Structural and biochemical investigation of a multiprotein complex between human ribonucleotide reductase and a protein-based activity regulator
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perezgoncalves-gmperez-phd-biology-2025-thesis.pdf
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Author(s)
Perez Goncalves, Gerardo Manuel
Advisor(s)
Drennan, Catherine L.
Date Issued
February 2025
Publisher
Massachusetts Institute of Technology
Abstract
Genomic instability is a common trait in many human cancers and can be driven by imbalances in deoxyribonucleotide triphosphate (dNTP) pools. A major regulator of the dNTP pools in humans is ribonucleotide reductase (RNR), the sole enzyme capable of de novo generation of deoxyribonucleotides from ribonucleotide substrates via a radical mechanism. Human RNR is composed of two subunits: the catalytic α subunit and the radical-generating β subunit. The two subunits must physically interact to be able to form a state competent for radical transfer. dATP acts as the native inhibitor of human RNR, forming α6-dATP rings that do not allow for the proper interaction between α and β subunits for radical transfer.
Recent work has also defined the IP₃ receptor binding protein released by inositol-1,4,5-triphoshate (IRBIT) protein in metazoans as a regulator of RNR activity. In this thesis, we investigate the interaction between human RNR and IRBIT, using a mixture of biochemical and structural biology techniques. We find that the inhibited α₆-dATP rings interact with IRBIT to form a large, >1 MDa complex that is distinct from α₆-dATP rings at the resolution of negative stain electron microscopy (EM). We observe a novel multiprotein complex via single-particle cryo-EM, with 2D-class averages suggesting the complex is composed of stacked α₆ hexamers. Rigid body docking into 3D-maps indicates a stoichiometry of (α₆-dATP)₂(IRBIT₂)₃, with the (IRBIT)2 molecules bridging the two RNR α₆ hexamers. Finally, analysis of our cryo-EM data suggests that the complex has conformational heterogeneity in which the distance between rings is influenced by the IRBIT₂ occupancy.
Methods for obtaining high yielding insect cell expression of human RNRs are also presented. We and others have carried out biochemical studies on human RNR with protein that were expressed in E. coli, but humans have post-translational modification pathways that E. coli lack, meaning that activity numbers might be artificial low for human RNR that is expressed in E. coli. We test this possibility, finding no substantial difference in enzymatic activity between insect cell expressed and E. coli bacteria expressed human RNR. Finally, we close the thesis with further analysis of the (α₆-dATP)₂(IRBIT₂)₃ complex, observing signs of conformational and compositional heterogeneity within our cryo-EM data that suggest reasons for the lack of high-resolution details in many of our reconstructions.
MIT Department
Massachusetts Institute of Technology. Department of Biology
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