Autoinhibition of a clamp-loader ATPase revealed by deep mutagenesis and cryo-EM.
Nat Struct Mol Biol
; 31(3): 424-435, 2024 Mar.
Article
em En
| MEDLINE
| ID: mdl-38177685
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 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Adenosina Trifosfatases
/
Replicação do DNA
Idioma:
En
Revista:
Nat Struct Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Estados Unidos