Physics of Complex Biosystems, Technical University of Munich, Garching, Germany.
Microbiology, Ludwig-Maximilians-University Munich, Martinsried, Germany.
PLoS Comput Biol. 2021 Feb 4;17(2):e1008680. doi: 10.1371/journal.pcbi.1008680. eCollection 2021 Feb.
Membrane proteins account for about one third of the cellular proteome, but it is still unclear how dynamic they are and how they establish functional contacts with cytoplasmic interaction partners. Here, we consider a membrane-integrated one-component receptor that also acts as a transcriptional activator, and analyze how it kinetically locates its specific binding site on the genome. We focus on the case of CadC, the pH receptor of the acid stress response Cad system in E. coli. CadC is a prime example of a one-component signaling protein that directly binds to its cognate target site on the chromosome to regulate transcription. We combined fluorescence microscopy experiments, mathematical analysis, and kinetic Monte Carlo simulations to probe this target search process. Using fluorescently labeled CadC, we measured the time from activation of the receptor until successful binding to the DNA in single cells, exploiting that stable receptor-DNA complexes are visible as fluorescent spots. Our experimental data indicate that CadC is highly mobile in the membrane and finds its target by a 2D diffusion and capture mechanism. DNA mobility is constrained due to the overall chromosome organization, but a labeled DNA locus in the vicinity of the target site appears sufficiently mobile to randomly come close to the membrane. Relocation of the DNA target site to a distant position on the chromosome had almost no effect on the mean search time, which was between four and five minutes in either case. However, a mutant strain with two binding sites displayed a mean search time that was reduced by about a factor of two. This behavior is consistent with simulations of a coarse-grained lattice model for the coupled dynamics of DNA within a cell volume and proteins on its surface. The model also rationalizes the experimentally determined distribution of search times. Overall our findings reveal that DNA target search does not present a much bigger kinetic challenge for membrane-integrated proteins than for cytoplasmic proteins. More generally, diffusion and capture mechanisms may be sufficient for bacterial membrane proteins to establish functional contacts with cytoplasmic targets.
膜蛋白约占细胞蛋白质组的三分之一，但目前尚不清楚它们的动态性质以及它们如何与细胞质相互作用伙伴建立功能联系。在这里，我们考虑一种膜整合的单组分受体，该受体也作为转录激活剂，并且分析了它如何在动力学上定位其在基因组上的特定结合位点。我们专注于 CadC 的情况，即大肠杆菌酸应激反应 Cad 系统的 pH 受体。CadC 是一种直接与其在染色体上的同源靶位点结合以调节转录的单组分信号蛋白的典型示例。我们结合荧光显微镜实验、数学分析和动力学蒙特卡罗模拟来探测这种靶标搜索过程。使用荧光标记的 CadC，我们在单个细胞中测量了从受体激活到成功与 DNA 结合的时间，这是因为稳定的受体-DNA 复合物可以作为荧光斑点可见。我们的实验数据表明，CadC 在膜中具有高度的流动性，并通过 2D 扩散和捕获机制找到其靶标。由于整个染色体的组织，DNA 的流动性受到限制，但靠近靶位点的标记 DNA 位点似乎具有足够的流动性，可以随机靠近膜。将 DNA 靶标位点迁移到染色体的遥远位置对平均搜索时间几乎没有影响，在这两种情况下均介于四到五分钟之间。然而，具有两个结合位点的突变株的平均搜索时间减少了约两倍。这种行为与用于细胞体积内 DNA 与表面上蛋白质的耦合动力学的粗粒度晶格模型的模拟一致。该模型还解释了实验确定的搜索时间分布。总体而言，我们的发现表明，与细胞质蛋白相比，DNA 靶标搜索对膜整合蛋白并没有更大的动力学挑战。更一般地说，扩散和捕获机制可能足以使细菌膜蛋白与细胞质靶标建立功能联系。
