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Journal of Bacteriology, June 2006, p. 4464-4473, Vol. 188, No. 12
0021-9193/06/$08.00+0     doi:10.1128/JB.01992-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Structure of the Subunit of Escherichia coli DNA Polymerase III in Complex with the Subunit

Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia,¹ Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden²

Received 29 December 2005/ Accepted 23 March 2006

	ABSTRACT

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

	INTRODUCTION

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The holoenzyme DNA polymerase III (Pol III) is the main replicative polymerase in Escherichia coli (37). It is a remarkable multisubunit enzyme that is capable of extraordinary speed and fidelity of action. Pol III is composed of 10 different subunits, 7 of which act as accessory subunits for a catalytic core composed of 3 tightly bound subunits. The polymerase active site is in the large subunit (130 kDa) of the core (33, 34). The 3'-5' proofreading exonuclease activity is located in the subunit (28 kDa) (48), specifically, in the N-terminal domain (186) (21 kDa), which also contains the binding site of the subunit (9 kDa). The subunit has no unambiguously designated function. The three subunits are arranged linearly such that the subunit occupies the central position, binding to both and (51).

The possibility of understanding the detailed function of the Pol III holoenzyme has provoked interest in the structure of this enzyme since its discovery more than 25 years ago. Unfortunately, neither the complete holoenzyme complex nor the catalytic core has been crystallized yet. Instead, there has been considerable effort to solve the structures of individual subunits by both X-ray crystallographic and nuclear magnetic resonance (NMR) methods, and there has been substantial success recently for two of the three elements of the core. The structure of 186 has been determined by X-ray crystallography (20) and has been modeled from NMR data (8). This structure led to a detailed understanding of the mechanism of the exonuclease activity (19, 20). NMR studies showed that the structure of the subunit in aqueous buffer contains three helices, a short stretch of ß structure, and large segments of flexible polypeptide chains (26). Preliminary spectral data suggested that some of the poorly structured regions attain structure on binding to 186. The refined structure of in a mixed alcohol-water buffer solution showed that this subunit is a three-helix bundle (39). The bacteriophage P1 homolog of , HOT, has the same three-helix bundle fold with minor structural variations (7). No atomic resolution structure for the subunit or any related polymerase belonging to the PolC family is known.

The role of in Pol III function beyond stabilizing the structure of is unclear. is not required for the polymerase or exonuclease activities of the core or the holoenzyme (35, 50). It does, however, stimulate the exonuclease activity of two- to fourfold (44, 51), enhances the interaction of with (52), and stabilizes 186 under thermal inactivation (19) and chemical denaturation (17) conditions. The biochemical evidence for a direct interaction of with the DNA substrate or template strand is ambiguous. HOT plays a similar role when it is substituted for , and there has been a report that it has an even greater stabilizing effect on (3). The structure of 186 changes little when it binds to (6), whereas some of the poorly structured regions of become more ordered (26).

The -binding epitope on 186 was defined using NMR chemical shift mapping experiments and was localized to a mainly hydrophobic surface encompassing strands ß2 and ß3, helices 1 and 2, and the N terminus of helix 7 (6). This places the -binding site on a face of 186 far (approximately 15 to 20 Å) from the active site. Identification of the 186-binding epitope on by similar experiments is more ambiguous because chemical shift changes in occur for most of the molecule when it binds to 186 (26).

One of the goals of structural genomics is to assign functions based on studies of the three-dimensional structures of proteins. With this in mind, and knowing that has a modest effect on the activity of , we have performed structural studies with the -186 complex to clarify the source of the effect on . This study is the latest in a series of studies directed at elucidating the interactions between , , and the DNA substrate. Here, we report the three-dimensional structure of when it is complexed with 186; the results were refined using distance and angular restraints supplemented with restraints derived from pseudocontact shifts (PCS) that were induced by inserting a single lanthanide ion into the metal-binding site of 186 (45). We compared the structure of in complex with 186 with the structure of in mixed alcohol-water buffer and with the structure of HOT in aqueous buffer. Below we also report the results of nuclear Overhauser enhancement (NOE) difference spectroscopy experiments that defined the contact surface of on 186 and compare the results with a model of the - complex that was assembled using only the PCS data (46).

	MATERIALS AND METHODS

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Proteins. Methods for overproduction and purification of and 186 and for production and purification of the -186 complex have been described in detail previously (19, 20). ¹³C/¹⁵N-labeled and ¹⁵N-labeled were produced using minimal media supplemented with L-(+)-[6-¹³C]glucose and ¹⁵NH₄Cl (Cambridge Isotope Laboratories) as described previously (26). Concentrations of -186 were determined spectrophotometrically using an ₂₈₀ value of 14,650 M^–1 cm^–1.

