Structural analysis of the inactive state of the Escherichia coli DNA polymerase clamp-loader complex

Steven L. Kazmirski*†‡, Marjetka Podobnik*‡§, Tanya F. Weitze*†, Mike O’Donnell¶, and John Kuriyan*†

*Department of Molecular and Cell Biology and Department of Chemistry, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720; †Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and ¶Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021

Article's page in PNAS - Local copy

Abstract / Figures from the paper / PDB Coordinates / Supplemental Materials


Clamp loader complexes are hetero-pentameric AAA+ ATPases that load sliding clamps onto DNA. The structure of the nucleotide-free E. coli clamp loader had been determined previously, and led to the proposal that the clamp loader cycles between an inactive state, in which the ATPase domains form a closed ring, and an ATP bound active state that opens up to form a “C” shape. The crystal structure was interpreted as being closer to the active state than the inactive state. The crystal structure of a nucleotide bound eukaryotic clamp loader (RFC) revealed a quite different and more tightly packed spiral organization of the ATPase domains, raising questions about the significance of the conformation seen earlier for the bacterial clamp loader. We now describe crystal structures of the E. coli clamp loader complex bound to the ATP analogs ATPγS (at 3.5 Å resolution) and ADP (at 4.1 Å). These structures are similar to that of the nucleotide-free clamp loader complex. Only two of the three functional ATP binding sites are occupied by ATPγS or ADP in these new structures, and the bound nucleotides make no interfacial contacts in the complex. These results, along with data from isothermal titration calorimetry, molecular dynamics simulations and comparison with the RFC structure suggest that the more open form of the E. coli clamp loader seen earlier and in the present work corresponds to a stable inactive state of the clamp loader in which the ATPase domains are prevented from engaging the clamp in the highly cooperative manner seen in the fully ATP-loaded RFC-clamp structure.


Figure 1. Structure of the clamp loader complex. (A) Two views of the structure of the ATPγS complex are shown. The A subunit is δ, which is primarily responsible for opening the β clamp. The ATP binding subunits, γ, are labeled B, C and D. The E subunit is δ’. (B) Schematic diagram showing a suggested mechanism for β clamp binding by the clamp loader complex. Illustrations of protein structures were generated using PyMOL (43).

Figure2. Ribbon representation of the nucleotide binding sites in the three γ subunits. (A) Structure of the three γ subunits. ATPγS is observed in γB and γD and a phosphate ion is bound to γC. (B) Electron density in a map calculated with amplitudes (|FO| - |FC|) and phases from a model in which nucleotide is omitted, for the ATPγS complex, site γD (blue mesh = 3σ, red mesh = 5σ). Electron density in γB is shown in (C).

Figure 3. Isothermal titration calorimetry (ITC) binding curves for the clamp loader complex in combination with various nucleotides.

Figure 4. Results of the molecular dynamics simulation. The number of atomic overlaps (defined as the number of interatomic contacts with distances less than 2.5 Å) between the β clamp and clamp loader are displayed for the conformations of clamp loader adopted during the MD simulation (red). Instantaneous structures of the clamp loader from the trajectory were individually docked onto the clamp such that domain I of δA matched δ in the crystal structure of the δ:β complex (14). Also shown is the distance (green) between the centers of mass of domain I of δA and δ’E.

Supplemental Figure 1. Surface representations of the nucleotide-free and nucleotide bound crystal structures of the clamp loader complex. In black are residues that are involved in crystal contacts in the lattice. Each of the crystal structures has a unique pattern of crystal contacts.

Supplemental Figure 2. Superposition of domain I of γC from the nucleotide-free and ATPγS bound crystal structures. γC is shown in red. γD for the ATPγS bound clamp loader (green) shows very little change in position with respect to γD of the nucleotide-free clamp loader (gray). This suggests that binding of ATP alone is not responsible for opening the ATP binding site on γC and other factor such as the β clamp may be necessary to cause the conformational change.

Supplemental Figure 3. Root mean square deviation in Cα positions with respect to the crystal structure for each of the domains of the clamp loader crystal structure: (A) domain I, (B) domain II, (C) domain III. For domain I the N-terminal 20 residues and the zinc-finger have been removed. From domain III, the C-terminal five residues have been removed. The colors for the subunits are δA magenta, γB blue, γC red, γD green, δ’E orange.

PDB Coordinates

1XXH - Atpgs Bound E. Coli Clamp Loader Complex
1XXI - ADP Bound E. Coli Clamp Loader Complex

Supplemental Material