Grants and Contributions:

Grant or Award spanning more than one fiscal year. (2017-2018 to 2018-2019)

Iron is a requisite component of most life forms on Earth. The molecule melanotransferrin (MTf) is a member of the transferrin (Tf) family of molecules. It is the most ancient member of the Tf family, dating back over 670 million years. In many species, it exists in both secreted and membrane-anchored forms and has been shown to play a role in transporting iron. Serum Tf carries iron throughout the circulatory system but the unique function(s) and characteristics of MTf in iron transport remain less defined. The 3D structure of MTf has never been determined, and while it is useful to draw comparisons with available crystal structure data for Tf, it is clear that MTf does not bind to the same receptors as Tf. We have evidence that MTf is taken up by a caveolae-dependent pathway rather than the Tf-receptor, clathrin-coated pit mechanism. By comparing the structure of MTf to Tf, we may identify the unique aspects of this ancient iron-binding protein and gain a better understanding of this alternate pathway for iron uptake.s

Hypothesis: The information obtained from the MTf crystal structures will be useful in determining iron and other heavy metal binding sites, and lead to insight into receptor-binding capacity.

Specific Aims:
1) To crystallize iron-bound, human MTf and determine its structure by x-ray crystallography. We have cloned, expressed and purified human MTf, which has been used to generate small pseudo-crystals. We propose refining the crystallization conditions to generate diffraction quality crystals for structural determination.
2) To characterize the interaction between MTf and candidate receptors. A number of candidate receptors have been noted for MTf in the literature, including our own unpublished findings. These candidate receptors will be cloned, expressed and subjected to binding assays with MTf (surface plasmon resonance and isothermal titration calorimetry). The structure of the confirmed MTf receptor(s) will then be solved both in the presence and absence of MTf.

Significance: These studies are part of our larger theme to analyze the ability of MTf to act as an iron-binding molecule, and to determine the role of MTf in biology. This work is important since the function, structure and behaviour of MTf are different from those of the well-studied Tf. Findings regarding MTf with its receptor(s) can convey important information about function and provide insights as to the physical interaction between the two molecules.