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Integrative Modelling Coupled with Ion Mobility Mass Spectrometry Reveals Structural Features of the Clamp Loader in Complex with Single-Stranded DNA Binding Protein
- Politis, Argyris, Park, Ah Young, Hall, Zoe, Ruotolo, Brandon T., Robinson, Carol V.
- Journal of Molecular Biology 2013 v.425 pp. 4790-4801
- DNA-binding proteins, DNA-directed DNA polymerase, Escherichia coli, chromosomes, mass spectrometry, models, single-stranded DNA
- DNA polymerase III, a decameric 420-kDa assembly, simultaneously replicates both strands of the chromosome in Escherichia coli. A subassembly of this holoenzyme, the seven-subunit clamp loader complex, is responsible for loading the sliding clamp (β2) onto DNA. Here, we use structural information derived from ion mobility mass spectrometry (IM-MS) to build three-dimensional models of one form of the full clamp loader complex, γ3δδ′ψχ (254kDa). By probing the interaction between the clamp loader and a single-stranded DNA (ssDNA) binding protein (SSB4) and by identifying two distinct conformational states, with and without ssDNA, we assemble models of ψχ–SSB4 (108kDa) and the clamp loader–SSB4 (340kDa) consistent with IM data. A significant increase in measured collision cross-section (~10%) of the clamp loader–SSB4 complex upon DNA binding suggests large conformational rearrangements. This DNA bound conformation represents the active state and, along with the presence of ψχ, stabilises the clamp loader–SSB4 complex. Overall, this study of a large heteromeric complex analysed by IM-MS, coupled with integrative modelling, highlights the potential of such an approach to reveal structural features of previously unknown complexes of high biological importance.