Electrostatic Interactions at the Dimer Interface Stabilize the E. coli β Sliding Clamp

Sliding clamps are ring-shaped oligomeric proteins that encircle DNA and associate with DNA polymerases for processive DNA replication. The dimeric Escherichia coli β-clamp is closed in solution but must adopt an open conformation to be assembled onto DNA by a clamp loader. To determine what factors...

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Bibliographic Details
Published in:Biophysical journal 2017-08, Vol.113 (4), p.794-804
Main Authors: Purohit, Anirban, England, Jennifer K., Douma, Lauren G., Tondnevis, Farzaneh, Bloom, Linda B., Levitus, Marcia
Format: Article
Language:English
Subjects:
Amino acids
Clamps
Conformation
Deoxyribonucleic acid
DNA
DNA biosynthesis
DNA Polymerase III - chemistry
DNA Polymerase III - genetics
DNA Polymerase III - metabolism
DNA-directed DNA polymerase
Dose-Response Relationship, Drug
E coli
Electrostatic properties
Escherichia coli - enzymology
Experiments
Fluctuations
Fluorescence
Hydrogen-Ion Concentration
Hydrophobicity
Interface stability
Interfaces
Kinetics
Mutation
Protein Multimerization
Protein Stability - drug effects
Protein Structure, Quaternary
Proteins
Salts
Salts - pharmacology
Sliding
Static Electricity
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Summary:Sliding clamps are ring-shaped oligomeric proteins that encircle DNA and associate with DNA polymerases for processive DNA replication. The dimeric Escherichia coli β-clamp is closed in solution but must adopt an open conformation to be assembled onto DNA by a clamp loader. To determine what factors contribute to the stability of the dimer interfaces in the closed conformation and how clamp dynamics contribute to formation of the open conformation, we identified conditions that destabilized the dimer and measured the effects of these conditions on clamp dynamics. We characterized the role of electrostatic interactions in stabilizing the β-clamp interface. Increasing salt concentration results in decreased dimer stability and faster subunit dissociation kinetics. The equilibrium dissociation constant of the dimeric clamp varies with salt concentration as predicted by simple charge-screening models, indicating that charged amino acids contribute to the remarkable stability of the interface at physiological salt concentrations. Mutation of a charged residue at the interface (Arg-103) weakens the interface significantly, whereas effects are negligible when a hydrophilic (Ser-109) or a hydrophobic (Ile-305) amino acid is mutated instead. It has been suggested that clamp opening by the clamp loader takes advantage of spontaneous opening-closing fluctuations at the clamp’s interface, but our time-resolved fluorescence and fluorescence correlation experiments rule out conformational fluctuations that lead to a significant fraction of open states.
ISSN:0006-3495
1542-0086
DOI:10.1016/j.bpj.2017.06.057