title: Native mass spectrometry provides direct evidence for DNA mismatch-induced regulation of asymmetric nucleotide binding in mismatch repair protein MutS.
abstract: The DNA mismatch repair protein MutS recognizes mispaired bases in DNA and initiates repair in an ATP-dependent manner. Understanding of the allosteric coupling between DNA mismatch recognition and two asymmetric nucleotide binding sites at opposing sides of the MutS dimer requires identification of the relevant MutS.mmDNA.nucleotide species. Here, we use native mass spectrometry to detect simultaneous DNA mismatch binding and asymmetric nucleotide binding to Escherichia coli MutS. To resolve the small differences between macromolecular species bound to different nucleotides, we developed a likelihood based algorithm capable to deconvolute the observed spectra into individual peaks. The obtained mass resolution resolves simultaneous binding of ADP and AMP.PNP to this ABC ATPase in the absence of DNA. Mismatched DNA regulates the asymmetry in the ATPase sites; we observe a stable DNA-bound state containing a single AMP.PNP cofactor. This is the first direct evidence for such a postulated mismatch repair intermediate, and showcases the potential of native MS analysis in detecting mechanistically relevant reaction intermediates.
authors: Biomolecular Mass Spectrometry and Proteomics Group, and Center for Biomedical Genetics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
authors: Maria Chiara
authors: MC
authors: Monti
authors: Serge X
authors: SX
authors: Cohen
authors: Alexander
authors: A
authors: Fish
authors: Herrie H K
authors: HH
authors: Winterwerp
authors: Arjan
authors: A
authors: Barendregt
authors: Peter
authors: P
authors: Friedhoff
authors: Anastassis
authors: A
authors: Perrakis
authors: Albert J R
authors: AJ
authors: Heck
authors: Titia K
authors: TK
authors: Sixma
authors: Robert H H
authors: RH
authors: van den Heuvel
authors: Joyce H G
authors: JH
authors: Lebbink
mesh_terms: D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000465:Algorithms; D020137:Base Pair Mismatch; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000378:metabolism*; D051719:MutS DNA Mismatch-Binding Protein / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D029968:Escherichia coli Proteins; D009711:Nucleotides; D000266:Adenylyl Imidodiphosphate; D004247:DNA; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli