Understanding iron acquisition within a bacterial iron-megastore

Iron is essential for the function of ten per cent of all enzyme families, and is responsible for the ability of haemoglobin to transport gasses in the blood. Iron is highly reactive with oxygen, which is very useful in the case of haemoglobin, but this is dangerous when the iron is in solution as it produces damaging free radicals. These free radicals can damage proteins and DNA in cells, and in serious cases this can cause mutations in the DNA and even kill cells. To help manage the balance between the demand for iron and the danger of free iron, cells have evolved different types of iron stores. These iron stores are called ferritins and all living organisms have at least one type of ferritin. Deletion of the ferritin genes is lethal in mammals and it significantly reduces the fitness of bacteria.

Ferritins are remarkable proteins whose form is essential for their function. They form small cages that enclose a central cavity where iron can be safely stored away from the rest of the cell. The ferritin cage is made up of multiple copies of a single protein, each with an active site that can safely catalyse the reaction of iron with oxygen to produce an iron mineral that is stored within the cavity. Different organisms have different types of ferritin with different sizes and thus capacity for storing iron. Some ferritins can even protect DNA from damage by directly binding to it and wrapping it around their shell.

We have started the study of a new family of ferritin proteins from bacteria and archaea that do not have a cage-like structure and instead look like ring-doughnuts. These doughnut ferritins are usually found within a cage, formed by another protein, that is twice as large as any other ferritin cage studied so far; this combination is known as a bacterial nanocompartment, or encapsulin. To be able to store iron ferritins absolutely have to have a cage-like structure, so this new arrangement of a doughnut-ferritin inside a cage protein is particularly interesting. We do not know how these proteins work together to sequester iron. In this project we will use structural biology methods, such as X-ray crystallography, mass spectrometry, and electron microscopy, coupled with metal analysis and biochemistry, to investigate the structure and function of this new iron storage system.

The remarkable ability of the encapsulin protein cages to bind and enclose their specific cargo protein has great potential to be exploited in biotechnology. Proteins and drug molecules that are toxic to bacterial cells could be produced and safely stored in these cages and only released when separated from the cells. To be able to fully understand the mechanism by which proteins are captured by the cage, we will reconstitute the encapsulin shell with enzymes and fluorescent proteins that are not normally found within it. Using experiments to separate proteins still left in solution from those within the cage, we will be able to measure the ability of the cage to bind to proteins of various sizes and properties. This information will allow us to make genetic systems for the production of these in bacteria for use in biotechnology.
Finally, we will study the metal binding ability of the new ferritin protein to determine how strongly it binds to iron and whether other metals can bind to the protein. We will change the amino acids present in the metal-binding site to alter the specificity of the protein to explore the potential for using this protein as a sensor for heavy metals, or for the production of metal nanoparticles that could be used as contrast agents in medical imaging.

The knowledge gained from this programme of work will give us a comprehensive understanding of this new system for iron storage and detoxification that we can use to begin to engineer nanocompartment systems. This work will lay a foundation for applications of nanocompartments in the healthcare and industrial biotechnology industries.

While iron is required for the function of nearly ten per cent of all enzyme families, in its ferrous form it is highly reactive and in the presence of hydrogen peroxide produces damaging hydroxyl radicals via the Fenton reaction. To balance their demand for iron and the dangerous consequences of free iron, cells have evolved iron storage systems known as ferritins. The cage-like structure of these proteins allows them to safely oxidise ferrous iron to ferric iron and store this as a ferrihydrite mineral within the cage. Our project is focused on a new family of ferritin-like proteins found in bacteria and archaea that do not form their own cage, but require a virus capsid-like protein, known as an encapsulin, to function as iron storage systems. We have a preliminary X-ray crystal structure of the new ferritin-like protein (Flp), which adopts a ring-like conformation with ten subunits with putative ferroxidase sites between them. Flp has a short C-terminal tag that directs it to the interior of the capsid as it is formed.

We will take an integrative structural biology approach to the study of this intriguing iron megastore to understand its organisation and function. We will use X-ray crystallography and electron microscopy to determine the structure and metal binding properties of the Flp and encapsulin. Mass spectrometry will be used to understand the organisation of the Flp ring and the influence of metal binding on the stability its multimeric ring arrangement. These studies will be complemented with biochemical characterisation and metal-binding studies carried out by ICP-MS. Ultimately we will be able to rationally engineer these systems to store different metals; or encapsulate different proteins tagged with the localisation sequence, to protect sensitive and toxic proteins. This research will ultimately allow us to produce a synthetic biology toolkit for manipulating encapsulins for use in biotechnology applications.

Wider beneficiaries
The research carried out in this project will have far reaching impacts with a wide group of beneficiaries. As well as the training opportunities for the project staff in Integrated Structural Biology and the production of academic impacts, this work will have the following wider impacts:

Nanotechnology
Ferritin nanocages are already widely used in nanotechnology as biomineralisation scaffolds for metal ions. These have been used in research into semi-conductor patterning, the production of quantum dots and memory devices. Our Encapsulin nanocompartments, with their higher metal-loading capacity than classical ferritins, will provide a new tool for research in this field with. The ability to modify the proteins within the Encapsulin shell and the fact that they exist in variants with different sizes will make them highly attractive platforms for nanotechnology and micro fabrication.

Electrical Engineering
Our research programme is of interest to electrical engineers due the potential for use of our Encapsulins for patterning semi-conductors. Ferritin cages loaded with semi-conductor materials can be used to pattern silicon substrates, and the Encapsulin nanocompartments will add another research tool in this area. The unique structure of our ferritin-like protein lends itself to modification and the fact that it binds metal ions in a highly-ordered manner could be advantageous in the design of conductive protein arrays for use in bio-batteries and bio-inorganic hybrid devices, such as biosensors.

Medical
The market for tracers in medical imaging is worth $8.6bn a year and the use of super-paramagnetic iron oxide nanoparticles (SPIOs) as contrast agents in MRI imaging is well established. Iron-loaded ferritins are explored for use in MRI, although the signal they offer is limited due to their small size in comparison to SPIOs. Our Encapsulin nanoparticles have the capacity to store five to ten times more iron than ferritin nanocages and give a much higher MRI contrast. Because the Encapsulin shell can be modified with antigens they could be used for tracking the fate of stem cells and cancer cells. These applications will have significant economic and societal impact as laboratory research is translated to treatments and new medical interventions. Our research will be of interest to medical researchers, clinicians and radiologists.

Synthetic Biology
The results of this research will be of interest to the wider synthetic biology community as we formulate models for protein self-assembly and encapsulation based on our Encapsulin system. The library of Encapsulin variants and Flp mutants we produce will allow us to develop platform technologies for protein encapsulation which will be of interest to the synthetic biology and bioengineering communities.

Industrial Biotechnology
Companies involved in the production of protein-based biologics and bioprocess design will benefit from our research into the basis for protein sequestration within Encapsulin nanocompartments. Our research will lay the foundation of knowledge for the use of Encapsulins as platforms for maximising the yields of high-value protein products and protecting sensitive enzymes in industrial reactions.

The market for recombinant enzymes for industrial bioprocessed and biologics is one of the fastest growing sectors of the global economy. Development of Encapsulin nancompartments as platforms for producing toxic and unstable enzymes and biologics, could have significant economic impacts as they could help to drive down the production costs for these high-value products through enabling their production in industrial hosts such strains, rather than mammalian cell-lines.
Protecting enzymes within the stable Encapsulin nanocage may also reduce process costs related to enzyme degradation, recovery, and loss. This research will therefore have an impact on the economic sustainability of novel bioprocesses.