Molecular basis of substrate-induced permeation by an amino acid antiporter
Molecular basis of substrate-induced permeation by an amino acid antiporter
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DOI：
10.1073/pnas.1018081108
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发表时间：
2011-03-08
影响因子：
11.1
通讯作者：
Palacin, Manuel
中科院分区：
文献类型：
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作者：
Kowalczyk, Lukasz;Ratera, Merce;Palacin, Manuel
氨基酸、多胺和有机阳离子(APC)超家族的转运蛋白在细胞氧化还原平衡、癌症和氨基酸尿症中发挥着重要作用。细菌L-精氨酸/胍丁胺逆向转运蛋白是APC的主要结构模式，与其他家族如钠离子偶联神经递质转运蛋白一样具有“5+5反向重复”的折叠结构。现有的ADIC晶体结构捕获了其运输周期的两种状态：开放到外部的apo和面向外的Arg(+)结合的闭塞。然而，Arg(+)在这两种状态之间的转换过程中的作用尚不清楚。在这里，我们报道了一个Arg(+)结合的ADIC突变体(N101a)在3.0埃分辨率下的开放构象的晶体结构，完成了5+5反向重复折叠转运体运输周期中的主要构象状态的图像。N101a结构是介于先前已知的ADIC构象之间的中间状态。目前结构中的Arg(+)-Gu基团表现出高的迁移率和离域性，阻碍了底物的闭塞，导致移位率低。进一步的分析支持该基团与Asn101和Trp293残基的适当配合才能过渡到闭塞状态，为底物诱导FIT在5+5反向重复折叠转运体中的分子机制提供了第一条线索。在ADIC和所有折叠相关转运体中发现的重复之间的假对称，抑制了在运输周期中发生的构象变化，特别是跨膜螺旋重排。在ADIC中，这些运动发生在远离二聚体界面的地方，解释了每个亚单位的独立功能。
Transporters of the amino acid, polyamine and organocation (APC) superfamily play essential roles in cell redox balance, cancer, and aminoacidurias. The bacterial L-arginine/agmatine antiporter, AdiC, is the main APC structural paradigm and shares the "5 + 5 inverted repeat" fold found in other families like the Na+-coupled neurotransmitter transporters. The available AdiC crystal structures capture two states of its transport cycle: the open-to-out apo and the outward-facing Arg(+)-bound occluded. However, the role of Arg(+) during the transition between these two states remains unknown. Here, we report the crystal structure at 3.0 angstrom resolution of an Arg(+)-bound AdiC mutant (N101A) in the open-to-out conformation, completing the picture of the major conformational states during the transport cycle of the 5 + 5 inverted repeat fold-transporters. The N101A structure is an intermediate state between the previous known AdiC conformations. The Arg(+)-guanidinium group in the current structure presents high mobility and delocalization, hampering substrate occlusion and resulting in a low translocation rate. Further analysis supports that proper coordination of this group with residues Asn101 and Trp293 is required to transit to the occluded state, providing the first clues on the molecular mechanism of substrate-induced fit in a 5 + 5 inverted repeat fold-transporter. The pseudosymmetry found between repeats in AdiC, and in all fold-related transporters, restraints the conformational changes, in particular the transmembrane helices rearrangements, which occur during the transport cycle. In AdiC these movements take place away from the dimer interface, explaining the independent functioning of each subunit.