DATP binding

Abstract: The apoptotic protease-activating factor 1 (Apaf-1) controls the onset of many known forms of intrinsic apoptosis in mammals. Apaf-1 exists in normal cells as an autoinhibited monomer. Upon binding to cytochrome c and dATP, Apaf-1 oligomerizes into a heptameric complex known as the apoptosome, which recruits and activates cell-killing caspases. Here we present an atomic structure of an intact mammalian apoptosome at 3.8 A resolution, determined by single-particle, cryo-electron microscopy (cryo-EM). Structural analysis, together with structure-guided biochemical characterization, uncovered how cytochrome c releases the autoinhibition of Apaf-1 through specific interactions with the WD40 repeats. Structural comparison with autoinhibited Apaf-1 revealed how dATP binding triggers a set of conformational changes that results in the formation of the apoptosome. Together, these results constitute the molecular mechanism of cytochrome c- and dATP-mediated activation of Apaf-1.

Abstract: Reduction of NDPs by murine ribonucleotide reductase (mRR) requires catalytic (mR1) and free radical-containing (mR2) subunits and is regulated by nucleoside triphosphate allosteric effectors Here we present a new, comprehensive, and quantitative model for allosteric control of mRR enzymatic activity based on molecular mass, ligand binding, and enzyme activity studies In this model, nucleotide binding to the specificity site (s-site) drives formation of an active R12R22 dimer, ATP or dATP binding to the adenine-specific site (a-site) results in formation of an inactive tetramer, and ATP binding to the newly described hexamerization site (h-site) drives formation of active R16R26 hexamer In contrast, an earlier phenomenological model [Thelander, L, and Reichard, P (1979) Annu Rev Biochem 67, 71−98] (the “RT” model) ignores aggregation state changes and mistakenly rationalizes ATP activation versus dATP inhibition as reflecting different functional consequences of ATP versus dATP binding to the a-si

Abstract: Reduction of NDPs by murine ribonucleotide reductase (mRR) requires catalytic (mR1) and free radical-containing (mR2) subunits and is regulated by nucleoside triphosphate allosteric effectors. Here we present the results of several studies that refine the recently presented comprehensive model for the allosteric control of mRR enzymatic activity [Kashlan, O. B., et al. (2002) Biochemistry 41, 462−474], in which nucleotide binding to the specificity site (s-site) drives formation of an active R12R22 dimer, ATP or dATP binding to the adenine site (a-site) drives formation of a tetramer, mR14a, which isomerizes to an inactive form, mR14b, and ATP binding to the hexamerization site (h-site) drives formation of an active R16R26 hexamer. Analysis of the a-site D57N variant of mR1, which differs from wild-type mR1 (wt-mR1) in that its RR activity is activated by both ATP and dATP, demonstrates that dATP activation of the D57N variant RR arises from a blockage in the formation of mR14b from mR14a, and provides st...