And they're out of the gate.
And they're out of the gate.
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他们已经出了门了。
DOI：
10.1113/jp281784
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Dale N
中科院分区：
文献类型：
--
作者：
Dale N
我们已经进入了缝隙连接样蛋白结构生物学的黄金时代。连接蛋白的第一个高分辨率结构于2009年发表（Maeda et al. 2009）。下一个进展花了近10年的时间，这是在cryoEM已经成熟成为确定膜蛋白结构的首选方法之后才发生的。最近，并在快速连续，高分辨率结构的Innexin 6，Pannexin 1，CalHM 2，Cx46和Cx 50（Myers et al. 2018）和Cx 31。3（Leeet al. 2020）已发表。为了继续我的赛马类比，Cx 46/50的最新结构（弗洛雷斯等人，2020），分辨率为1.9 μ m，勉强领先该领域。在差距连接构型中，连接蛋白形成由两个端对端对接的六聚体通道组成的十二聚体复合物。每个连接蛋白亚基具有四个跨膜区段和细胞内C末端。螺旋N-末端位于孔内。由于有六个这样的N-末端，一个长期存在的想法是，它们形成了一个门控通道的塞子。Yue et al（2021）发表在本期《生理学杂志》上的论文，在原子水平上对Cx46/50中N端通道门控的详细机制理解方面迈出了实质性的一步。CryoEM提供了结构的快照。虽然可以直接从cryoEM数据中收集一些动态信息，但分子动力学（MD）模拟通过将生命带入静态图像，有助于加强结构和功能之间的联系。MD考虑到三级结构的限制内的残基的运动，以及残基之间的相互作用的能量和可能性，以产生它们如何争夺位置的动态模拟。显然，输入结构的质量越好，MD的输出就越好。幸运的是，Cx46/50的N-末端的残基即使在用于本研究的3.4 bp结构中也被很好地解析，这为本文中获得的见解提供了基础。由于作者有两种结构的非常密切相关的Cx46和Cx 50在他们的处置，他们交换了Cx46和Cx 50的N-末端，使嵌合结构和比较的N-末端的序列，以确定差异，从而使相关的突变，可以改变功能。虽然他们模拟了许多不同的变体，他们对第九个残基（Cx 50中的Asn和Cx46中的Arg）的研究非常有趣。他们的MD模拟显示，在Cx46中，Arg 9可以与邻近亚基N末端的Glu 12形成盐桥，这是模拟中的主要构象。另一种构象也存在（但不太频繁），其中与相邻亚基的Arg 9的π键稳定了相同亚基的N-末端内的Arg 9和Glu 12的相互作用。显然，这些相互作用不存在于Cx 50的N-末端，而存在不带电荷的残基Asn 9。然而，它们在一定程度上存在于Asn 9突变为精氨酸的Cx 50中。这些相互作用的净效应是将Arg 9移动到远离渗透路径的孔的一侧，而不是如cryoEM结构中所建模的那样粘在孔的中心。当离子穿过通道孔时离子所经历的自由能也可以在MD模拟中计算。作者发现，Cl-的自由能景观在Cx46和50中的整个孔隙中相当恒定（且相似）。然而，Cx46的N-末端，而不是Cx 50，提供了一个显着的障碍K+渗透。尽管Arg 9被压在...
We have entered a golden age for the structural biology of gap junction-like proteins. The first high-resolution structure of a connexin was published in 2009 (Maeda et al. 2009). It took nearly 10 years for the next advance, which occurred only after cryoEM had matured to become the method of choice to determine the structures of membrane proteins. Recently, and in quick succession, high-resolution structures for Innexin6, Pannexin1, CalHM2, Cx46 and Cx50 (Myers et al. 2018) and Cx31. 3 (Leeet al. 2020) have been published. To continue my horse-racing analogy, the most recent structures for Cx46/50 (Flores et al. 2020), at a resolution of 1.9 Å, narrowly lead the field. In the gap junction configuration, connexins form a dodecameric complex consisting of two hexameric channels docked end to end. Each connexin subunit has four transmembrane segments, and an intracellular C-terminus. The helical N-terminus resides within the pore. As there are six of these N-termini, a long-standing idea has been that they form a plug that gates the channel. The paper by Yue et al (2021) published in this issue of the Journal of Physiology has made substantive steps towards detailed mechanistic understanding of channel gating by this N-terminus in Cx46/50 at an atomic level. CryoEM provides snapshots of structure. Although some dynamic information can be gleaned directly from cryoEM data, molecular dynamics (MD) simulations, by bringing life into what would otherwise be static images, help to reinforce the link between structure and function. MD takes into account the movements of residues within the constraints of the tertiary structure, and the energies of, and thus likelihood of, interactions between residues to produce a dynamic simulation of how they jockey for position. Clearly the better the quality of the input structure, the better the output from the MD. Fortunately, the residues in the N-terminus of Cx46/50 are well resolved even in the 3.4 Å structures used for this study, and this provides the basis for the insights gained in the current paper. As the authors had two structures of the very closely related Cx46 and Cx50 at their disposal, they swapped the N-termini of Cx46 and Cx50 to make chimaeric structures and compared the sequences of the N-termini to identify differences and thus make the relevant mutations that could alter function.While they simulated many different variants, their studies on the ninth residue (Asn in Cx50 and Arg in Cx46) are highly interesting. Their MD simulations revealed that in Cx46, Arg9 can form a salt bridge with Glu12 of the N-terminus of the neighbouring subunit and this was a predominant conformation in the simulations. An alternative conformation also existed (but less frequently), in which a pi-bond with Arg9 of the neighbouring subunit stabilized an interaction of Arg9 and Glu12 within the N-terminus of the same subunit. Obviously, these interactions are not present for the N-termini of Cx50 where the uncharged residue Asn9 is present instead. However, they are present to some degree for Cx50 where Asn9 has been mutated to Arginine. The net effect of these interactions is to move Arg9 to the side of the pore away from the permeation pathway, rather than sticking into the centre of the pore as modelled in the cryoEM structure. The free energy experienced by ions as they traverse the channel pore can also be calculated in MD simulations. The authors found that the free energy landscape for Cl–is fairly constant (and similar) throughout the pore in both Cx46 and 50. However, the N-terminus of Cx46, but not Cx50, offers a significant barrier to K+ permeation. Despite the fact that Arg9 is pressed against the side of the …