Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry.
Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry.
复制标题
DOI：
10.1093/nar/gkl407
复制
发表时间：
2006
影响因子：
14.9
通讯作者：
Friedhoff P
中科院分区：
文献类型：
--
作者：
Ahrends R;Kosinski J;Kirsch D;Manelyte L;Giron-Monzon L;Hummerich L;Schulz O;Spengler B;Friedhoff P
为了研究DNA错配修复系统中的蛋白质-蛋白质相互作用位点，我们开发了一种交联/质谱技术，采用了一种市售的三功能交联剂，该交联剂具有硫醇特异性甲乙硫磺酸基团、光活化二苯甲酮片段和生物素亲和标签。XACM方法结合了光交联(X)，交联混合物的溶出，通过生物素处理的亲和纯化(A)，交联产物的化学编码(C)，然后是MALDI-TOF质谱(M)。我们利用同型二聚体DNA错配修复蛋白MutL的单半胱氨酸变体说明了该方法的可行性。此外，我们成功地应用该方法鉴定了单半胱氨酸MutH变体A223C与其激活蛋白MutL之间形成的光交联，在c端螺旋上标记了三功能交联剂。鉴定的交联多肽映射到MutL c端二聚化结构域的一个保守表面斑块。这些观察结果被额外的突变和化学交联研究证实。我们的结果揭示了MutL全酶和MutH-MutL-DNA复合物的潜在结构。
To investigate protein–protein interaction sites in the DNA mismatch repair system we developed a crosslinking/mass spectrometry technique employing a commercially available trifunctional crosslinker with a thiol-specific methanethiosulfonate group, a photoactivatable benzophenone moiety and a biotin affinity tag. The XACM approach combines photocrosslinking (X), in-solution digestion of the crosslinked mixtures, affinity purification via the biotin handle (A), chemical coding of the crosslinked products (C) followed by MALDI-TOF mass spectrometry (M). We illustrate the feasibility of the method using a single-cysteine variant of the homodimeric DNA mismatch repair protein MutL. Moreover, we successfully applied this method to identify the photocrosslink formed between the single-cysteine MutH variant A223C, labeled with the trifunctional crosslinker in the C-terminal helix and its activator protein MutL. The identified crosslinked MutL-peptide maps to a conserved surface patch of the MutL C-terminal dimerization domain. These observations are substantiated by additional mutational and chemical crosslinking studies. Our results shed light on the potential structures of the MutL holoenzyme and the MutH–MutL–DNA complex.