Design and engineering of human transferrin receptor 1 and its binding proteins

Sammanfattning: The human transferrin receptor 1 (TfR) is central in maintaining adequate cellular iron levels with the iron carrier proteins transferrin (Tf) and ferritin (Ft). Known to bind TfR is also HFE, another iron regulatory protein, competing with Tf for receptor binding. Non-functional HFE leads to hereditary hemochromatosis, a disorder leading to unsustainable iron accumulation in most organs of the body. TfR is also expressed at the blood-brain barrier at high levels, therefore being one attractive target for development of non-invasive pharmaceutics delivery applications to the brain. Several diseases are caused by pathogens binding TfR. Among them malaria, caused by the parasite Plasmodium vivax, and several hemorrhagic fevers caused by zoonotic new world arenaviruses with rodent reservoirs in South America.The TfR extracellular unit is structurally categorized in three domains, the helical, protease-like and apical domains. The helical domain is responsible for receptor dimerization. Transferrin is binding to the helical and protease-like domains, with an overlapping binding interface with HFE at the helical domain. The apical domain has structurally been shown to bind Ft, theattachment protein of P. vivax and Machupo virus glycoprotein 1 (MGP1).This thesis focus is protein design and engineering of TfR and proteins interacting with TfR. The receptor domain where pathogens and Ft bind to TfR has been decoupled from the receptor and expressed in bacteria after utilizing computational protein design. The binding characteristics of MGP1 to TfR has been studied and optimized with directed evolution, showing a mutation that enhances binding affinity by 30-fold. Two small proteins have been designed with two different computational design strategies to bind TfR, at the pathogen and HFE binding sites. The two proteins, TB08 and TB14, are after directed evolution and mutational optimization binding to the TfR in a yeast surface display system.