y253C), for which rates of modification were almost fully reversed ( except Y253C) upon inclusion of bile acid substrates or removal of Na+ from the MTS preincubation medium. Activity assessments at equilibrative [Na+] revealed numerous Na+-sensitive residues, suggesting their proximity in or around Na+ interaction sites. In silico modeling reveals the intimate and potentially cooperative orientation of MTS-accessible TM6 residues toward functionally important TM7 amino acids, substantiating TM6 participation during the transport cycle. We conclude a functional requirement for helical flexibility imparted by Pro234, Gly237, and Gly241, probably forming a "conformational switch" requisite for substrate turnover; meanwhile, MTSaccessible residues, which line a helical face spatially distinct from this switch, may participate during substrate permeation.