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

doi:10.1073/pnas.0407904101

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

^*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

Contributed by John Kuriyan, October 22, 2004

Abstract

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 ATPγS (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 ATPγS 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.

Footnotes

↵ ∥ To whom correspondence should be addressed. E-mail: kuriyan{at}berkeley.edu.
↵ ‡ S.L.K. and M.P. contributed equally to this work.
↵ § Present address: National Institute of Chemistry, Laboratory for Biosynthesis and Biotransformation, P.O. Box 660, Hajdrihova 19, 1001 Ljubljana, Slovenia.
Abbreviations: RFC, replication factor C; ITC, isothermal titration calorimetry; MD, molecular dynamics.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 1XXH and 1XXI).
Freely available online through the PNAS open access option.