Structure of the θ subunit of Escherichia coli DNA polymerase III in complex with the ε subunit

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the alpha, E, and 0 subunits. The 0 subunit is the smallest and least understood subunit. The three-dimensional structure of 0 in a complex with the unlabeled N-terminal domain of the epsilon subunit, epsilon 186, 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 epsilon 186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of 0 in a methanol-water buffer and of the bacteriophage PI homolog, HOT, in aqueous buffer. Conserved nuclear Overhauser enhancement (NOE) patterns obtained for free and complexed 0 show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free 0 (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 0 in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of epsilon 186 on 0 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 0 are located about 15 angstrom or farther from the lanthanide ion in the active site of epsilon 186, in agreement with the extensive biochemical data for the 0-epsilon system.