Structural characterisation of the complete cycle of sliding clamp loading in Escherichia coli

Abstract

Ring-shaped DNA sliding clamps are essential for DNA replication and genome maintenance. Clamps need to be opened and chaperoned onto DNA by clamp loader complexes (CLCs). Detailed understanding of the mechanisms by which CLCs open and place clamps around DNA remains incomplete. Here, we present a series of six structures of the Escherichia coli CLC bound to an open or closed clamp prior to and after binding to a primer-template DNA, representing the most significant intermediates in the clamp loading process. We show that the ATP-bound CLC first binds to a clamp, then constricts to hold onto it. The CLC then expands to open the clamp with a gap large enough for double-stranded DNA to enter. Upon binding to DNA, the CLC constricts slightly, allowing clamp closing around DNA. These structures provide critical high-resolution snapshots of clamp loading by the E. coli CLC, revealing how the molecular machine works.

Fig. 1. The clamp loading process and its participants.

a Sliding clamps from E. coli (PDB 1MMI (E. coli β₂ clamp)), phage T4 (1CZD (T4 gp45 clamp)) and human (1VYJ (human PCNA clamp)) are closed rings containing six structurally similar domains. The E. coli clamp is dimeric, while T4 and eukaryotic clamps are trimers. The N- and C-termini of the proteins are labelled as N and C, respectively. b Views of the clamp loader complex (CLC) from collar to AAA+ domain (top view, top panel) and front to back (side view, bottom panel). The E. coli CLC core subunits, δ, γ1, γ2, γ3 and δ’ (coloured salmon, medium pink, medium blue, deep sky blue and plum, respectively), are arranged anticlockwise in canonical positions A−E. ATP molecules (or analogues) are bound between Domains I and II of γ subunits at the γ1−γ2, γ2−γ3 and γ3−δ’ interfaces. c Domain organisation of E. coli CLC core subunits. All subunits contain AAA+ ATPase domains (I and II) and a Collar domain (III). The τ subunits contain an additional Domain IV that interacts with the DnaB helicase and Domain V that binds tightly to the Pol III α subunit. d Schematic of clamp loading in E. coli. To load a clamp, the ATP-bound CLC (state A) first binds to a β₂ clamp (B)^,– and opens it (C)^–. Alternatively, the β₂ clamp may open spontaneously and the CLC trap it in an open state (C). Then primer-template (p/t) DNA passes through the gap of the clamp and binds in the chamber of the CLC (D) either via its ssDNA or dsDNA portion depending on the size of the gap in β₂. Upon binding and recognition of a p/t DNA junction, ATP hydrolysis occurs as the β₂ clamp closes on DNA (E)^, and the CLC is ejected leaving the loaded clamp on p/t DNA for binding of the Pol III αεθ core to the clamp (F)^,. Structures already reported for the E. coli CLC are in colours (states A and D without β₂); structures only available for other organisms are in grey (B, yeast and human; C, yeast; D and E, phage T4 and yeast).

Fig. 2. Clamp binding by the E. coli clamp loader complex (CLC) is highly dynamic.

a Cryo-EM map of E. coli CLC bound to a closed β₂ clamp (CLC•β₂^closed) in the absence of p/t DNA. Cryo-EM densities corresponding to the δ, γ1, γ2, γ3 and δ’ subunits of the CLC are coloured salmon, medium purple, medium blue, deep sky blue and plum, respectively. The β-I subunit of β₂ is coloured yellow and β-II orange. The colouring scheme is kept throughout unless stated otherwise. b The AAA+ modules (shown without the Collar domain) of γ1 and δ in CLC•β₂^closed encroach into the gap between δ and δ’. Structures are aligned on γ2, which is barely perturbed among aligned structures. The AAA+ domains of the free δγ₃δ’ complex (PDB 1XXH (ATPγS bound E. coli clamp loader complex)) are disorganised, while those of CLC•β₂^closed and CLC•β₂^open are more regularly packed. c CLC−β interfaces on β₂ (left) and CLC (right) of CLC•β₂^closed. CLC−β interactions are mediated by peptides of the CLC subunits that are located on an α-helix and the following loop in their Domains I, which are presented as white ribbons on the surfaces of β₂. β₂ and the CLC are presented side-by-side by separating and turning them 90° in opposite directions. Buried areas are coloured as for their interacting counterparts; those coloured in salmon (δ) and deep sky blue (γ3) are in the two symmetry-related canonical peptide binding sites of β₂. d Dynamics of clamp binding revealed by 3D variability analysis (3DVA). The resolution at which eigenvectors of 3DVA are filtered was set to 6 Å. Volumes of the first (brick red) and last (blue) frames of principal component_001 of 3DVA that show the most significant and biologically relevant conformational changes are presented.