NMR spectroscopy. NMR experiments with complexes of ¹³C/¹⁵N- or ¹⁵N-labeled and unlabeled 186 were carried out at 25 or 30°C using a Varian 600-MHz INOVA spectrometer equipped with a 5-mm PENTA probe, a Varian 800-MHz INOVA spectrometer equipped with a 5-mm triple-resonance probe, and a Bruker Avance 800 spectrometer equipped with a 5-mm TXI probe. Each probe was equipped with triple-axis actively shielded gradients. Each protein sample was extensively dialyzed and then concentrated by ultrafiltration to obtain a concentration of 1 mM in 20 mM sodium phosphate (pH 7.0) buffer containing 100 mM NaCl and 0.1 mM dithiothreitol, and D₂O was added to a concentration of 10% (vol/vol) before NMR spectra were acquired. Sequential backbone and C^ß resonances were assigned by combined analysis of the results of the following experiments: HNCA (55), HN(CO)CA (55), C^ß-decoupled HNCA (36), C^ß-decoupled HN(CO)CA (36), HNCO (41), HCACO (15), HN(CA)CO (11), HNCACB (55), CBCA(CO)NH (14), three-dimensional (3D) sensitivity-enhanced ¹⁵N-separated total correlation spectroscopy (TOCSY) (56) with a mixing time of 70 ms, and 3D sensitivity-enhanced ¹⁵N-separated nuclear Overhauser enhancement spectroscopy (NOESY) (56) and ¹³C-separated NOESY (40) with mixing times of 80 ms. Side chain ¹³C and ¹H resonances were assigned based on the results of HCCH-TOCSY (25), H(CCO)NH (13), and C(CO)NH (13) experiments. The results of all the experiments mentioned above were recorded at a ¹H frequency of 600 MHz. The results of an additional 3D sensitivity-enhanced ¹⁵N-separated NOESY experiment and a ¹³C-separated NOESY experiment, each with a mixing time of 80 ms, were recorded at a ¹H frequency of 800 MHz. The results of ¹³C/¹⁵N-half-filtered and double-half-filtered NOESY experiments (43) were recorded (at a ¹H frequency of 800 MHz) to establish the residues in ¹³C/¹⁵N-labeled that have NOEs to residues in unlabeled 186. The acquisition and processing parameters were similar to those described previously (26).

The 186 epitope on was determined by NOE difference spectroscopy by using the method of Eichmüller et al. (10), except that the experiment was not performed in the constant-time mode. The experiment created parallel ( mode) and antiparallel ( mode) alignments of the longitudinal magnetization of unlabeled 186 and ¹³C/¹⁵N-labeled . Two separate experiments were performed at a ¹H frequency of 600 MHz, and in each experiment the and modes were acquired in an interleaved fashion as described previously (10), using mixing times of 300 and 0 ms. The cross-peak intensities of the two-dimensional maps for the four data sets (₃₀₀, ₃₀₀, ₀, and ₀) were quantified using SPARKY (12), and the ratio of the intensities from these data sets, = (₃₀₀/₃₀₀)/(₀/₀), was used to verify the proximity of each cross-peak to the interface. A ratio of <0.5 was taken to indicate close proximity to the interface, and a ratio of >0.8 was taken to indicate that a side chain was not at the interface.

¹³C/¹⁵N-labeled -186 with a single lanthanide ion (Dy³⁺, Er³⁺, or Ho³⁺) in the active site of 186 was prepared as described previously (45). For these experiments, the buffer was exchanged with 20 mM Tris and 100 mM NaCl (pH 7.0) by ultrafiltration because of the limited solubility of lanthanide phosphates. Pseudocontact shifts induced when the lanthanide ion bound to the -186 complex were measured and evaluated by using ¹⁵N and ¹³C heteronuclear single quantum correlation (HSQC) spectra. Well-resolved shifted cross-peaks in the ¹⁵N-HSQC spectra of lanthanide-labeled -186 were initially assigned by inspection since they were displaced from their diamagnetic counterparts along approximately diagonal lines. The remaining cross-peaks were assigned using predicted PCS calculated from a preliminary model of that was derived from NOE and angular restraints. The assignments were verified by an iterative process that involved comparing the calculated PCS following structure refinement with the measured PCS derived from inspection of cross-peak positions in the diamagnetic and paramagnetic two-dimensional HSQC spectra. In addition, diamagnetic and paramagnetic 3D HNCO spectra were recorded for the -186-Dy³⁺ complex, and the PCS were observed in all three dimensions. This facilitated assignment of PCS that were larger in this complex than in the complexes with other lanthanide ions.

