Integrative modelling coupled with ion mobility mass spectrometry reveals structural features of the clamp loader in complex with single-stranded DNA binding protein

Argyris Politis*, Ah Young Park, Zoe Hall, Brandon T. Ruotolo, Carol V. Robinson

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

50 Citations (Scopus)

Abstract

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 (254 kDa). 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 (108 kDa) and the clamp loader-SSB4 (340 kDa) 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-SSB 4 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. 

Original languageEnglish
Pages (from-to)4790-4801
Number of pages12
JournalJournal of Molecular Biology
Volume425
Issue number23
DOIs
Publication statusPublished - 29 Nov 2013

Keywords

  • clamp loader
  • DNA replication
  • integrative modelling
  • ion mobility-mass spectrometry
  • single-stranded DNA binding protein (SSB)

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