Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 28 (12), 1792-802

Structural Basis for ADP-mediated Transcriptional Regulation by P1 and P7 ParA

Affiliations

Structural Basis for ADP-mediated Transcriptional Regulation by P1 and P7 ParA

Thomas D Dunham et al. EMBO J.

Abstract

The accurate segregation of DNA is essential for the faithful inheritance of genetic information. Segregation of the prototypical P1 plasmid par system requires two proteins, ParA and ParB, and a centromere. When bound to ATP, ParA mediates segregation by interacting with centromere-bound ParB, but when bound to ADP, ParA fulfils a different function: DNA-binding transcription autoregulation. The structure of ParA is unknown as is how distinct nucleotides arbitrate its different functions. To address these questions, we carried out structural and biochemical studies. Crystal structures show that ParA consists of an elongated N-terminal alpha-helix, which unexpectedly mediates dimerization, a winged-HTH and a Walker-box containing C-domain. Biochemical data confirm that apoParA forms dimers at physiological concentrations. Comparisons of four apoParA structures reveal a strikingly flexible dimer interface that allows ParA to adopt multiple conformations. The ParA-ADP structure shows that ADP-binding activates DNA binding using a bipartite mechanism. First, it locks in one specific dimer conformation, and second, it induces the folding of two DNA-binding basic motifs that we show are critical for operator binding.

Figures

Figure 1
Figure 1
Sequence homology of P1 and P7 ParA proteins and overall structure. (A) Sequence alignment of the P1 and P7 ParA proteins. Secondary structural elements are indicated over the sequences and the three structural regions are coloured yellow (N-terminal α-helical region), red (winged-HTH region) and cyan (Walker-box C-domain). Regions that fold into helices on ADP binding are indicated by pink cylinders. The HTH, Walker A, Walker A′, Walker B and ParA-specific regions are labelled. Residues mutated in the study are also labelled. (B) Ribbon diagram of the P7 apoParA dimer. The molecule is coloured coded as in (A). This figure and Figures 2, 3A–C, 4A–C, 5 and 6A were made with PyMOL (Delano, 2002).
Figure 2
Figure 2
Four structures of apoParA reveal that ParA is a conformationally flexible dimer. (A) Structure of the P7 apoParA C2221 dimer A. One subunit is coloured green and the other magenta. The magenta subunit is shown in the same orientation in (B–D). The α1–C-domain′ interaction is the only conserved dimer interaction. (B) Structure of the P7 apoParA C2221 dimer B. (C) Structure of the P7 apoParA P6222 dimer. (D) Structure of the P1 apoParA-domain-swapped dimer. The swapped regions are labelled.
Figure 3
Figure 3
The conserved ParA α1–C-domain′ dimer interface. (A) Superimposition of β8, α14, β9 and α15 of the P7 apoParA and the P1 apoParA structures, which results in a superimposition of α1′. α1 is coloured grey, whereas β8, α14, β9 and α15 are coloured green for the P7 apoParA (C2221 A dimer), blue for the P7 apoParA (C2221 B dimer), yellow for the P7 apoParA P6222 structure and salmon for the P1 apoParA structure. α15 of the P1 structure is from the other subunit due to domain swapping. (B) α1–C-domain′ contacts in the P7 apoParA structure. Shown as sticks are the residues mediating interactions. Residues Ala12 and Ala15 corresponding to Ala11 and Ala14 in P1 ParA are labelled in black. (C) α1–C-domain′ contacts in the P1 apoParA structure. (D) SEC-LS analysis of ParA oligomer state. For clarity, the portion of the chromatogram used for the analysis for the monomer and dimer states has been shown. Samples fall into two groups characterized by their molecular masses (mw) as calculated using ASTRA software; a dimeric state: P1 ParA, wt (jade; mw=8.86±0.02 104 g/mol), P7 ParA, wt (black; mw=8.73±0.07 104 g/mol), P1 ParA (T278W) (green; mw=1.00±0.01 105 g/mol), P1 ParA with C-terminal tag (light green; mw=9.16±0.82 104 g/mol) and ParA (A277Y) (grey; mw=1.00±0.01 105 g/mol); or monomeric state: ParA (Δnt20) (red; mw=4.99±0.03 104 g/mol), ParA (A14S) (blue; mw=5.14±0.11 104 g/mol) and ParA (A11S) (aqua; mw=6.27±0.05 104 g/mol). (E) SEC-LS carried out at the lower limit of detection showing ParA dimers. P1 ParA was measured at 50 μM (green), 25 μM (grey), 10 μM (black) and 5 μM (blue).
Figure 4
Figure 4
ParA–ADP structure: conformational locking and local refolding. (A) Comparison of apoParA C2221 A and ParA–ADP dimers. One subunit is coloured magenta and one green. The magenta subunits are shown in the same orientation for reference. The ADPs are shown as CPK. The regions that become folded on ADP binding are coloured black. (B) Superimposition of all corresponding Cα atoms of the two ParA–ADP dimers, space group P212121 (cyan) and P41212 (yellow), showing that, unlike the apo structures, ADP-binding locks in one dimer conformation. (C) Comparison of the apo ParA and ParA–ADP monomer structures. The monomers are shown in the same orientation and the regions that fold on ADP binding are coloured black and labelled in the ParA–ADP monomer. (D) Partial proteolysis assays. Each reaction mixture (10 μl) contained 3 μg of the indicated ParA and trypsin as indicated. ADP, when present, was at 2 mM. The mixtures were analysed by electrophoresis on 12% SDS–polyacrylamide gels. The trypsin:parA ratio (w/w) and the presence of ADP are indicated above each lane.
Figure 5
Figure 5
ADP-binding interactions: Walker motifs and a second ADP-binding pocket. (A) Overall structure of the P212121 ParA–ADP structure. The α1 helices and the Walker motifs, A, A′ and B are labelled. The Walker motifs are coloured green (Walker A), yellow (Walker A′) and dark blue (Walker B). The ParA-specific motif is cyan. The second ADP-binding site is coloured salmon and the ADP is shown as CPK. Inset to the upper right is a close-up view of the interactions between the two ParA subunits and the second (non-primary)-bound ADP molecule. (B) Close-up of the ‘primary' ADP-binding site and a 2.05 Å resolution FoFc map (magenta mesh), calculated after omitting the ADP, and contoured at 4.5σ. The nucleotide-binding motifs are coloured as in (A).
Figure 6
Figure 6
ADP-mediated folding of basic regions/ATP-induced polymer formation. (A) ParA–ADP–DNA model predicting the role of basic regions (coloured red) that fold and are stabilized on ADP binding in DNA binding. The ADP molecules are shown as blue sticks. (B) DNase I protection of parOP site. Shown are footprint experiments for wild type P1 ParA and ParA proteins in which basic motif residues, R351, S370, K375 and R378 were mutated to alanines. (C) Schematic of the proposed ADP/ATP switching mechanism mediating the dual roles of ParA. ParA is coloured according to Figure 1A. The dimer interface is shown to morph in between multiple conformations indicative of the plasticity of the apo dimer. ADP-binding locks in a specific dimer state active for DNA binding, whereas ATP binding is predicted to stabilize a different dimeric state that stimulates filament formation leading to plasmid separation.

Similar articles

See all similar articles

Cited by 27 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback