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首页> 外文期刊>Journal of Molecular Biology >Dynamics and DNA substrate recognition by the catalytic domain of lambda integrase.
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Dynamics and DNA substrate recognition by the catalytic domain of lambda integrase.

机译：动态和DNA底物识别的lambda整合酶的催化域。

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摘要

Bacteriophage lambda integrase (lambda-Int) is the prototypical member of a large family of enzymes that catalyze site-specific DNA recombination via the formation of a Holliday junction intermediate. DNA strand cleavage by lambda-Int is mediated by nucleophilic attack on the scissile phosphate by a conserved tyrosine residue, forming an intermediate with the enzyme covalently attached to the 3'-end of the cleaved strand via a phosphotyrosine linkage. The crystal structure of the catalytic domain of lambda-Int (C170) obtained in the absence of DNA revealed the tyrosine nucleophile at the protein's C terminus to be located on a beta-hairpin far from the other conserved catalytic residues and adjacent to a disordered loop. This observation suggested that a conformational change in the C terminus of the protein was required to generate the active site in cis, or alternatively, that the active site could be completed in trans by donation of the tyrosine nucleophile from a neighboring molecule in the recombining synapse. We used NMR spectroscopy together with limited proteolysis to examine the dynamics of the lambda-Int catalytic domain in the presence and absence of DNA half-site substrates with the goal of characterizing the expected conformational change. Although the C terminus is indeed flexible in the absence of DNA, we find that conformational changes in the tyrosine-containing beta-hairpin are not coupled to DNA binding. To gain structural insights into C170/DNA complexes, we took advantage of mechanistic conservation with Cre and Flp recombinases to model C170 in half-site and tetrameric Holliday junction complexes. Although the models do not reveal the nature of the conformational change required for cis cleavage, they are consistent with much of the available experimental data and provide new insights into the how trans complementation could be accommodated.
机译：噬菌体λ整合酶（lambda-Int）是一大类酶的原型成员，这些酶通过形成霍利迪连接中间物催化位点特异性DNA重组。 Lambda-Int切割DNA链的过程是通过保守的酪氨酸残基对易裂磷酸盐的亲核攻击介导的，形成了一种中间体，该酶与酶通过磷酸酪氨酸键共价连接到切割链的3'末端。在没有DNA的情况下获得的Lambda-Int（C170）催化结构域的晶体结构表明，该蛋白C末端的酪氨酸亲核试剂位于一个β-发夹结构上，与其他保守的催化残基相距较远，且与无序环相邻。该观察结果提示，需要在蛋白质的C末端进行构象变化才能在顺式中生成活性位点，或者可以通过从重组突触中邻近分子的酪氨酸亲核捐赠而反过来完成该活性位点。 。我们使用NMR光谱结合有限的蛋白水解作用来研究存在和不存在DNA半位底物的情况下lambda-Int催化域的动力学，目的是表征预期的构象变化。尽管在没有DNA的情况下C末端确实具有柔性，但我们发现含酪氨酸的β-发夹结构改变并不与DNA结合有关。为了获得有关C170 / DNA复合物的结构见解，我们利用Cre和Flp重组酶的机械保守作用在半位点和四聚体霍利迪连接复合物中模拟C170。尽管模型没有揭示顺式切割所需的构象变化的性质，但它们与许多可用的实验数据一致，并为如何适应反式互补提供了新的见识。

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