Binding of allosteric effectors to ribonucleotide reductase protein R1: reduction of active-site cysteines promotes substrate binding
Binding of allosteric effectors to ribonucleotide reductase protein R1: reduction of active-site cysteines promotes substrate binding
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DOI：
10.1016/s0969-2126(97)00259-1
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发表时间：
1997-08-15
期刊：
影响因子：
5.7
通讯作者：
Eklund, H
中科院分区：
文献类型：
--
作者：
Eriksson, M;Uhlin, U;Eklund, H
背景：核糖核苷酸还原酶（Ribonucleotide reductase，RNR）是DNA合成过程中的一种重要酶，催化脱氧核糖核苷酸的从头合成。该酶由两个二聚体组成，称为R1和R2，并含有氧化还原活性半胱氨酸残基Cys 462和Cys 225。核糖核苷酸还原为脱氧核糖核苷酸涉及自由基的转移。自由基的途径先前已建议从晶体学结果，并支持定点诱变研究。大多数RNR通过两个不同的核苷酸结合位点进行变构调节：一个位点控制一般活性，另一个控制底物特异性。我们的目的是从晶体学上证明底物结合，并找到两个效应器结合sites.Results：我们在这里报告的第一个晶体结构的RNR Rf的减少形式。结构表明，在还原氧化还原活性半胱氨酸时，Cys 462的硫原子变得深埋。更容易接近的Cys 225移动到Cys 462的先前位置，为底物腾出空间。此外，R1与效应物、效应物类似物和效应物加底物的复合物的结构提供了关于这些结合位点的信息。底物GDP结合在两个结构域之间的裂缝中，其β-磷酸结合到两个螺旋的N末端;核糖与保守残基形成氢键。dTTP在变构底物特异性位点的结合稳定了靠近二聚体界面和活性位点的三个环，而一般的变构结合位点位于远离活性位点的位置。正确底物的结合受变构效应物的结合调节，而实际底物的结合主要发生在活性位点半胱氨酸被还原时。其中一个环在与dTTP结合后稳定，通过与核苷酸碱基的直接相互作用参与底物结合位点的形成。一般变构效应位点，位于远离活性位点，似乎调节全酶内的亚基相互作用。
Background: Ribonucleotide reductase (RNR) is an essential enzyme in DNA synthesis, catalyzing all de novo synthesis of deoxyribonucleotides. The enzyme comprises two dimers, termed R1 and R2, and contains the redox active cysteine residues, Cys462 and Cys225. The reduction of ribonucleotides to deoxyribonucleotides involves the transfer of free radicals. The pathway for the radical has previously been suggested from crystallographic results, and is supported by site-directed mutagenesis studies. Most RNRs are allosterically regulated through two different nucleotide-binding sites: one site controls general activity and the other controls substrate specificity. Our aim has been to crystallographically demonstrate substrate binding and to locate the two effector-binding sites.Results: We report here the first crystal structure of RNR Rf in a reduced form. The structure shows that upon reduction of the redox active cysteines, the sulfur atom of Cys462 becomes deeply buried. The more accessible Cys225 moves to the former position of Cys462 making room for the substrate. In addition, the structures of R1 in complexes with effector, effector analog and effector plus substrate provide information about these binding sites. The substrate GDP binds in a cleft between two domains with its beta-phosphate bound to the N termini of two helices; the ribose forms hydrogen bonds to conserved residues. Binding of dTTP at the allosteric substrate specificity site stabilizes three loops close to the dimer interface and the active site, whereas the general allosteric binding site is positioned far from the active site.Conclusions: Binding of substrate at the active site of the enzyme is structurally regulated in two ways: binding of the correct substrate is regulated by the binding of allosteric effecters, and binding of the actual substrate occurs primarily when the active-site cysteines are reduced. One of the loops stabilized upon binding of dTTP participates in the formation of the substrate-binding site through direct interaction with the nucleotide base. The general allosteric effector site, located far from the active site, appears to regulate subunit interactions within the holoenzyme.