METALLOCHAPERONES: The partitioning of metals to delivery pathways

A large proportion of the reactions of life are catalysed by metals. Yet most of the enzymes driving these reactions prefer to associate with metals that prevent their activity rather than with the correct metals. Cells must therefore help enzymes to acquire the correct metals.

Many metal-catalysed reactions are of high value to biotechnology and/or are the targets of antimicrobial treatments (immune systems have evolved to exploit metals to control microbes in so-called nutritional immunity, while metal-chelants and metal-ionophores have empirically been used as antimicrobials widely across the bioeconomy). For these reasons we have worked with Industry (Lonza, Syngenta, Procter and Gamble) via recent and/or on-going collaborative projects, plus with more than 480 members (about a third from outside academia) of our BBSRC Metals in Biology Network in Industrial Biotechnology and Bioenergy.

Our overarching goal has always been to understand the cellular logic for metals: That is, how do cells enable proteins to acquire the correct metals? Central to this understanding is an observation that the cytosol buffers metals in an order of concentrations which is the inverse of metal-binding preferences: Thus tight-binding metals such as copper, zinc and nickel are buffered to low concentrations while weak-binding metals like magnesium, manganese and ferrous-iron, are buffered to higher concentrations. Metal-sensors are tuned to these concentrations to prevent the buffers from becoming depleted or saturated (Nature Chemical Biology, currently embargoed and in press). However this, in turn, raises an inevitable next question as to how the metal-sensor proteins themselves, along with other proteins of metal homeostasis, select the correct metals.

Recently we have been able to answer the question of metal-specificity of metal-sensors by comparing properties within a set of sensors from a common cell (Salmonella). In short, the correct sensor for each metal is simply whichever one is the most sensitive in the set for that particular metal. From these recent studies we now know what factors determine the most sensitive sensor in the set. We also now know how to calculate the metal concentration that triggers each Salmonella metal sensor, and thus have unique insight into the buffered concentrations of each metal in a common cytosol. It is hypothesised that, akin to the metal-sensors, other proteins of metal-homeostasis are also tuned to these same metal concentrations. This study will now seize the opportunity to apply similar approaches to understand how a set of metal-delivery proteins select the correct metals.

About one third of metalloenzymes are at the end of specialised metal delivery pathways. This solves the challenge of metal-selectivity for these metalloenzymes provided the correct metals partition onto the delivery pathways in the first place (otherwise the delivery-pathways will propagate mismetalation, and indeed there is evidence that such aberrations can occur). By making similar measurements of the metal-affinities and abundances of metal delivery proteins, it will now become possible to identify (1) which delivery protein is the best in the set for each metal and (2) which delivery proteins have affinities above or below the inferred buffered concentrations for each metal, as estimated from the metal-concentrations which trigger the metal-sensors. These data will reveal which pathways may be vulnerable to mismetalation and hence inform upon how to generate formulations which subvert the metal-handling systems (for use as antimicrobials). These data will also inform on how to enhance enzyme metalation via these pathways in support of synthetic biological approaches to biotechnology.

A set of metal-binding proteins of metal-delivery pathways including metallochaperones, chelatases, and metal-stores (plus homologues and partner proteins), have been identified in Salmonella from past work, from reviews of the literature and by bioinformatics. The individual proteins are known, or proposed, to handle iron (for heme, siroheme, iron sulphur clusters) nickel (Ni,Fe hydrogenase), cobalt (B12), copper (export via CopA), manganese (export via MntP), molybdenum (molybdopterin) or zinc. The aim is to discover how the correct metal partitions onto the respective metal-delivery routes.

These proteins will be expressed, purified and prepared in a manner suitable for the determination of affinities for cognate and non-cognate metals. The number of molecules of each protein per cell will be determined using quantitative mass spectrometry. A ranking of the set of delivery proteins will show where the cognate metal is likely to partition to the correct delivery protein. By comparing the delivery proteins to recent estimates of buffered metal concentrations (generated from the metal-concentrations which trigger Salmonella metal sensors) it should again be possible to infer where metals are, or are not, likely to partition to the correct delivery pathway. For some delivery pathways, complexes with other molecules will be necessary to enable correct metal partitioning, and this will be assessed. Changes in protein abundance when the metal-delivery pathway operates (for example under anaerobic conditions) will be measured and may enable a sufficient fraction of the correct metal to partition onto a delivery pathway.

The data will show where mismetalation is liable to occur suggesting where there is a need for additional check-points for metal-selectivity. Opportunities to subvert the pathways to develop antimicrobials, and constraints in engineering these pathways of metal-supply to enzymes of value to biotechnology, will be revealed.

The impact of this research cuts across multiple aspects of the bioeconomy and so non-academic beneficiaries will be from a diversity of sectors (with interests in exploiting metal-related antimicrobials, industrial-scale biological processes, bioremediation, metal-related nutrition and supplements as examples). The work also relates to the goals of the Metals in Biology BBSRC NIBB which is one of three so-called cross-cutting networks. This research will provide the underpinning understanding of the cellular handling of metals which will permit either (i) the subversion- or (ii) the enhancement- of metal supply to a large number of metalloproteins: The former will, for example, enable the development of new antimicrobials (for agriculture and food production, consumer goods, industrial-biotechnology and health care) while the latter will inform synthetic biological approaches to engineering increased metalloenzyme activity, for example, for bioprocessing and biotranformations, thus supporting the manufacture of diverse industrial biotechnology products that depend, directly or indirectly, on efficient metalloenzymes.

The PI has current, or recently-completed, collaborations with potential commercial beneficiaries outside of academia within the agriculture, consumer-goods, healthcare and industrial biotechnology (biologics production) sectors, providing some routes for realising the impact of this research. Members of Durham research commercialisation team in collaboration with legal services will advise where there may be opportunities (and/or commitments) to offer these existing Industrial collaborators first refusal to exploit outcomes of the new research program. The Metals in Biology BBSRC NIBB has about a third of its membership from outside academia and this provides many established routes for disseminating the findings to relevant individuals. This dissemination will be either under the protection of non-disclosure agreements pre-publication, and/or via more general routes for circulation maintained by the BBSRC NIBB manager. The vital mechanisms are in place to provide pathways to identify and exploit commercial opportunities arising from this research.

In addition to publication of the results of the research in high quality, open access, Journals, plus conference presentations (generally invited and sometimes invited several years in advance), at the end of this programme a review article will be written describing the diversity of metallochaperones, and proteins of metal-delivery pathways, placed in the context of advances in understanding the cellular logic for metals. The PI and CoI's will inspire interest in the sub-discipline through engagement and outreach activities, with some exemplars described in the impact plan.

The personnel, specifically the postdoctoral researcher, will receive rigorous training in protein biochemistry, bio-inorganic chemistry and the cell biology of metals and more broadly in transferable skills to the benefit of future employers. As evidence of the quality of such training, the greatest proportion of staff from the PI's laboratory have progressed to successful research careers in academia or industry. Through a global network of contacts the PI has, and will continue to, support former staff both individually and more generally by promoting the sub-discipline (advising major conferences, reviewing for discovery journals, writing recommendations for promotions and prizes as examples).