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-Structure paper
Title | Autoinhibition of a clamp-loader ATPase revealed by deep mutagenesis and cryo-EM. |
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Journal, issue, pages | Nat Struct Mol Biol, Vol. 31, Issue 3, Page 424-435, Year 2024 |
Publish date | Jan 4, 2024 |
Authors | Kendra Marcus / Yongjian Huang / Subu Subramanian / Christine L Gee / Kent Gorday / Sam Ghaffari-Kashani / Xiao Ran Luo / Lisa Zheng / Michael O'Donnell / Sriram Subramaniam / John Kuriyan / |
PubMed Abstract | Clamp loaders are AAA+ ATPases that facilitate high-speed DNA replication. In eukaryotic and bacteriophage clamp loaders, ATP hydrolysis requires interactions between aspartate residues in one ...Clamp loaders are AAA+ ATPases that facilitate high-speed DNA replication. In eukaryotic and bacteriophage clamp loaders, ATP hydrolysis requires interactions between aspartate residues in one protomer, present in conserved 'DEAD-box' motifs, and arginine residues in adjacent protomers. We show that functional defects resulting from a DEAD-box mutation in the T4 bacteriophage clamp loader can be compensated by widely distributed single mutations in the ATPase domain. Using cryo-EM, we discovered an unsuspected inactive conformation of the clamp loader, in which DNA binding is blocked and the catalytic sites are disassembled. Mutations that restore function map to regions of conformational change upon activation, suggesting that these mutations may increase DNA affinity by altering the energetic balance between inactive and active states. Our results show that there are extensive opportunities for evolution to improve catalytic efficiency when an inactive intermediate is involved. |
External links | Nat Struct Mol Biol / PubMed:38177685 / PubMed Central |
Methods | EM (single particle) / X-ray diffraction |
Resolution | 2.628 - 3.21 Å |
Structure data | EMDB-42399, PDB-8unf: EMDB-42402, PDB-8unh: PDB-8uh7: PDB-8uk9: |
Chemicals | ChemComp-08T: ChemComp-MG: ChemComp-ADP: ChemComp-HOH: ChemComp-AF3: ChemComp-AGS: |
Source |
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Keywords | REPLICATION/DNA / Clamp loader / T4 / AAA+ATPase / REPLICATION / REPLICATION-DNA complex / DNA BINDING PROTEIN/DNA / DNA Replication / AAA+ ATPase / Bacteriophage / Complex / DNA / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex / active / DNA-bound / autoinhibited / DNA-free / catalytically inactive / stable |