Tangled Up in Knots: Structures of Inactivated Forms of E. coli Class Ia Ribonucleotide Reductase

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Structure (Impact Factor: 5.62). 06/2012; 20(8):1374-83. DOI: 10.1016/j.str.2012.05.009
Source: PubMed


Ribonucleotide reductases (RNRs) provide the precursors for DNA biosynthesis and repair and are successful targets for anticancer drugs such as clofarabine and gemcitabine. Recently, we reported that dATP inhibits E. coli class Ia RNR by driving formation of RNR subunits into α4β4 rings. Here, we present the first X-ray structure of a gemcitabine-inhibited E. coli RNR and show that the previously described α4β4 rings can interlock to form an unprecedented (α4β4)2 megacomplex. This complex is also seen in a higher-resolution dATP-inhibited RNR structure presented here, which employs a distinct crystal lattice from that observed in the gemcitabine-inhibited case. With few reported examples of protein catenanes, we use data from small-angle X-ray scattering and electron microscopy to both understand the solution conditions that contribute to concatenation in RNRs as well as present a mechanism for the formation of these unusual structures.

Download full-text


Available from: Nozomi Ando, May 20, 2014
1 Follower
34 Reads
  • [Show abstract] [Hide abstract]
    ABSTRACT: Ribonucleotide reductases (RRs) catalyze a crucial step of de novo DNA synthesis by converting ribonucleoside diphosphates to deoxyribonucleoside diphosphates. Tight control of the dNTP pool is essential for cellular homeostasis. The activity of the enzyme is tightly regulated at the S-phase by allosteric regulation. Recent structural studies by our group and others provided the molecular basis for understanding how RR recognizes substrates, how it interacts with chemotherapeutic agents, and how it is regulated by its allosteric regulators ATP and dATP. This review discusses the molecular basis of allosteric regulation and substrate recognition of RR, and particularly the discovery that subunit oligomerization is an important prerequisite step in enzyme inhibition.
    Progress in molecular biology and translational science 05/2013; 117:389-410. DOI:10.1016/B978-0-12-386931-9.00014-3 · 3.49 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates (dNDPs). The Escherichia coli class Ia RNR uses a mechanism of radical propagation by which a cysteine in the active site of the RNR large (α2) subunit is transiently oxidized by a stable tyrosyl radical (Y•) in the RNR small (β2) subunit over a 35-Å pathway of redox-active amino acids: Y• ↔ [W?] ↔ Y in β2 to Y ↔ Y ↔ C in α2. When 3-aminotyrosine (NHY) is incorporated in place of Y, a long-lived NHY• is generated in α2 in the presence of wild-type (wt)-β2, substrate, and effector. This radical intermediate is chemically and kinetically competent to generate dNDPs. Herein, evidence is presented that NHY• induces formation of a kinetically stable α2β2 complex. Under conditions that generate NHY•, binding between YNHY-α2 and wt-β2 is 25-fold tighter (K = 7 nM) than for wt-α2|wt-β2 and is cooperative. Stopped-flow fluorescence experiments establish that the dissociation rate constant for the YNHY-α2|wt-β2 interaction is ∼10-fold slower than for the wt subunits (∼60 s). EM and small-angle X-ray scattering studies indicate that the stabilized species is a compact globular α2β2, consistent with the structure predicted by Uhlin and Eklund's docking model [Uhlin U, Eklund H (1994) Nature 370(6490):533-539]. These results present a structural and biochemical characterization of the active RNR complex "trapped" during turnover, and suggest that stabilization of the α2β2 state may be a regulatory mechanism for protecting the catalytic radical and ensuring the fidelity of its reactivity.
    Proceedings of the National Academy of Sciences 03/2013; 110(10):3835-40. DOI:10.1073/pnas.1220691110 · 9.67 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: CS2 hydrolase, a zinc-dependent enzyme that converts carbon disulfide to carbon dioxide and hydrogen sulfide, exists as a mixture of octameric ring and hexadecameric catenane forms in solution. A combination of size exclusion chromatography, multi-angle laser light scattering, and mass spectrometric analyses revealed that the unusual catenane structure is not an artefact, but a naturally occurring structure.
    Chemical Communications 06/2013; 49(71). DOI:10.1039/c3cc43219j · 6.83 Impact Factor
Show more