Metallochaperones and metal-sensors in metal-allocation

'To begin with, for you to be here now trillions of drifting atoms had somehow to assemble in an intricate and curiously obliging manner to create you', 'why atoms take this trouble is a bit of a puzzle' [Bill Bryson (2003), taken from the opening lines of the Introduction to 'A short history of nearly everything', winner of the 2004 Aventis prize for science books]. Among the assembled atoms are essential metals including copper, zinc, cobalt and iron. It is estimated that approximately one third of all gene products need one or other metal to function and we do not understand how each protein acquires the correct metal. A naive expectation was that proteins would tightly bind the correct metal and only bind all others weakly, or not at all. However, from our recent work we have reported two proteins, one that senses surplus zinc atoms within a bacterial cell and the other that transports surplus zinc atoms out of the cell, that bind the wrong metal, copper, much more tightly than zinc. R.J.P. Williams (now Emeritus Professor of Chemistry in Oxford) notes that the cell faces a problem because the order of affinities for metals follows a general series which for these four atoms is copper>zinc>cobalt>iron. How can a cell contain proteins that require the most competitive metals, such as copper, while simultaneously containing proteins that require less competitive atoms, such as cobalt or iron? A simplistic prediction is that copper will bind to them all rendering the non-copper proteins non-functional. It is hypothesised that somehow or other metals such as copper are kept away from the metal-binding sites of all but the correct proteins. We have developed an ideal experimental system in which to test this. Part of the answer to Bill Bryson's puzzle is that much of the work to assemble these atoms is done for you by plants and some microbes, which build inorganic elements into primary organic molecules using sunlight as energy. In plant chloroplasts, and ancestrally related cyanobacteria, this vital energy conversion involves electrons flowing through copper atoms bound to plastocyanin. It could therefore be argued that plastocyanin is the most important destination for copper on Earth. Copper atoms must be delivered to internal compartments, thylakoids, to reach plastocyanin but if the above hypothesis is correct then this exposes the fundamental problem. How can copper be prevented from erroneously binding to all other proteins while en route to plastocyanin? We discovered a pathway involving an importer that brings copper into the cell, a second protein that transports it into the thylakoids and a third protein, a metallochaperone, which shuttles copper between the other two. By handing copper from one protein to the next in the pathway it is hypothesised that this metal can be kept away from all other proteins, but this implies that the protein-interactions now dictate which metals are acquired by metalloproteins rather than their inherent metal-binding preferences. We have recently visualised the metallochaperone-transporter interaction at a molecular level of detail and can now test what selects the interacting partners. We will test whether the specificity of this interaction keeps copper away from the zinc transporter and away from sensors that detect other metals. Metal-sensors are of special use because they can report when they acquire a metal inside a living cell. If correct, the hypothesis implies that metals such as copper have the potential to associate tightly with the wrong proteins. This has implications across biology because it creates a risk for aberrations. It is likely to be a key feature of cellular responses to metal nutrient-excess, -limitation and other conditions which could promote metal-release from bona fide sites, including senescence and oxidative stress.

We will examine metal-selectivity in proteins involved in the homeostasis of copper, zinc and cobalt in Synechocystis PCC 6803. Proteins involved in metal-homeostasis lie near the apex of the selectivity hierarchy since the fidelity of their discrimination between metals determines the number of atoms of each metal in cells. This, in turn, has implications for metal-occupancy by other proteins. This research will (i) provide evidence that cell physiology acts to overcome inherent metal-affinities to prevent the wrong metals from occupying metallo-proteins, and (ii) expose mechanisms involved. The structure of an Atx1-PacS complex has been resolved. This indicates why Atx1 does not interact with the similar ferredoxin-fold of ZiaA. We will investigate the mechanism, and importance, of metallochaperone partner selection, by generating mutants. We will also test whether Atx1 contributes to the recycling of endogenous copper. We recently characterised a zinc-sensor that tightly binds copper to prevent zinc-perception in vitro. We will test whether this is true of ZiaR and Zur, both in vitro and in vivo. We will test whether zinc prevents cobalt binding by CoaR. We will test whether the copper-trafficking pathway is needed to keep copper away from these metal-sensors and examine whether metal imported by known transporters is channelled towards, or away from, these sensors. In the course of the work these sensors will be further characterised.