ABC transporters are found in all organisms and almost every cellular compartment. They mediate the transport of various solutes across membranes, energized by ATP binding and hydrolysis. Dysfunctions can result in severe diseases, such as cystic fibrosis or antibiotic resistance. In type IV ABC transporters, each of the two nucleotide-binding domains is connected to a transmembrane domain by two coupling helices, which are part of cytosolic loops. Although there are many structural snapshots of different conformations, the interdomain communication is still enigmatic. Therefore, we analyzed the function of three conserved charged residues in the intracytosolic loop 1 of the human homodimeric, lysosomal peptide transporter TAPL (transporter associated with antigen processing-like). Substitution of D278 in coupling helix 1 by alanine interrupted peptide transport by impeding ATP hydrolysis. Alanine substitution of R288 and D292, both localized next to the coupling helix 1 extending to transmembrane helix 3, reduced peptide transport but increased basal ATPase activity. Surprisingly, the ATPase activity of the R288A variant dropped in a peptide-dependent manner, whereas ATPase activity of wildtype and D292A was unaffected. Interestingly, R288A and D292A mutants did not differentiate between ATP and GTP in respect of hydrolysis. However, in contrast to wildtye TAPL, only ATP energized peptide transport. In sum, D278 seems to be involved in bidirectional interdomain communication mediated by network of polar interactions, whereas the two residues in the cytosolic extension of transmembrane helix 3 are involved in regulation of ATP hydrolysis, most likely by stabilization of the outward-facing conformation.

中文翻译：

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同二聚体 ABC 转运蛋白中的域间通信


ABC 转运蛋白存在于所有生物体和几乎每个细胞区室中。它们通过 ATP 结合和水解来介导各种溶质的跨膜运输。功能障碍可能导致严重的疾病，例如囊性纤维化或抗生素耐药性。在 IV 型 ABC 转运蛋白中，两个核苷酸结合结构域中的每一个都通过两个偶联螺旋连接到跨膜结构域，这两个偶联螺旋是胞质环的一部分。尽管有许多不同构象的结构快照，但域间通信仍然是个谜。因此，我们分析了人同型二聚体溶酶体肽转运蛋白 TAPL（与抗原加工样相关的转运蛋白）胞质内环 1 中三个保守带电残基的功能。用丙氨酸取代偶联螺旋 1 中的 D278，通过阻碍 ATP 水解来中断肽转运。 R288 和 D292 的丙氨酸取代（均位于延伸到跨膜螺旋 3 的偶联螺旋 1 旁边）减少了肽转运，但增加了基础 ATP 酶活性。令人惊讶的是，R288A变体的ATP酶活性以肽依赖性方式下降，而野生型和D292A的ATP酶活性不受影响。有趣的是，R288A 和 D292A 突变体在水解方面没有区分 ATP 和 GTP。然而，与野生型 TAPL 不同，只有 ATP 为肽转运提供能量。总之，D278 似乎参与了由极性相互作用网络介导的双向域间通讯，而跨膜螺旋 3 的胞质延伸中的两个残基则参与了 ATP 水解的调节，最有可能是通过稳定向外的构象。