Substrate-induced changes in the structural properties of LacY
Substrate-induced changes in the structural properties of LacY
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DOI：
10.1073/pnas.1404446111
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发表时间：
2014-04-22
影响因子：
11.1
通讯作者：
Mueller, Daniel J.
中科院分区：
文献类型：
--
作者：
Serdiuk, Tetiana;Madej, M. Gregor;Mueller, Daniel J.
大肠埃希菌的乳糖渗透酶(Lacy)是主要促进剂超家族的典范，它催化半乳糖苷和H+在细胞膜上的偶联化学计量转位。为了催化转运，Lacy经历了很大的构象变化，允许糖和H+结合部位交替进入膜的两侧。尽管有强有力的证据表明存在交替的访问机制，但仍不清楚H+和糖结合如何触发导致交替构象状态的一连串相互作用。在这里，我们使用动态单分子力谱来研究底物结合是如何诱导这种现象的。半乳糖苷结合强烈改变Lacy的N-端6-螺旋束的动力学、能量和机械性质，而C-端6-螺旋束在很大程度上不受影响。在N-末端6-螺旋束中，包含糖结合关键残基的螺旋V的性质变化最大。特别是，形成N-端结构域的二级结构在未结合状态下表现出机械脆性，但在底物结合状态下具有高度柔性的构象，显著延长了寿命和能量稳定性。因此，糖结合调节N-末端结构域的性质以启动半乳糖苷/H+共转运蛋白。与野生型Lacy相反，构象受限突变体Cys154->；Gly的性质不会随着糖的结合而改变。还观察到，Cys154->；Gly的单一突变改变了分子内的相互作用，从而使单个跨膜螺旋表现出不同的性质。结果支持Lacy的工作模型，在该模型中，底物结合诱导交替的构象状态，并提供对其特定的动力学、能量和机械性能的洞察。
The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H+ across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugarand H+-binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H+-and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H+ symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154 -> Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154 -> Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties.