Analysis of the LAGLIDADG interface of the monomeric homing endonuclease I-DmoI.
Analysis of the LAGLIDADG interface of the monomeric homing endonuclease I-DmoI.
复制标题
单体归巢核酸内切酶 I-DmoI 的 LAGLIDADG 界面分析。
DOI：
10.1093/nar/gkh618
复制
发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Belfort,Marlene
中科院分区：
文献类型：
--
作者：
Silva,GeorgeH;Belfort,Marlene
LAGLIDADG核酸内切酶家族的一般结构折叠由两个相似的α/β结构域（αββαββα）组成，它们组装为同源二聚体或单体，其中结构域通过假二重对称性相关。这种对称性的中心是紧密排列的LAGLIDADG双螺旋束，分别形成单基序或双基序蛋白质结构域之间的主要分子间或分子内接触区域。在这项工作中，我们进一步研究了LAGLIDADG残基参与螺旋-螺旋相互作用的作用。通过将来自同二聚体I-CreI的界面残基接枝到单体I-DmoI中的相应位置来探索LAGLIDADG螺旋相互作用的可重复性。产生的LAGLIDADG交换突变体是部分活性的，更喜欢切口dsDNA，而不是使常规的双链断裂。突变的LAGLIDADG区域内的一系列部分回复突变体显示出不同程度地恢复切割活性，导致一种比野生型I-DmoI更有活性的I-DmoI突变体。这些突变体中的一些的表型是调和的基础上发现跨膜蛋白的GxxxG螺旋相互作用相似。此外，还产生了I-DmoI的分裂变体，证明I-DmoI的LAGLIDADG螺旋能够形成并维持蛋白质-蛋白质界面，从而产生活性异源二聚体。
The general structural fold of the LAGLIDADG endonuclease family consists of two similar α/β domains (αββαββα) that assemble either as homodimers or monomers with the domains related by pseudo‐two‐fold symmetry. At the center of this symmetry is the closely packed LAGLIDADG two‐helix bundle that forms the main inter‐ or intra‐molecular contact region between the domains of single‐ or double‐motif proteins, respectively. In this work, we further examine the role of the LAGLIDADG residues involved in the helix–helix interaction. The interchangeability of the LAGLIDADG helix interaction was explored by grafting interfacial residues from the homodimeric I‐CreI into the corresponding positions in the monomeric I‐DmoI. The resulting LAGLIDADG exchange mutant is partially active, preferring to nick dsDNA rather than making the customary double‐strand break. A series of partial revertants within the mutated LAGLIDADG region are shown to restore cleavage activity to varying degrees resulting in one I‐DmoI mutant that is more active than wild‐type I‐DmoI. The phenotype of some of these mutants was reconciled on the basis of similarity to the GxxxG helix interaction found in transmembrane proteins. Additionally, a split variant of I‐DmoI was created, demonstrating that the LAGLIDADG helices of I‐DmoI are capable of forming and maintaining the protein–protein interfacein transto create an active heterodimer.