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Autoinhibition of a clamp-loader ATPase revealed by deep mutagenesis and cryo-EM.
Marcus, Kendra; Huang, Yongjian; Subramanian, Subu; Gee, Christine L; Gorday, Kent; Ghaffari-Kashani, Sam; Luo, Xiao Ran; Zheng, Lisa; O'Donnell, Michael; Subramaniam, Sriram; Kuriyan, John.
Afiliación
  • Marcus K; Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA.
  • Huang Y; Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA.
  • Subramanian S; Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA.
  • Gee CL; Howard Hughes Medical Institute, University of California, Berkeley, CA, USA.
  • Gorday K; Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
  • Ghaffari-Kashani S; Biophysics Graduate Group, University of California, Berkeley, CA, USA.
  • Luo XR; Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
  • Zheng L; Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
  • O'Donnell M; Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
  • Subramaniam S; Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA.
  • Kuriyan J; Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
Nat Struct Mol Biol ; 31(3): 424-435, 2024 Mar.
Article en 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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Adenosina Trifosfatasas / Replicación del ADN Idioma: En Revista: Nat Struct Mol Biol Asunto de la revista: BIOLOGIA MOLECULAR Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
Texto completo
Añadir a Mi BVS
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PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Adenosina Trifosfatasas / Replicación del ADN Idioma: En Revista: Nat Struct Mol Biol Asunto de la revista: BIOLOGIA MOLECULAR Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos