Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides, making it an essential enzyme to the life of all living organisms. As such, RNR has been a successful drug target for treating a wide range of cancers. However, only in a few cases such as the RNR from the Herpes virus has the ?2?2 subunit interaction been utilized as a drug target. This is largely due to the lack of structural information on the RNR ?2?2 subunit interface, despite the fact that the crystal structures of the individual ?2 and ?2 homodimers of the E. coli class Ia RNR have been known for more than two decades. The relative orientations at which ?2 and ?2 bind to form the complete ?2?2 complex is generally understood, but there is little atomic resolution information on the subunit interaction, as many of the residues at the interface are unresolved in crystal structures. The objective of this work is to use the E. coli class Ia RNR as a model system to solve the structure of the ?2?2 interface region by magnetic resonance spectroscopy. This structural information will be important for understanding the complicated proton-coupled electron transfer (PCET) processes that take place across the subunit interface, and lays a foundation for the development of drugs designed to inhibit PCET between ?2 and ?2 in class Ia RNRs. While ?2?2 formation normally occurs only transiently during turnover, we demonstrate that a stable ?2?2 complex can be trapped on a timescale and protein concentration sufficient for nuclear magnetic resonance (NMR) measurements. One subunit will be 13C-labeled, and the other 15N-labeled, allowing for an investigation of the intersubunit heteronuclear dipolar contacts unique to the interface region between ?2 and ?2. The sensitivity of the experiment will be enhanced by dynamic nuclear polarization experiments utilizing the biradical polarizing agent AMUPol (5-10 mM). 13C-15N contacts will be probed by the FS-REDOR and ZF-TEDOR pulse sequences. The final modeling of the ?2?2 interface region will make use of molecular dynamics simulations of the available crystal structures constrained to previous cryo-electron microscopy data, previous pulsed electron paramagnetic resonance data, and the 13C-15N distance constraints determined in this work. Obtaining a structural model for the class Ia ?2?2 subunit interface region of RNR is expected to aid in understanding the mechanism of proton-coupled electron transfer between ?2 and ?2, opening the door for drug development efforts targeting the RNR class Ia ?2?2 subunit interaction.
Ribonucleotide Reductase is an essential enzyme to the life of all living organisms, and is a drug target for a wide range of cancers. Solving the structure of the model E. coli class Ia subunit interface will facilitate drug development efforts to inhibit the enzyme activity.
| Greene, Brandon L; Taguchi, Alexander T; Stubbe, JoAnne et al. (2017) Conformationally Dynamic Radical Transfer within Ribonucleotide Reductase. J Am Chem Soc 139:16657-16665 |
