Structural investigations into the regulatory states of human Ribonucleotide Reductase - PROJECT SUMMARY/ABSTRACT Maintaining the equilibrium of the various ribonucleoside and deoxyribonucleoside triphosphates (NTPs and dNTPs) within cells is critical for homeostasis. As the only de novo mechanism for dNTP synthesis, ribonucleotide reductases (RNRs) are essential enzymes in all forms of life. These metalloenzymes catalyze the reduction of ribonucleotides into deoxyribonucleotides, which is essential for maintaining the dNTP pools for DNA synthesis and repair. Dysregulation of RNR activity is associated with tumorigenesis, and cancer cells are overly reliant on the enzyme as a result of their rampant proliferation. Therefore, inhibitors of RNR proteins are effective anti-cancer therapeutics and have been proposed as potential antibiotics. However, to date, there is no structure of human RNR in the active state, and many aspects of the protein’s regulation are still unknown. Recent structural work on RNR proteins has resulted in high-resolution structures of the active complex of the E. coli enzyme, which is a class Ia RNR like the human enzyme. These enzymes consist of two dimeric subunits referred to as α2 and β2. Although these enzymes are similar, there are also significant differences between the species. For example, while both proteins form rings when inactivated, the content of these rings are different, and preliminary data indicates that the inactivation mechanism is also distinct. The goal of this project is to use cryogenic electron microscopy (cryo-EM) to determine structures of the human RNR protein in both active and inactive states. My first aim is to determine cryo-EM structures of the α2 subunit bound to both ATP and dATP in the allosteric activity effector pocket within the regulatory domain. These data will reveal any conformational changes of the regulatory domain due to the differential binding and ATP and dATP that could not be observed in the crystal structures due to lattice contacts. I will then determine the structure of the human enzyme in the active α2β2 conformation to identify key features and interactions within the human enzyme. Together, these structures will uncover details about this integral protein and how it functions, which I will interrogate using site-directed mutagenesis and biochemical assays. Our findings will aid in the development of novel RNR inhibitors for the treatment of cancer or bacterial infections. As one of the world’s most well-renowned research institutions, MIT is an ideal environment to complete these aims. My mentor, Dr. Catherine Drennan, is an international leader in the field of structural biology with a focus on the study of metalloenzymes. Her mentorship will enable me to excel in my postdoctoral research and allow me to reach my best as a scientist moving forward.