Spectral analysis and experimental restraints. Spectral weighting followed by multidimensional Fourier transformation employing linear prediction was carried out with Varian VNMR version 6.1 software (Varian Associates, Palo Alto, CA) or NMRPipe (5) as described previously (26). After transformation, the processed data from all experiments were converted into the XEASY (1) and/or SPARKY (12) format. Peak picking, spectral analysis, and semiautomated resonance assignment were performed with XEASY, SPARKY, and CANDID (22). Three-dimensional NOESY cross-peaks were integrated using the program SPSCAN v1.0.53 (R. W. Glaser and K. Wüthrich, http://www.mol.biol.ethz.ch/wuthrich/software/spscan/). CALIBA (part of the CYANA suite of programs) (16) was used to convert NOESY cross-peak volumes into upper distance bounds. A total of 106 chemical shift index (CSI)-derived and dihedral angle restraints from the program SHIFTY (42) were used. In addition to the NOE restraints, 48 hydrogen bond restraints (r_NH-O = 1.5 to 2.4 Å, rN-O = 2.4 to 3.4 Å) were included based on the CSI and other indicators of -helical structure and where a single hydrogen bond acceptor was identified in preliminary structure calculations. A total of 162 pseudocontact shifts of amide protons derived from separate experiments with ¹³C/¹⁵N-labeled -186-Dy³⁺, ¹³C/¹⁵N-labeled -186-Er³⁺, and ¹³C/¹⁵N-labeled -186-Ho³⁺ were used in the final structural refinement with the program XPLOR-NIH (49).

Structure calculations. Initial structures were calculated using CYANA (16) and CANDID (22) with distance and dihedral angle restraints. Typically, each CYANA run was composed of 100 steps of minimization, followed by 4,000 molecular dynamics steps and then by 1,000 energy minimization steps. An iterative process involving several rounds of calculations was employed to assign previously ambiguous NOE cross-peaks using the program module CANDID (22). During final structure refinement with the program XPLOR-NIH (49), the ¹H-¹H NOEs and angular restraints were supplemented with 162 PCS estimated from the chemical shifts of backbone resonances in ¹⁵N-HSQC experiments with ¹³C/¹⁵N-labeled -186 recorded with and without a lanthanide ion (Dy³⁺, Er³⁺, or Ho³⁺). The parameters of the magnetic susceptibility anisotropy tensor () were determined using the PCS values obtained for samples of -186 complexes when each leucine and phenylalanine in 186 had been selectively ¹⁵N labeled as described previously (45). The values for the tensor anisotropy parameter _ax for Dy³⁺, Er³⁺, and Ho³⁺ used in the calculations were 40.3, –10.9, and 14.7 M³ x 10^–32, respectively, and the values for the tensor anisotropy parameter _rh used in the calculations were 4.5, –5.1, and 3.2 m³ x 10^–32, respectively. Each XPLOR-NIH run was composed of 18,000 molecular dynamics steps, followed by 5,000 energy minimization steps. The quality of the structures was evaluated with PROCHECK-NMR (28). Experimental restraints were analyzed using the programs XPLOR-NIH and AQUA (28). Figures 2, 3, and 6 were prepared with MOLMOL (27).

View larger version (43K): [in this window] [in a new window]   FIG. 2. NMR-determined structures of the subunit in complex with 186. (A) Stereo view of superposition of the backbone heavy atoms from the final ensemble of the 12 lowest-energy structures refined with distance, torsion angle, and pseudocontact shift restraints. Superposition was accomplished using the backbone atoms of Q10 to L66. (B and C) Calculated charge distribution mapped onto the surface of the subunit (residues 10 to 66). The regions with basic potential are indicated by blue, the regions with acidic potential are indicated by red, and white regions are uncharged regions of the surface. The mean structure is displayed, and the molecule in panel B has the same orientation as the stereo views in panels A and D, with helix 3 on the left, helix 1 in the center, and helix 2 on the right, whereas the molecule in panel C is rotated 180° about the vertical axis. (D) Stereo view of 186-bound , showing some methyl-bearing side chains and the calculated position of the lanthanide ion in the active site of 186. The side chains of methyl-bearing residues that showed substantial differential attenuation in the NOE difference experiment (>0.5) are blue. The approximate point of closest approach of the metal ion to the backbone is indicated. The coordinates are available from the PDB (PDB code 2AXD).

View larger version (27K): [in this window] [in a new window]   FIG. 3. Comparison of the structures of the lowest-energy structure of 186-bound (A), the lowest-energy uncomplexed structure (B), and the lowest-energy HOT structure (C). All structures have the same overall fold, but the angles between helices 1 and 3 are different. The ribbons trace the backbones of 186-bound , free (PDB code 2AE9), and HOT (PDB code 1SE7), respectively.

