Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry.
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.
Ahrends
Robert
R
Institut für Biochemie (FB 08), Justus-Liebig-Universität, D-35392 Giessen, Germany.
Kosinski
Jan
J
Kirsch
Dieter
D
Manelyte
Laura
L
Giron-Monzon
Luis
L
Hummerich
Lars
L
Schulz
Oliver
O
Spengler
Bernhard
B
Friedhoff
Peter
P
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001577:Benzophenones / Q000737:chemistry; D001665:Binding Sites; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000235:genetics; D045643:DNA Repair Enzymes / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D008027:Light; D008301:Maleimides / Q000737:chemistry; D000070956:MutL Proteins; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019809:Streptavidin / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry
D001577:Benzophenones; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008301:Maleimides; C074770:MutL protein, E coli; D010455:Peptides; D013438:Sulfhydryl Compounds; D019809:Streptavidin; C046109:benzophenone-4-maleimide; D004706:Endodeoxyribonucleases; C059682:methyl-directed mismatch repair protein, E coli; D010447:Peptide Hydrolases; D000251:Adenosine Triphosphatases; D000070956:MutL Proteins; D045643:DNA Repair Enzymes; D003545:Cysteine