title: Integrative modelling coupled with ion mobility mass spectrometry reveals structural features of the clamp loader in complex with single-stranded DNA binding protein.
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-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.
authors.affiliation: Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK. Electronic address: argyris.politis@chem.ox.ac.uk.
authors.forename: Argyris
authors.initials: A
authors.lastname: Politis
authors.forename: Ah Young
authors.initials: AY
authors.lastname: Park
authors.forename: Zoe
authors.initials: Z
authors.lastname: Hall
authors.forename: Brandon T
authors.initials: BT
authors.lastname: Ruotolo
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
mesh_terms: D004258:DNA Polymerase III / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D021621:Imaging, Three-Dimensional; D013058:Mass Spectrometry; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009097:Multienzyme Complexes; D004258:DNA Polymerase III
keywords: ATD; CCS; CG; DNA replication; IM; MS; PA; PDB; Protein Data Bank; USR; arrival time distribution; clamp loader; coarse grained; collision cross-section; integrative modelling; ion mobility; ion mobility-mass spectrometry; mass spectrometry; projection approximation; single-stranded DNA; single-stranded DNA binding protein (SSB); ssDNA; ultrafast shape recognition