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Please use this identifier to cite or link to this item: http://arks.princeton.edu/ark:/88435/dsp01s7526g502
Title: Convergent allostery in Bacillus subtilis ribonucleotide reductase studied by SAXS, crystallography, and Cryo-EM
Authors: Thomas, William Carey
Advisors: Ando, Nozomi
Contributors: Chemistry Department
Keywords: allostery
cryo-EM
enzymes
regulation
ribonucleotide reductase
SAXS
Subjects: Biochemistry
Biophysics
Issue Date: 2021
Publisher: Princeton, NJ : Princeton University
Abstract: Regulation of enzymatic activity is crucial for life, and this regulation often proceeds through allosteric transitions involving dynamic conformational change. One important enzyme that exhibits intricate regulation is ribonucleotide reductase (RNR), which uses a conserved, radical-based mechanism to catalyze the de novo conversion of ribonucleotides to deoxyribonucleotides. RNR allostery is complex and varied throughout the tree of life, frequently involving multiple lev-els of allosteric control. In this thesis, we reexamine regulation in class Ib RNRs, a major subclass of the enzyme family found only in bacteria, which were long thought to lack activity regulation due to a characteristic truncation of the canonical regulatory domain. Using small-angle X-ray scat-tering (SAXS), X-ray crystallography, and cryo-electron microscopy (cryo-EM), we show that the class Ib RNR of Bacillus subtilis has independently evolved a form of activity regulation. We de-scribe two new allosteric sites that bind both conventional allosteric effectors like dATP and ATP as well as novel activity effectors dAMP and GTP. These effectors control the reversible intercon-version of six different active and inhibited oligomeric structures. Under inhibiting conditions, the catalytic α subunit forms non-canonical dimers that are building blocks for larger inhibited oligo-mers. These oligomers are then characterized at near-atomic resolution by cryo-EM, revealing the surprising formation of helical α filaments in which the radical-generating β subunit can be seques-tered to prevent productive turnover. Crystal structures of the canonical α2 dimer provide detailed views of ligand-binding modes at allosteric sites that dictate association or dissociation of oligo-meric interfaces. These structures also suggest the functional importance of the C-termini of both RNR subunits in the full catalytic cycle. Finally, SAXS and cryo-EM are used to investigate the dynamics of the active RNR α2β2 tetramer and provide evidence that under activating conditions, the β2 subunit is highly mobile. Combined, these results illustrate the essential role that both oligo-meric and conformational flexibility play in the regulation of RNR activity. Further, these results show how convergent allosteric mechanisms can emerge within this diverse enzyme family.
URI: http://arks.princeton.edu/ark:/88435/dsp01s7526g502
Alternate format: The Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog: catalog.princeton.edu
Type of Material: Academic dissertations (Ph.D.)
Language: en
Appears in Collections:Chemistry

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