posted on 2024-11-14, 22:44authored byMax A Keniry, Ah-Young Park, Elisabeth A Owen, Samir M Hamdan, Guido Pintacuda, Gottfried Otting, Nicholas DixonNicholas Dixon
The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the α, ε, and θ subunits. The θ subunit is the smallest and least understood subunit. The three-dimensional structure of θ in a complex with the unlabeled N-terminal domain of the ε subunit, ε186, was determined by multidimensional nuclear magnetic resonance spectroscopy. The structure was refined using pseudocontact shifts that resulted from inserting a lanthanide ion (Dy3+, Er3+, or Ho3+) at the active site of ε186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of θ in a methanol-water buffer and of the bacteriophage P1 homolog, HOT, in aqueous buffer. Conserved nuclear Overhauser enhancement (NOE) patterns obtained for free and complexed θ show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free θ (Keniry et al., Protein Sci. 9:721-733, 2000) were attributed to errors in the latter structure. The present structure satisfies the pseudocontact shifts better than either the structure of θ in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of ε186 on θ was mapped by NOE difference spectroscopy and was found to involve helix 1 and the C-terminal part of helix 3. The pseudocontact shifts indicated that the helices of θ are located about 15 Å or farther from the lanthanide ion in the active site of ε186, in agreement with the extensive biochemical data for the θ-ε system.
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Citation
Keniry, M., Park, A., Owen, E. A., Hamdan, S. M., Pintacuda, G., Otting, G. & Dixon, N. E. (2006). Structure of the theta subunit of Escherichia coli DNA polymerase III in complex with the epsilon subunit. Journal of Bacteriology, 188 (12), 4464-4473.