Peptide transporter DtpA has two alternate conformations, one of which is promoted by inhibitor binding
Peptide transporter DtpA has two alternate conformations, one of which is promoted by inhibitor binding
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DOI：
10.1073/pnas.1312959110
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发表时间：
2013-10-15
影响因子：
11.1
通讯作者：
Mueller, Daniel J.
中科院分区：
文献类型：
--
作者：
Bippes, Christian A.;Ge, Lin;Mueller, Daniel J.
大PTR家族的肽转运蛋白（PTR）促进二肽和三肽的摄取，为细胞提供蛋白质合成和代谢中间体所需的氨基酸。虽然一些PTRs的结构和功能的特点，药物如何调节肽转运仍然不清楚。为了深入了解这一机制，我们表征抑制剂结合大肠杆菌PTR二肽和三肽通透酶A（DtpA），这表明底物特异性类似于其人类同源hPEPT 1。在证明了hPEPT 1的最强抑制剂Lys[Z-NO2]-瓦尔也作为DtpA的高亲和力抑制剂后，我们使用单分子力谱来定位稳定肽转运蛋白的结构片段，并研究了这些结构片段中的哪些在抑制剂结合后改变了稳定性。用包埋在脂质膜中并暴露于生理相关条件的DtpA进行该表征。在未结合状态下，DtpA采用两种主要的交替构象，其中跨膜α-螺旋（TMH）2是稳定的（类似于43%的DtpA分子）或不稳定的（类似于57%的DtpA分子）。这两种构象被理解为代表转运蛋白的内向和外向构象状态。随着抑制剂浓度的增加，以稳定的TMH 2为特征的构象变得越来越普遍，在饱和时达到92%。我们的测量进一步表明，赖氨酸[Z-NO2]-瓦尔与TMH 2中的离散残基相互作用，这些残基对于配体结合和底物亲和力是重要的。这些相互作用反过来稳定TMH 2，从而促进DtpA的抑制构象.
Peptide transporters (PTRs) of the large PTR family facilitate the uptake of di- and tripeptides to provide cells with amino acids for protein synthesis and for metabolic intermediates. Although several PTRs have been structurally and functionally characterized, how drugs modulate peptide transport remains unclear. To obtain insight into this mechanism, we characterize inhibitor binding to the Escherichia coli PTR dipeptide and tripeptide permease A (DtpA), which shows substrate specificities similar to its human homolog hPEPT1. After demonstrating that Lys[Z-NO2]-Val, the strongest inhibitor of hPEPT1, also acts as a high-affinity inhibitor for DtpA, we used single-molecule force spectroscopy to localize the structural segments stabilizing the peptide transporter and investigated which of these structural segments change stability upon inhibitor binding. This characterization was done with DtpA embedded in the lipid membrane and exposed to physiologically relevant conditions. In the unbound state, DtpA adopts two main alternate conformations in which transmembrane a-helix (TMH) 2 is either stabilized (in similar to 43% of DtpA molecules) or not (in similar to 57% of DtpA molecules). The two conformations are understood to represent the inward- and outward-facing conformational states of the transporter. With increasing inhibitor concentration, the conformation characterized by a stabilized TMH 2 becomes increasingly prevalent, reaching similar to 92% at saturation. Our measurements further suggest that Lys[Z-NO2]-Val interacts with discrete residues in TMH 2 that are important for ligand binding and substrate affinity. These interactions in turn stabilize TMH 2, thereby promoting the inhibited conformation of DtpA.