Mapping protein-protein interactions between MutL and MutH by cross-linking.
Strand discrimination in Escherichia coli DNA mismatch repair requires the activation of the endonuclease MutH by MutL. There is evidence that MutH binds to the N-terminal domain of MutL in an ATP-dependent manner; however, the interaction sites and the molecular mechanism of MutH activation have not yet been determined. We used a combination of site-directed mutagenesis and site-specific cross-linking to identify protein interaction sites between the proteins MutH and MutL. Unique cysteine residues were introduced in cysteine-free variants of MutH and MutL. The introduced cysteines were modified with the cross-linking reagent 4-maleimidobenzophenone. Photoactivation resulted in cross-links verified by mass spectrometry of some of the single cysteine variants to their respective Cys-free partner proteins. Moreover, we mapped the site of interaction by cross-linking different combinations of single cysteine MutH and MutL variants with thiol-specific homobifunctional cross-linkers of varying length. These results were used to model the MutH.MutL complex and to explain the ATP dependence of this interaction.
Giron-Monzon
Luis
L
Institut für Biochemie (FB 08), Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany.
Manelyte
Laura
L
Ahrends
Robert
R
Kirsch
Dieter
D
Spengler
Bernhard
B
Friedhoff
Peter
P
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D020137:Base Pair Mismatch; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005816:Genetic Complementation Test; D006639:Histidine / Q000737:chemistry; D008027:Light; D013058:Mass Spectrometry; D008956:Models, Chemical; D008958:Models, Molecular; D015233:Models, Statistical; D008969:Molecular Sequence Data; D000070956:MutL Proteins; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014357:Trypsin / Q000494:pharmacology
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C074770:MutL protein, E coli; D006639:Histidine; D000255:Adenosine Triphosphate; D004706:Endodeoxyribonucleases; C059682:methyl-directed mismatch repair protein, E coli; D014357:Trypsin; D000251:Adenosine Triphosphatases; D000070956:MutL Proteins; D045643:DNA Repair Enzymes; D003545:Cysteine