The discernment of metals by a set of DNA-binding transcriptional regulators

据最近估计，所有酶中有47%需要铜、锌、镍、钴、铁、锰、钙和镁等金属。平均而言，几乎一半的操纵细胞活动的尝试（例如在代谢工程中）将涉及必须以某种方式获得正确金属的酶。酶对正确金属的选择基本上取决于蛋白质折叠位点的金属可用性，而细胞中的金属可用性又基本上取决于检测每种金属过量或不足的传感器。至关重要的是，这些传感器以某种方式区分不同的无机元素。一种名为MTF 1的锌传感器是目前人类已知的唯一一种DNA结合、金属结合、金属传感器。然而，在过去的二十年里，我们和其他许多人发现了一个不断扩大的细菌DNA结合金属敏感蛋白的库。这些传感器打开或关闭基因;每个传感器对特定的金属做出反应。已经发现了一些传感器调节的基因，并确定了它们对金属的反应。在受调控的基因中，有一些基因编码进口者，他们获取更多的所需金属，而出口者则输出过剩的金属和/或有毒的金属。传感器以几种不同的方式工作。有些结合到DNA上，实际上关闭了一个基因。当传感器与金属结合时，其结构发生变化，使其不再与DNA紧密结合，基因变得活跃。其他金属传感器则相反。它们的结构在结合金属时发生变化，只有在这种条件下，它们才能与DNA紧密结合并关闭基因。最后，一些传感器在有金属和没有金属的情况下都与DNA结合，但结合金属引起的蛋白质结构变化会扭曲DNA以激活基因表达。这些分类的金属传感器的特性提供了一个机会，探索如何识别金属。一个天真的期望是，每个传感器将紧密结合它检测到的金属，而结合所有其他金属很弱或根本不结合。但事实并非如此，事实上，生物无机化学的基本规则意味着，对于柔性蛋白质来说，情况很少如此。因此，问题就变成了，不管基因调控的机制如何，每种金属是如何触发正确的传感蛋白的？为了回答这个问题，我们需要考虑一组来自单个细菌的传感器。我们需要考虑它们对不同金属的亲和力，不是孤立的，而是在同一细胞中所有其他金属传感器的金属亲和力的背景下。一个简单的解释可能是，传感器给出正确的积分响应作为其“相对”金属亲和力的函数，而不是其“绝对”金属亲和力：钴传感器是该组中最紧密的钴结合剂，锌传感器是该组中最紧密的锌结合剂，等等。为这项工作选择的生物体，蓝细菌，有一套金属传感器，这些传感器的特性特别适合于比较它们的金属亲和力，一个对另一个，使用我们在2007年开发并发表的方法。在本计划中，我们还将描述至少一种新的金属传感器。这是基础研究。识别金属是生命的基本要素。尽管如此，它在生物科学和生物技术中具有影响和应用，因此，我们和纽卡斯尔的“细胞中的金属”小组的其他成员积极与生物技术工业部门合作。影响计划中介绍了与该方案有关的工业联系。

It has recently been estimated that 47% of all enzymes require metals such as copper, zinc, nickel, cobalt, iron, manganese, calcium and magnesium. On average, almost half of all attempts to manipulate the activities of cells (for example in metabolic engineering) will involve an enzyme which must somehow acquire the correct metal. Selection of the correct metals by enzymes is substantially governed by metal-availability at the site of protein folding, and metal-availability in cells is, in turn, substantially governed by sensors that detect excess or deficiency of each metal. Crucially, these sensors somehow discern the different inorganic elements, one from another. A zinc-sensor called MTF1 is currently the only DNA-binding, metal-binding, metal-sensor known in humans. However, over the last two decades we, and many others, have discovered an expanding repertoire of bacterial DNA-binding metal-sensing proteins. These sensors turn genes on or off; each sensor acting in response to specific metals. The genes that some of the sensors regulate have been found and the metals they respond to identified. Among the regulated genes are ones encoding importers that acquire more of those metals which are needed and exporters that pump out metals that are surplus to requirements and/or solely toxic. The sensors work in several different ways. Some bind to DNA and, in effect, switch a gene off. When the sensor binds to the metal its structure changes such that it no longer binds tightly to the DNA and the gene becomes active. Other metal-sensors do the reverse. Their structure changes upon binding a metal such that only under these conditions do they bind tightly to DNA and switch a gene off. Finally, some sensors bind to DNA both with and without a metal but a change in protein structure caused by binding the metal distorts the DNA to activate gene expression. The characterisation of these assorted metal sensors has provided an opportunity to explore how metals are discerned. A naïve expectation was that each sensor would tightly bind the metal it detected and bind all other metals weakly or not at all. But this turns out not to be the case and indeed fundamental rules of bioinorganic chemistry imply that for flexible proteins it could rarely be the case. Thus the question becomes, regardless of the mechanism of gene regulation, how does each metal trigger the correct sensor protein? To answer this question we need to consider a set of sensors from a single bacterium. We need to consider their affinities for different metals, not in isolation, but in the context of the metal-affinities of all of the other metal-sensors in the same cell. A simple explanation could be that the sensors give the correct integrated response as a function of their 'relative' metal affinities rather than their 'absolute' metal affinities: the cobalt sensor being the tightest cobalt-binder of the set, the zinc-sensor being the tightest zinc-binder of the set, and so on. The organism chosen for this work, a cyanobacterium, has a set of metal-sensors with properties that are peculiarly well suited to comparing their metal-affinities, one against the other, using a method that we exploited and published in 2007. In this program we will also characterize at least one new metal-sensor. This is fundamental research. Discerning metals is, literally, elemental to life. Nonetheless, it has implications and applications across the biosciences and biotechnology and for this reason we, and the rest of the 'Metals in Cells' group at Newcastle, actively collaborate with the biotechnology industrial sector. Industrial links related to this programme are described in the impact plan.