Homology modeling and site-directed mutagenesis identify amino acid residues underlying the substrate selection mechanism of human monocarboxylate transporters 1 (hMCT1) and 4 (hMCT4)
Homology modeling and site-directed mutagenesis identify amino acid residues underlying the substrate selection mechanism of human monocarboxylate transporters 1 (hMCT1) and 4 (hMCT4)
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DOI：
10.1007/s00018-019-03151-z
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发表时间：
2019-05
影响因子：
8
通讯作者：
Yuya Futagi;Masaki Kobayashi;Katsuya Narumi;Ayako Furugen;K. Iseki
中科院分区：
文献类型：
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作者：
Yuya Futagi;Masaki Kobayashi;Katsuya Narumi;Ayako Furugen;K. Iseki
人类单羧酸转运体(HMCTS/SLC16As)介导单羧酸化合物跨质膜的转运。在HMCTs中，hMCT1和hMCT4在多种组织中表达，并参与能量代谢的运输底物。这两个转运体都转运乳酸，但是，尽管hMCT1也转运s1-5-羟脯氨酸(L-OPRO)，但这种化合物只被hMCT4最少转运。因此，我们对hMCT1和hMCT4底物特异性差异的分子机制感兴趣。因此，我们建立了hMCT1和hMCT4的3D结构模型，以确定参与这些转运蛋白底物专一性的氨基酸残基。我们发现hMCT1的底物专一性受涉及周转数(M69)和底物亲和力(F367)的残基调节，这些残基负责(直接或间接)识别l-OPro的-NH-部分。此外，我们的hMCT1同源模型预测M69和F367参与与hMCT1的另一个区域的疏水相互作用，强调了它在l-OPro结合和易位循环中的潜在重要作用。诱变实验支持这一模型，表明有效的l-OPro运输需要在第69位有一个疏水的长线性结构，在第367位有一个疏水的γ分支结构。我们的工作表明，氨基酸残基M69和F367是hMCT1转运l-OPro的关键分子元件。这两个残基可能参与底物识别和/或底物诱导的构象变化。
Human monocarboxylate transporters (hMCTs/SLC16As) mediate the transport of monocarboxylic compounds across plasma membranes. Among the hMCTs, hMCT1 and hMCT4 are expressed in various tissues, and transport substrates involved in energy metabolism. Both transporters mediatel-lactate transport, but, although hMCT1 also transportsl-5-oxoproline (l-OPro), this compound is minimally transported by hMCT4. Thus, we were interested in the molecular mechanism responsible for the difference in substrate specificity between hMCT1 and hMCT4. Therefore, we generated 3D structure models of hMCT1 and hMCT4 to identify amino acid residues involved in the substrate specificity of these transporters. We found that the substrate specificity of hMCT1 was regulated by residues involved in turnover number (M69) and substrate affinity (F367), and these residues were responsible for recognizing (directly or indirectly) the –NH– moiety ofl-OPro. Furthermore, our homology model of hMCT1 predicted that M69 and F367 participate in hydrophobic interactions with another region of hMCT1, emphasizing its potentially important role in the binding and translocation cycle ofl-OPro. Mutagenesis experiments supported this model, showing that efficientl-OPro transport required a hydrophobic, long linear structure at position 69 and a hydrophobic, γ-branched structure at position 367. Our work demonstrated that the amino acid residues, M69 and F367, are key molecular elements for the transport ofl-OPro by hMCT1. These two residues may be involved in substrate recognition and/or substrate-induced conformational changes.