Cale Hendricks

Structural Characterization of the Prolyl Hydroxylase Domain-2 (PHD2) Oxygen Transport Tunnel

The ability to maintain homeostasis with respect to oxygen concentration is essential for mammalian life. The pathway that regulates this process, which is upregulated in cancer, is known as the hypoxia signaling pathway. Hypoxia-inducible-factor-1a (HIF-1a) is an important transcriptional activator of this pathway that is regulated by an oxygen-sensitive protein, prolyl hydroxylase domain (PHD). Using oxygen as a substrate, PHD hydroxylates HIF-1a for degradation. However, the structural contribution to oxygen binding and sensitivity of PHD is relatively unexplored. Previous research using protein engineering identified a tunnel in PHD used for oxygen transport and mutations to the tunnel were able to down-regulate the signaling pathway during a state of hypoxia. This research aims to determine structural evidence of an oxygen-gas tunnel of wildtype (WT) PHD and changes to the tunnel caused by mutation using protein crystallization. Site directed mutagenesis of a recombinant plasmid of PHD2 was used to convert the tunnel residue tryptophan 389 to phenylalanine. With subsequent cell transformation, gene expression, protein purification via nickel-affinity and size exclusion chromatography, and analysis via UV-vis spectroscopy and gel-electrophoresis, pure WT and W389F PHD protein samples were produced. Optimization of hanging-drop protein crystallization led to promising micro-crystals that will be further analyzed using X-ray crystallography to create structural images with atomic resolution. Overall, the current and future results of this project, when paired with functional in-vivo studies, demonstrate the importance of the PHD oxygen transport tunnel in regulating the hypoxia signaling pathway.