Clamp-loader complexes are heteropentameric AAA ؉ ATPases that load sliding clamps onto DNA. The structure of the nucleotide-free Escherichia 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 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 [replication factor C (RFC)] revealed a 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 describe crystal structures of the E. coli clamp-loader complex bound to the ATP analog ATP␥S (at a resolution of 3.5 Å) and ADP (at a resolution of 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 ATP␥S or ADP in these 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 described 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.AAA ϩ ATPase ͉ clamp loader ͉ DNA polymerase III ͉ DNA replication ͉ replication factor C C hromosomal replicases in all of the branches of life achieve high processivity by tethering their catalytic subunits to sliding DNA clamps (1-3). The closed circular sliding clamps are loaded onto DNA by ATP-dependent clamp-loader complexes (4), which are conserved heteropentameric ATPase assemblies. Most sliding clamps are stable closed rings in solution, although recent studies suggest that the clamp in T4 bacteriophage may be open in solution and that the role of the clamp loader might be to stabilize the open form and guide it to DNA (5). The subunits of the clamp-loader complexes consist of three conserved domains, with the first two being closely related to the nucleotide-binding domains of AAA ϩ ATPases (6-10). The AAA ϩ domains of the clamp loader are suspended loosely from a circular helical collar that is formed by the third (C-terminal) domains.ATP binding to the clamp loader triggers a conformational change that allows the complex to bind to and open the clamp and load it onto DNA (11, 12). In an intriguing control mechanism, the interaction with DNA stimulates the otherwise suppressed ATPase activity of the clamp loader, leading to ATP hydrolysis (11, 13) and separation of the clamp-loader complex from the DNA-loaded clamp. How ATP binding is coupled to loading of the clamp on DNA is poorly understood, and eluc...

Sliding clamps are loaded onto DNA by ATP-driven clamp loader complexes. The structure of the E. coli clamp loader in a nucleotide-free state has been determined previously. We now report crystal structures of a truncated form of the isolated gamma-ATPase subunit, gamma(1-243), of the E. coli clamp loader, in nucleotide-free and bound forms. The gamma subunit adopts a defined conformation when empty, in which the nucleotide binding site is blocked. The binding of either ATPgammaS or ADP, which are shown to bind with equal affinity to gamma(1-243), induces a change in the relative orientation of the two domains such that nucleotides can be accommodated. This change would break one of the gamma:gamma interfaces seen in the empty clamp loader complex, and may represent one step in the activation process.