Identification of amino acids essential for estrone-3-sulfate transport within transmembrane domain 2 of organic anion transporting polypeptide 1B1.
Identification of amino acids essential for estrone-3-sulfate transport within transmembrane domain 2 of organic anion transporting polypeptide 1B1.
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有机阴离子转运多肽 1B1 跨膜结构域 2 内雌酮 3-硫酸盐转运必需氨基酸的鉴定
DOI：
10.1371/journal.pone.0036647
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Hong M
中科院分区：
文献类型：
--
作者：
Li N;Hong W;Huang H;Lu H;Lin G;Hong M
跨膜结构域作为膜蛋白的重要结构，对蛋白质正确靶向细胞膜以及在转运蛋白中发挥转运功能至关重要。OATP序列的计算机分析显示，跨膜结构域2（TM 2）是在不同家族成员中具有高度氨基酸同一性的跨膜结构域之一。在本研究中，我们确定了四种氨基酸（Asp 70，Phe 73，Glu 74和Gly 76），这些氨基酸对OATP 1B 1（一种在人肝脏中特异性表达的OATP成员）的转运功能至关重要。这四个氨基酸与丙氨酸的取代导致运输活性显着降低。进一步的突变显示Asp 70和Glu 74的带电性质对于转运蛋白的正常功能是至关重要的。动力学参数的比较表明，Asp 70可能与底物相互作用，而Glu 74可能通过形成盐桥参与稳定结合位点。Phe 73的芳香环结构似乎起着重要的作用，因为Phe 73与酪氨酸，另一种具有类似结构的氨基酸取代，导致部分恢复的运输功能。另一方面，用丙氨酸或缬氨酸取代Gly 76不能恢复转运蛋白的功能。考虑到跨膜螺旋的性质，我们提出Gly 76可能对维持蛋白质的正确结构很重要。有趣的是，当对对应于OATP 1B 1低亲和力结合位点的较高浓度的雌酮-3-硫酸酯（50 µM）进行转运功能分析时，Phe 73、Glu 74和Gly 76突变体均显示出与野生型相当的转运功能，表明这些氨基酸对OATP 1B 1内雌酮-3-硫酸酯的低亲和力组分的影响较小，而Asp 70似乎参与了这两个位点的相互作用。
As an important structure in membrane proteins, transmembrane domains have been found to be crucial for properly targeting the protein to cell membrane as well as carrying out transport functions in transporters. Computer analysis of OATP sequences revealed transmembrane domain 2 (TM2) is among those transmembrane domains that have high amino acid identities within different family members. In the present study, we identify four amino acids (Asp70, Phe73, Glu74, and Gly76) that are essential for the transport function of OATP1B1, an OATP member that is specifically expressed in the human liver. A substitution of these four amino acids with alanine resulted in significantly reduced transport activity. Further mutagenesis showed the charged property of Asp70 and Glu74 is critical for proper function of the transporter protein. Comparison of the kinetic parameters indicated that Asp70 is likely to interact with the substrate while Glu74 may be involved in stabilizing the binding site through formation of a salt-bridge. The aromatic ring structure of Phe73 seems to play an important role because substitution of Phe73 with tyrosine, another amino acid with a similar structure, led to partially restored transport function. On the other hand, replacement of Gly76 with either alanine or valine could not recover the function of the transporter. Considering the nature of a transmembrane helix, we proposed that Gly76 may be important for maintaining the proper structure of the protein. Interestingly, when subjected to transport function analysis of higher concentration of esteone-3-sulfate (50 µM) that corresponds to the low affinity binding site of OATP1B1, mutants of Phe73, Glu74, and Gly76 all showed a transport function that is comparable to that of the wild-type, suggesting these amino acids may have less impact on the low affinity component of esteone-3-sulfate within OATP1B1, while Asp 70 seems to be involved in the interaction of both sites.
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DOI：
10.1152/ajpgi.00458.2001
发表时间：
2002-04-01
影响因子：
4.5
作者：
Cai, SY;Wang, W;Boyer, JL
通讯作者：
Boyer, JL
影响因子：
25.7
作者：
Bossuyt, X;Muller, M;Meier, PJ
通讯作者：
Meier, PJ
影响因子：
2.4
作者：
Meier-Abt, F;Mokrab, Y;Mizuguchi, K
通讯作者：
Mizuguchi, K
影响因子：
7.3
作者：
Glaeser, H.;Mandery, K.;Koenig, J.
通讯作者：
Koenig, J.
影响因子：
4.8
作者：
Tirona, RG;Leake, BF;Kim, RB
通讯作者：
Kim, RB