Ferritins are a class of ubiquitous, globular proteins that can assemble into a highly symmetric nanocage (1) (2). Ferritins mineralize iron nanoparticles within their inner hollow shell and are responsible for iron homeostasis in organisms (3). Comprehending the molecular mechanisms behind the mineralization will help better understand the fundamental processes involved and also can help further engineer the same for various bio-material synthesis applications. Single particle cryogenic electron microscopy (Cryo-EM) is a powerful structural biology tool which images proteins in their native functional state and has been previously used to obtain an atomic resolution model of Apoferritin (4). In our previous work (5), using Cryo-EM for studying two different biomineralization cases (including Ferritin) we obtained a low resolution map in case of Ferritin and thus not much structural information at the mineralization interface could be obtained. In this study we use Cryo-EM data processing including nanoparticle masking, to study the biomineral formation in Human Light Chain Ferritin (HuLF). We present a 4.4 Å structure of the Ferritin-Iron nanoparticle complex. This resolution allowed us to fit the model of Apoferritin (PDB 2FFX) to the electron density map we obtained. The amino acid residues at the protein-nanoparticle interface can be observed and provide information regarding interacting molecules responsible for the mineralization. Previous X- ray crystallography studies have shown glutamic acid residues on the surface of the inner light chain Ferritin shell to be responsible for the formation of the initial iron nucleation cluster (6) (7). Our structure shows the formed iron nanoparticle to be centered around the above mentioned residues.

Thus this sets up the grounds for the next part of our study to involve collection of larger data sets. Data collection with more particles having the protein-nanoparticle complex and preferably using energy filters would facilitate higher resolution reconstruction, subsequently providing an atomic level understanding of the entire process. This study further sets the template for analysis of more protein mediated biomineralization cases which can lead to better rational design of bioengineered nanomaterials.

References:

1. Honarmand Ebrahimi, Hagedoorn, P.-L., & Hagen, W. R. (2015). Unity in the Biochemistry of the Iron-Storage Proteins Ferritin and Bacterioferritin. Chemical Reviews, 115(1), 295–326. https://doi.org/10.1021/cr5004908
   
2. Wang, Li, C., Ellenburg, M., Soistman, E., Ruble, J., Wright, B., Ho, J. X., & Carter, D. C. (2006). Structure of human ferritin L chain. Acta Crystallographica. Section D, Biological Crystallography., 62(7), 800–806. https://doi.org/10.1107/S0907444906018294
   
3. Zhang, Yu, X., Xie, J., & Xu, H. (2021). New Insights into the Role of Ferritin in Iron Homeostasis and Neurodegenerative Diseases. Molecular Neurobiology, 58(6), 2812–2823. https://doi.org/10.1007/s12035-020-02277-7
   
4. Irimia-Dominguez, Sun, C., Li, K., Muhoberac, B. B., Hallinan, G. I., Garringer, H. J., Ghetti, B., Jiang, W., & Vidal, R. (2020). Cryo-EM structures and functional characterization of homo- and heteropolymers of human ferritin variants. Scientific Reports, 10(1), 20666–20666. https://doi.org/10.1038/s41598-020-77717-4
   
5. Sen, Thaker, A., Sirajudeen, L., Williams, D., & Nannenga, B. L. (2022). Protein–Nanoparticle Complex Structure Determination by Cryo-Electron Microscopy. ACS Applied Bio Materials, 5(10), 4696–4700. https://doi.org/10.1021/acsabm.2c00130
   
6. Pozzi, Ciambellotti, S., Bernacchioni, C., Di Pisa, F., Mangani, S., & Turano, P. (2017). Chemistry at the protein–mineral interface in L-ferritin assists the assembly of a functional (μ3-oxo)Tris[(μ2-peroxo)] triiron(III) cluster. Proceedings of the National Academy of Sciences - PNAS, 114(10), 2580–2585. https://doi.org/10.1073/pnas.1614302114
7. Ciambellotti, Pozzi, C., Mangani, S., & Turano, P. (2020). Iron Biomineral Growth from the Initial Nucleation Seed in L‐Ferritin. Chemistry : a European Journal, 26(26), 5770–5773. https://doi.org/10.1002/chem.202000064