Elucidation of the epsilon-theta subunit interface of Escherichia coli DNA polymerase III by NMR spectroscopy.
The DNA polymerase III holoenzyme (HE) is the primary replicative polymerase of Escherichia coli. The epsilon (epsilon) subunit of HE provides the 3'-->5' exonucleolytic proofreading activity for this complex. Epsilon consists of two domains: an N-terminal domain containing the proofreading exonuclease activity (residues 1-186) and a C-terminal domain required for binding to the polymerase (alpha) subunit (residues 187-243). In addition to alpha, epsilon also binds the small (8 kDa) theta (theta) subunit. The function of theta is unknown, although it has been hypothesized to enhance the 3'-->5' exonucleolytic proofreading activity of epsilon. Using NMR analysis and molecular modeling, we have previously reported a structural model of epsilon186, the N-terminal catalytic domain of epsilon [DeRose et al. (2002) Biochemistry 41, 94]. Here, we have performed 3D triple resonance NMR experiments to assign the backbone and C(beta) resonances of [U-(2)H,(13)C,(15)N] methyl protonated epsilon186 in complex with unlabeled theta. A structural comparison of the epsilon186-theta complex with free epsilon186 revealed no major changes in secondary structure, implying that the overall structure is not significantly perturbed in the complex. Amide chemical shift comparisons between bound and unbound epsilon186 revealed a potential binding surface on epsilon for interaction with theta involving structural elements near the epsilon catalytic site. The most significant shifts observed for the epsilon186 amide resonances are localized to helix alpha1 and beta-strands 2 and 3 and to the region near the beginning of alpha-helix 7. Additionally, a small stretch of residues (K158-L161), which previously had not been assigned in uncomplexed epsilon186, is predicted to adopt beta-strand secondary structure in the epsilon186-theta complex and may be significant for interaction with theta. The amide shift pattern was confirmed by the shifts of aliphatic methyl protons, for which the larger shifts generally were concentrated in the same regions of the protein. These chemical shift mapping results also suggest an explanation for how the unstable dnaQ49 mutator phenotype of epsilon may be stabilized by binding theta.
DeRose
Eugene F
EF
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Box 12233, Research Triangle Park, North Carolina 27709, USA.
Darden
Thomas
T
Harvey
Scott
S
Gabel
Scott
S
Perrino
Fred W
FW
Schaaper
Roel M
RM
London
Robert E
RE
D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D004261:DNA Replication; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics; D005092:Exonucleases / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D021122:Protein Subunits; D017386:Sequence Homology, Amino Acid
D010446:Peptide Fragments; D021122:Protein Subunits; D005090:Exodeoxyribonucleases; D005092:Exonucleases; D043211:Exodeoxyribonuclease V