Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry

被引:36
作者
Ahrends, Robert
Kosinski, Jan
Kirsch, Dieter
Manelyte, Laura
Giron-Monzon, Luis
Hummerich, Lars
Schulz, Oliver
Spengler, Bernhard
Friedhoff, Peter [1 ]
机构
[1] Univ Giessen, Inst Biochem FB08, D-35392 Giessen, Germany
[2] Humboldt Univ, Inst Chem, D-12489 Berlin, Germany
[3] Int Inst Mol & Cell Biol, Lab Bioinformat & Prot Engn, PL-02109 Warsaw, Poland
[4] Univ Giessen, Inst Anorgan & Analyt Chem FB 08, D-35392 Giessen, Germany
关键词
D O I
10.1093/nar/gkl407
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 photocrossfinking (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.
引用
收藏
页码:3169 / 3180
页数:12
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