Ordered ATP hydrolysis in the gamma complex clamp loader AAA+ machine.
The gamma complex couples ATP hydrolysis to the loading of beta sliding clamps onto DNA for processive replication. The gamma complex structure shows that the clamp loader subunits are arranged as a circular heteropentamer. The three gamma motor subunits bind ATP, the delta wrench opens the beta ring, and the delta' stator modulates the delta-beta interaction. Neither delta nor delta' bind ATP. This report demonstrates that the delta' stator contributes a catalytic arginine for hydrolysis of ATP bound to the adjacent gamma(1) subunit. Thus, the delta' stator contributes to the motor function of the gamma trimer. Mutation of arginine 169 of gamma, which removes the catalytic arginines from only the gamma(2) and gamma(3) ATP sites, abolishes ATPase activity even though ATP site 1 is intact and all three sites are filled. This result implies that hydrolysis of the three ATP molecules occurs in a particular order, the reverse of ATP binding, where ATP in site 1 is not hydrolyzed until ATP in sites 2 and/or 3 is hydrolyzed. Implications of these results to clamp loaders of other systems are discussed.
Johnson
Aaron
A
Howard Hughes Medical Institute and the Rockefeller University, New York, New York 10021, USA. johnsoa@mail.rockefeller.edu
O'Donnell
Mike
M
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism*; D020816:Amino Acid Motifs; D001120:Arginine / Q000737:chemistry; D020134:Catalytic Domain; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D020035:Holoenzymes / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D009154:Mutation; D013997:Time Factors
D029968:Escherichia coli Proteins; D020035:Holoenzymes; D000255:Adenosine Triphosphate; D004247:DNA; D001120:Arginine; D000251:Adenosine Triphosphatases