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

据最近估计，47%的酶需要铜、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)将涉及一种酶，它必须以某种方式获得正确的金属。酶对正确金属的选择在很大程度上取决于蛋白质折叠部位的金属可获得性，而细胞中的金属可获得性在很大程度上又取决于检测每种金属过剩或不足的传感器。至关重要的是，这些传感器以某种方式区分了不同的无机元素。一种名为MTF1的锌传感器是目前已知的唯一一种DNA结合、金属结合、人类使用的金属传感器。然而，在过去的二十年里，我们和其他许多人已经发现了一个不断扩大的细菌DNA结合金属传感蛋白的谱系。这些传感器开启或关闭基因；每个传感器对特定的金属做出反应。一些传感器调节的基因已经被发现，它们对金属的反应也被识别出来。在受监管的基因中，有编码进口商获得更多所需金属的基因，以及输出过剩和/或纯粹有毒的金属的出口商。传感器有几种不同的工作方式。一些基因与DNA结合，实际上关闭了一个基因。当传感器与金属结合时，它的结构发生变化，不再与DNA紧密结合，基因变得活跃。其他金属传感器的情况正好相反。当它们与金属结合时，它们的结构会发生变化，只有在这些条件下，它们才会与DNA紧密结合，并关闭基因。最后，一些传感器与DNA结合，但结合金属引起的蛋白质结构变化会扭曲DNA，从而激活基因表达。这些分类金属传感器的特性为探索如何识别金属提供了机会。一种天真的预期是，每个传感器都会将它检测到的金属紧紧地结合在一起，而对所有其他金属的结合很弱，或者根本不结合。但事实证明并非如此，事实上，生物无机化学的基本规则表明，对于柔性蛋白质来说，情况几乎不是这样。因此，问题变成了，不管基因调控的机制是什么，每种金属是如何触发正确的传感器蛋白的？要回答这个问题，我们需要考虑一组来自单一细菌的传感器。我们需要考虑它们对不同金属的亲和力，而不是孤立地，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。一个简单的解释是，传感器给出正确的综合响应是因为它们的“相对”金属亲和力，而不是它们的“绝对”金属亲和力：钴传感器是组中最紧的钴粘合剂，锌传感器是组中最紧的锌粘合剂，以此类推。为这项工作选择的生物是蓝藻，它有一套金属传感器，其特性特别适合于使用我们开发并在2007年发表的方法来比较它们的金属亲和力。在本节目中，我们还将介绍至少一种新的金属传感器。这是基础研究。从字面上讲，辨别的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领域有着广泛的影响和应用，因此，我们和纽卡斯尔“细胞中的金属”小组的其他成员积极与生物技术行业合作。影响计划中描述了与该方案有关的产业联系。

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.