Photocycle-dependent conformational changes in the proteorhodopsin cross-protomer Asp–His–Trp triad revealed by DNP-enhanced MAS-NMR
Photocycle-dependent conformational changes in the proteorhodopsin cross-protomer Asp–His–Trp triad revealed by DNP-enhanced MAS-NMR
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DOI：
10.1073/pnas.1817665116
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发表时间：
2019-04
期刊：
影响因子：
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通讯作者：
Jakob Maciejko;J. Kaur;Johanna Becker‐Baldus;C. Glaubitz
中科院分区：
文献类型：
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作者：
Jakob Maciejko;J. Kaur;Johanna Becker‐Baldus;C. Glaubitz
意义 变形视紫红质 (PR) 存在于各种生态系统的海洋细菌中，是最丰富的光感受器之一。它将光转化为跨膜电化学梯度作为电池的能量来源。 PR 与许多微生物视紫红质一样，形成功能上未解析的高级寡聚物。在此，提出了 PR 五聚体内功能相关的跨原体相互作用的实验证据。 Asp-His-Trp 三联体在寡聚界面上形成，参与光循环，并可能充当 pH 传感器。这些发现为微生物视紫红质的机制提供了潜在的范式转变。这种见解只能通过将固态核磁共振波谱与动态核极化和光中间体的光诱导冷冻捕获相结合才能获得。蛋白视紫红质 (PR) 是一种高丰度、五聚体、光驱动质子泵。质子转移与微生物离子泵典型的典型光循环有关。尽管 PR 单体能够经历完整的光循环，但问题是，通过特定的跨启动子相互作用在膜中形成的五聚体复合物是否在其功能机制中发挥作用。在这里，我们使用动态核极化 (DNP) 增强的固态魔角旋转 (MAS) NMR 与光中间体的光诱导冷冻捕获相结合来解决这个主题。高度保守的残基 H75 位于原聚体界面处。我们发现它从 (τ)- 互变异构体转变为 (π)- 互变异构体，并改变了 M 状态下的环方向。它基于特定的 His/Trp 环方向跨寡聚界面与 W34 偶联，同时稳定同一原聚体内主要质子受体 D97 的 pKa。我们进一步表明，特定的 W34 突变对 D97 和通过 H75 介导的质子转移有巨大影响。残基 H75 定义了一个交叉原基体 Asp-His-Trp 三联体，它可能充当质子转移的 pH 依赖性调节剂。我们的数据代表了微生物视紫红质同源寡聚物原体之间光依赖性、功能相关的串扰。
Significance Proteorhodopsin (PR) is found in marine bacteria in various ecosystems and is one of the most abundant photoreceptors. It converts light into a transmembrane, electrochemical gradient as a source of energy for the cell. PR, like many microbial rhodopsins, forms functionally unresolved higher oligomers. Here, experimental evidence for functionally relevant cross-protomer interactions within the PR pentamer is presented. An Asp–His–Trp triad is formed across the oligomerization interface, participates in the photocycle, and potentially acts as a pH sensor. These findings provide a potentially paradigm shifting aspect for the mechanism of microbial rhodopsins. Such an insight could only be obtained by combining solid-state NMR spectroscopy with dynamic nuclear polarization and light-induced cryotrapping of photointermediates. Proteorhodopsin (PR) is a highly abundant, pentameric, light-driven proton pump. Proton transfer is linked to a canonical photocycle typical for microbial ion pumps. Although the PR monomer is able to undergo a full photocycle, the question arises whether the pentameric complex formed in the membrane via specific cross-protomer interactions plays a role in its functional mechanism. Here, we use dynamic nuclear polarization (DNP)-enhanced solid-state magic-angle spinning (MAS) NMR in combination with light-induced cryotrapping of photointermediates to address this topic. The highly conserved residue H75 is located at the protomer interface. We show that it switches from the (τ)- to the (π)-tautomer and changes its ring orientation in the M state. It couples to W34 across the oligomerization interface based on specific His/Trp ring orientations while stabilizing the pKa of the primary proton acceptor D97 within the same protomer. We further show that specific W34 mutations have a drastic effect on D97 and proton transfer mediated through H75. The residue H75 defines a cross-protomer Asp–His–Trp triad, which potentially serves as a pH-dependent regulator for proton transfer. Our data represent light-dependent, functionally relevant cross talk between protomers of a microbial rhodopsin homo-oligomer.