Fig. 3. The E. coli clamp loader complex (CLC) opens β₂ by crab claw-like motions.

a Cryo-EM map of CLC bound to an open clamp (CLC•β₂^open) coloured as in Fig. 2, and with the highest confidence regions of ψ(χ) in orange red. Density next to Domain I of δ’, presumed to be from the unstructured C-terminal region of a γ/τ subunit is coloured white and indicated by a red asterisk. Top right, close-up view showing the unassigned EM density near the N-terminus of δ’. A moving window of 8-residue peptides selected from the C-terminal flexible region of γ were docked into δ’ and scored against the density. The best scoring peptide corresponded to the moderately-conserved γ residues Val403−Thr409. This assignment is yet to be verified experimentally. Bottom right, unsharpened EM density of the CLC-binding peptide of ψ (Thr2–Glu28) that bridges all three γ subunits. b Interface areas between AAA+ modules increase significantly in the transition from CLC•β₂^closed to CLC•β₂^open. Source data are provided as a Source Data file. c Conformational changes of the AAA+ modules during transition from CLC•β₂^closed to CLC•β₂^open. While the AAA+ modules of γ2, γ3 and δ’ barely move, those of γ1 and δ swing outwards, resulting in expansion of the CLC and opening of β₂. Structures were superimposed by aligning the γ2 subunit using MatchMaker in Chimera. Arrows indicate movements of individual subunits. d Orthogonal views of conformational changes in β₂ during transition from CLC•β₂^closed to CLC•β₂^open. The clamp remains mostly flat after opening. Most obvious motions are on the β-I subunit with Domain I rotating around Domain III of β-II and Domains II–III around Domain I. e Conformational changes in the final stage of clamp opening revealed by 3D variability analysis. The resolution at which eigenvectors of 3DVA are filtered was set to 5 Å. Volumes of the first and last frames of principal component_000 of 3DVA that illustrate the most significant and biologically relevant conformational changes are presented.

Fig. 4. All clamp loader complex (CLC) core subunits bind to domains of the open clamp in a similar fashion.

a CLC−β interfaces on β₂ and the CLC of CLC•β₂^open. CLC−β interactions are mediated by peptides of the CLC subunits that are presented on the surfaces of β₂ with their secondary structures depicted (white). Interface areas are coloured as their interacting counterparts. Peptide-binding surfaces of the δ, γ3 and δ’ subunits on β₂ are more extensive than those of γ1 and γ2. b Close-up views of clamp-binding peptides (ribbons with side chains as sticks) in binding pockets of β₂ (surface representation). Sequences of peptides involving in clamp binding are shown above. Important residues, Leu73 and Phe74 of δ and Tyr113 of γ subunits that anchor the peptides in the binding sites, are labelled. The surface of β₂ is coloured by hydrophobicity with hydrophobic areas in orange, neutral areas in white and hydrophilic areas in blue.

Fig. 5. The β₂ clamp partially or completely closes on loading onto primer-template DNA.

a Cryo-EM map of the clamp loader complex (CLC) bound to a partially open clamp and p/t DNA (CLC•DNA•β₂^open). Densities corresponding to individual subunits are coloured as in Fig. 2. Density of the template DNA strand is coloured gold and the primer stand pink. Grey density at the top right corner belongs to SSB. b Cryo-EM map of the CLC bound to a closed clamp and p/t DNA (CLC•DNA•β₂^closed). c Cryo-EM map of the CLC bound to p/t DNA (CLC•DNA). d The CLC constricts slightly on binding of p/t DNA. The AAA+ modules of CLC•β₂^open (coloured grey) and CLC•DNA•β₂^open are superimposed to show small inward movements of the CLC upon p/t DNA binding. Arrows point to the tips of the δ subunits. e Orthogonal views showing β₂ partially closes on p/t DNA and also becomes spiral. β₂ of CLC•β₂^open in the left pane is coloured grey and CLC•DNA•β₂^open yellow and orange. Most movement occurs in the β-I subunit. β₂ of CLC•DNA•β₂^open becomes lock washer-like (right). f,g CLC−β interfaces on β₂ and CLC of CLC•DNA•β₂^open and CLC•DNA•β₂^closed, respectively, showing that the latter has much reduced contacts. h Mg•ADP•AlF₄⁻ molecules are present in all three ATP-binding sites of CLC•DNA•β₂^closed and the arginine finger residues: R169 of γ2 and γ3 and R158 of δ’, coordinate the AlF₄⁻ groups that mimic the γ-phosphates of ATP. Mg²⁺ (spheres) and AlF₄^– ions are coloured in shades of green and EM densities of Mg•ADP•AlF₄⁻ are shown as mesh. Hydrogen bonds between ADP•AlF₄⁻ and residues of the CLC are shown as red lines.

Fig. 6. Structural views of clamp loading in E. coli.

Schematic of the clamp loading pathway in E. coli. To load a clamp, the ATP-bound clamp loader complex (CLC) (state A) first binds to it via the δ subunit. The CLC then constricts, holds onto the clamp by simultaneous binding of the δ, γ1, γ3 and δ’ subunits (B). The CLC next expands and opens the clamp (C). The CLC−open clamp complex then directly binds to a p/t DNA. The CLC slightly constricts, and the clamp first partly closes (D) before complete closure around the p/t DNA junction (E). ATP hydrolysis, which may occur at any stage after p/t junction recognition, is presumed to accelerate clamp closing and trigger ejection of the CLC to allow a DNA polymerase to bind to the loaded clamp for DNA synthesis.