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

Lead Research Organisation: Newcastle University
Department Name: Inst for Cell and Molecular Biosciences

Abstract

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.

Technical Summary

The aim is to discover how a cell discerns metals. At least seven families of DNA-binding, cytosolic metal sensors have been characterised in bacteria (Fur, DtxR, MerR, CsoR-RcnR, ArsR-SmtB, CopY, NikR). The exact complement of metal-sensors varies between bacteria with different representatives of each sensor-family responding to different metals. We need to understand how the correct sensor among the complement of metal sensors in a given cytosol responds to the correct metal. A novel hypothetical explanation for metal-specificity will be tested. The cyanobacterium Synechocystis PCC 6803 has been selected for this work due to (i) properties of its set of known, or deduced, metal-sensors which are peculiarly favourable for some of the planned experiments (ii) our background knowledge, biological reagents and technical expertise, (iii) the special physiological significance and biotechnological implications of metals such as copper, iron, nickel and cobalt in these organisms. This is an opportunity to obtain a comprehensible answer to the question of how a network of receptors is integrated to give rise to the correct cellular responses. The actions of at least one new metal-sensor will also be characterised in the course of this research.

Planned Impact

By controlling the levels of metals in cells metal sensors affect metal occupancy of metallo-proteins. Since some 47% of enzymes require metals, understanding how sensors discern metals, one from another, has broad implications across the biosciences and broad applications in biotechnology. Considerable time and energy has been expended in the past ~ six months to establish valuable industrial links and some of these are described in the (confidential) impact summary. Letters of support are attached. The most imminent of the anticipated applications relates to bio-processing and the production of recombinant metallo-proteins. Here the timescale for possible marketed products could be five years. Many synthetic biology projects are liable to require the manipulation of metal supply. Of these, engineering the supply of nickel for hydrogenase for biohydrogen production by photosynthetic bacteria is perhaps the most urgent. We know that nickel supply is poor in cyanobacteria and that this limits hydrogenase activity. The PI is a member of the Sir Joseph Swan Institute for Energy Research and collaborations have been established with individuals and networks involved in developing the photoproduction of hydrogen. Here the timescales for benefits to be fully realised, in terms of hydrogen fuel, are presumed to exceed ten years. The detection of metals by pathogens is central to virulence. Some metals and metal-chelators are already in use both as agrochemicals for crop and livestock protection, and in therapies related to human health. It is expected that the knowledge gained from this project could be applied to the development of new agrochemicals and new therapeutics. The development cycle for new agrochemicals and therapeutics is long and time to market is unlikely to be less than ten years. Nonetheless, there is scope for optimising use of existing treatments, perhaps in novel combinations, and here the lead time may be much swifter.

Publications

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Related Projects

Project Reference Relationship Related To Start End Award Value
BB/H006052/1 17/05/2010 16/01/2011 £365,836
BB/H006052/2 Transfer BB/H006052/1 01/01/2011 31/05/2013 £296,372
 
Description The specific detection of nickel and zinc was shown to be a function of relative metal-affinity in a set of cytosolic metal sensors. This relationship does not apply to the detection of cobalt.



A sensor that detects surplus nickel in a cyanobacterial cytoplasm and triggers nickel-export was characterised. The cells' cobalt-sensor binds nickel more tightly than cobalt. However, because the newly described nickel-sensor binds nickel even more tightly (than the cobalt-sensor or sensors for other metals in the same cell) it will trigger nickel-export below the concentration for aberrant binding to the other-sensors. Thus the specific detection of nickel can be explained based upon relative nickel-affinities. The hypothesis that the research programme set out to test is therefore true for nickel, showing how metal-specificity can become a combined function of a cells set of metal-sensors. The papers describing these discoveries are recommended by Faculty 1000.



The nickel-sensor was also shown to be triggered by zinc such that the purified protein is driven off its target DNA sequence by zinc. Inside cells the nickel-sensor does not 'normally' (at steady state) respond to zinc under conditions when the bona fide zinc-sensors respond. The zinc-sensors have a zinc-affinity about two orders of magnitude tighter than the zinc-affinity of the nickel-sensor. The zinc-sensors (which control zinc import and export) must therefore maintain a zinc concentration within this two-order of magnitude range. Most importantly, metal-selectivity in favour of zinc also correlates with the relative zinc-affinities of the sensors, again consistent with the central hypothesis for this programme.



