Zinc binding alters the conformational dynamics and drives the transport cycle of the cation diffusion facilitator YiiP.
Zinc binding alters the conformational dynamics and drives the transport cycle of the cation diffusion facilitator YiiP.
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DOI：
10.1085/jgp.202112873
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发表时间：
2021-08-02
期刊：
影响因子：
--
通讯作者：
Stokes DL
中科院分区：
文献类型：
--
作者：
Lopez-Redondo M;Fan S;Koide A;Koide S;Beckstein O;Stokes DL
Lopez-Redondo等人使用cryo-EM和分子动力学来评估Zn 2+结合对锌/质子反向转运蛋白YiiP的构象和动力学的影响。去除Zn 2+导致增强的构象动力学，并导致过渡到运输周期中的中间状态。YiiP是将Zn 2+转运与质子动力偶联的次级转运蛋白。来自原核生物的YiiP和来自人类的Znt 8的结构研究揭示了三个不同的Zn 2+位点和保守的同源二聚体结构。这些结构定义了面向内和面向外的状态，这些状态表征了原型的交替访问传输机制。为了研究Zn 2+结合对构象转变的影响，我们采用cryo-EM结合分子动力学模拟，比较了在Zn 2+存在和不存在的情况下，希瓦氏菌（Shewanella oneidensis）YiP的结构。为了实现单颗粒冷冻EM，我们使用噬菌体展示文库来开发对YiiP具有高亲和力的Fab抗体片段，从而产生YiiP/Fab复合物。为了进行MD模拟，我们开发了一个非键虚拟模型的Zn 2+和验证其性能与已知的Zn 2+结合蛋白。使用这些工具，我们发现，在Zn 2+的存在下，YiiP采用与以前在管状晶体中看到的一致的面向内的构象。在用高亲和力螯合剂去除Zn 2+后，YiiP表现出增强的灵活性，并采用了一种新的构象，该构象似乎是向内和向外状态之间的中间体。这种构象涉及一个疏水门的关闭，该疏水门被假定为控制进入主要转运位点。几个独立的冷冻EM地图的比较表明，从面向内的状态的过渡是由一个次要的Zn 2+网站在细胞质膜界面的占用控制。这项工作增强了我们对单个Zn 2+结合位点及其在运输循环构象动力学中的作用的理解。
Lopez-Redondo et al. use cryo-EM and molecular dynamics to evaluate the effects of Zn2+ binding on the conformation and dynamics of the zinc/proton antiporter YiiP. Removal of Zn2+ leads to enhanced conformational dynamics and causes a transition to an intermediate state in the transport cycle. YiiP is a secondary transporter that couples Zn2+ transport to the proton motive force. Structural studies of YiiP from prokaryotes and Znt8 from humans have revealed three different Zn2+ sites and a conserved homodimeric architecture. These structures define the inward-facing and outward-facing states that characterize the archetypal alternating access mechanism of transport. To study the effects of Zn2+ binding on the conformational transition, we use cryo-EM together with molecular dynamics simulation to compare structures of YiiP from Shewanella oneidensis in the presence and absence of Zn2+. To enable single-particle cryo-EM, we used a phage-display library to develop a Fab antibody fragment with high affinity for YiiP, thus producing a YiiP/Fab complex. To perform MD simulations, we developed a nonbonded dummy model for Zn2+ and validated its performance with known Zn2+-binding proteins. Using these tools, we find that, in the presence of Zn2+, YiiP adopts an inward-facing conformation consistent with that previously seen in tubular crystals. After removal of Zn2+ with high-affinity chelators, YiiP exhibits enhanced flexibility and adopts a novel conformation that appears to be intermediate between inward-facing and outward-facing states. This conformation involves closure of a hydrophobic gate that has been postulated to control access to the primary transport site. Comparison of several independent cryo-EM maps suggests that the transition from the inward-facing state is controlled by occupancy of a secondary Zn2+ site at the cytoplasmic membrane interface. This work enhances our understanding of individual Zn2+ binding sites and their role in the conformational dynamics that govern the transport cycle.
DOI：
10.1007/s10969-010-9088-5
发表时间：
2010-06
期刊：
Journal of structural and functional genomics
影响因子：
--
作者：
Kim, Changki;Vink, Martin;Hu, Minghui;Love, James;Stokes, David L;Ubarretxena-Belandia, Iban
通讯作者：
Ubarretxena-Belandia, Iban