When cells are initially (after 1 h) transferred to elevated zinc, the nickel-sensor does transiently detect surplus zinc. This dis-regulation shows that there is a pulse in the internal zinc concentration sufficient to exceed the zinc-affinity of the nickel-sensor (Figure 1, final manuscript in draft). In addition to providing insight into the nature of intracellular metal-pools, these data show how a dependence of metal-selectivity upon relative metal-affinity can lead to aberrant gene expression. In this example a transient excess of zinc would trigger a disadvantageous efflux of nickel, potentially making cells susceptible to nickel deficiency.



Finally, we have shown that the hypothesis is untrue for cobalt. The cobalt-sensor has the weakest affinity for cobalt among the set of sensors. Moreover, purified zinc-sensors can be triggered by cobalt yet do not normally (at steady state) respond to surplus cobalt inside cells when the cobalt-sensor is activated. Thus, cobalt is channelled to the cobalt-sensor (away from the zinc-sensors). The penultimate publication reported how channelling to the sensor could be mediated by components of the vitamin B12 biosynthetic pathway
Exploitation Route With joint funding from industrial partners we are now teasing out details of the cross-talk between metal-sensory circuits to identify opportunities to subvert the sensory networks. There is a history of using metals and metal-chelants as antimicrobials. The human immune system has also evolved to exploit metal-excess, and metal-deficiency, to control pathogens (so called, nutritional immunity). Understanding how metal-sensing circuits are integrated should enable the development of antimicrobial combinations of chelants and ionophores. The discovery that metal-sensing can be a function of relative metal-affinities has made this possible.


Nickel is required by hydrogenases and nickel supply for this enzyme is inadequate inside photosynthetic cyanobacteria. It has been suggested that cyanobacterial hydrogenase might be exploited to generate hydrogen biofuel (from sunlight). It should now be possible to adjust the nickel-affinity of the newly characterised nickel-sensor for the purpose of increasing nickel availability to hydrogenase. Such experiments will test the dogma that there is (commonly though clearly not universally and not for cobalt) an inverse relationship between the buffered concentration of metals (such as nickel and zinc) and the metal-affinity of the respective metal sensors. This has broad applicability (due to the prevalence of metalloenzymes) to engineering metal-supply for synthetic biology.
Sectors Other

 
Description refer to BB/H006052/2
Sector Other
 
Description Salmonella metal sensory network with Procter and Gamble
Amount £760,000 (GBP)
Funding ID BB/JO17787/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2012 
End 09/2017
 
Title Clones expressing metal sensor proteins 
Description Vectors containing cyanobacterial sensor sequences 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2014 
Provided To Others? Yes  
Impact Enabled studies of metal coordination sites by XAS and of binding affinities (as examples). 
 
Title Cyanobacteria deleted in metal sensors including InrS-mutants 
Description Mutants with altered metal homeostasis, most notably nickel 
Type Of Material Cell line 
Year Produced 2014 
Provided To Others? Yes  
Impact Cells provide a model for how to adjust the set point for a metal which is being disseminated at multiple industrial biotechnology events in 2014 
 
Description Invited lecture Trace Elements in Biology & Medicine, CO USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited lecture at FASEB meeting in Colorado Springs

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Invited lecture at the University of Sheffield 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach local
Primary Audience Participants in your research or patient groups
Results and Impact Invited seminar to Molecular Biology and Biotechnology

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Invited lecture at the University of Warwick, Chemistry 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach local
Primary Audience Participants in your research or patient groups
Results and Impact Seminar to the Chemistry Department in Warwick

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Invited seminar at the University of Warwick, Biology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Lecture to Biologists in Warwick

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Invited seminar in Seville 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited lecture to Instituto de Bioquimica Vegetal y Fotosintesis, Seville

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Invited talk at International Conference on BioInorganic Chemistry, Interlakken, Switzerland 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The results of our research were described which sparked questions and discussions immediately afterwards and ongoing by e-mail.
Year(s) Of Engagement Activity 2019
URL https://www.chem.uzh.ch/dam/jcr:d809e5d0-e81b-42d0-a1c9-175c8e13e958/ICBIC19_ScientificProgram_v5.pd...
 
Description Luigio Sacconi memorial lecture in Chemistry, Florence 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Invited lecture as part of the year of chemistry celebrations

no actual impacts realised to date
Year(s) Of Engagement Activity 2011