View larger version (38K): [in this window] [in a new window]   FIG. 6. Stereo representation of the structure of the -186 complex as determined from pseudocontact shifts induced by Dy3+ and Er3+ ions (46). Residues 10 to 66 are included in the ribbon representation of , and the amino and carboxy termini are indicated by blue and red, respectively. The position of the lanthanide ion is indicated by the magenta sphere. The amide proton of H49 in 186, which makes an NOE with the amide proton of A6 in (46), is indicated by a blue ball-and-stick diagram. The N and C termini of 186 are labeled.

	RESULTS

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View larger version (28K): [in this window] [in a new window]   FIG. 1. Effect of Dy3+ on the 15N-HSQC spectrum of the 13C/15N-labeled subunit complexed with 186. Spectra were recorded in 90% H2O-10% D2O and 100 mM NaCl-20 mM Tris buffer (pH 7.0) at 25°C. Spectra recorded with Dy3+ (blue peaks) and in the absence of paramagnetic ion (red peaks) are superimposed. Lines connect pairs of diamagnetic and paramagnetic cross-peaks. Red lines indicate a negative pseudocontact shift, and blue lines indicate a positive pseudocontact shift. Cross-peaks are labeled with the residue assignment. For clarity, not all peaks are connected or labeled.

Structure calculations. The input data for structure calculations included ¹H NOEs from ¹⁵N- and ¹³C-separated NOESY spectra, angular data from chemical shifts, and PCS as described in Materials and Methods. The PCS data were particularly valuable as the number of long-range NOEs was relatively small (Table 1), in part because most long-range contacts involved poorly resolved side chain resonances and most of the resonances from the aromatic rings of F27, Y31, and F52 were broad.

View this table: [in this window] [in a new window]   TABLE 1. Structural statistics and root mean square deviations for the 12 NMR structure conformers representing the subunit of DNA polymerase III in complex with 186

The quality of the structure was supported by the appearance of the Ramanchandran plot, generated by PROCHECK-NMR (28), which showed that all the residues in the generously allowed and disallowed regions are in disordered parts of the protein (mostly the C terminus), for which there were no meaningful NOE data to restrict the protein backbone to any region of conformational space.

Description of the solution structure and comparison with uncomplexed and HOT. The NMR-derived structure of bound to 186 is a three-helix bundle (Fig. 2 and 3). Helix 1 is approximately antiparallel to helix 2 and parallel to helix 3, making angles of 170° and 50°, respectively. Helix 2 makes an angle of about 130° with helix 3. A hydrophobic core between the helices is populated mostly by short- and long-chain aliphatic residues. Several side chain resonances of K15 are shifted upfield, in agreement with the location of K15 between helices 1 and 3 and packed above W51. D19 and R55 are adjacent and partially protected from the solvent, and they may form a salt bridge. Several basic residues in helices 2 and 3 are oriented away from the protein core into the solution and are clustered on one side of . These residues are arginine residues at positions 42, 49, 53, 60, and 68.

The overall fold of the structure (Fig. 3A) is different from the fold described previously for free in aqueous buffer (26) and is more similar to the fold of free in a mixed methanol-H₂O buffer (39) and the fold of HOT in aqueous buffer (7). Although the number of long-range NOEs for complexed with 186 is fairly small, the NOEs are more uniformly distributed over the sequence than they were for the calculation of free in aqueous buffer (26). Since several of the characteristically ring current shifted resonances and the patterns of NOEs are very similar in complexed and free in aqueous buffer, we concluded that the structure described here and the structure of in the mixed solvent (39) reflect the structure of free in aqueous buffer more closely than the previously published structure (26). The most significant difference between free and 186-bound appears to be in different mobilities that cause line broadening for different segments of the polypeptide chain, particularly at the interface between helix 1 and helix 3. Binding to 186 stabilizes the structure rather than inducing any large changes.

HOT, which exhibits 53% sequence identity with (29), also folds into a three-helix bundle (7). Ribbon diagrams of 186-bound , uncomplexed in mixed buffer (39), and uncomplexed HOT are shown in Fig. 3. Each structure has helices that are approximately the same length, and each structure has loop regions that reverse the direction of the peptide chain. The orientations of helices 1 and 3 differ by about 10° for complexed and uncomplexed (Fig. 3A and B) and by 20° for complexed and HOT (Fig. 3A and C). A DALI (23) search of the Protein Data Bank (PDB) revealed that of the proteins in the PDB, HOT is the protein most closely related to , with a Z score of 5.8 for the structured regions.

The structures of complexed and uncomplexed in the mixed-solvent buffer are very similar, and the greatest differences are the orientation of helix 1 with respect to the other two helices and the conformation of loop L1. These differences may be due to structural changes on complexation with or to the use of long-range PCS restraints. The latter better define the orientation of helices and loops, especially where there are few long-range distance restraints. Mueller et al. (39) commented on the paucity of long-range restraints in the vicinity of helix 2 and loop L2 of uncomplexed .

Electrostatic surface of . Figure 2B and C show opposing faces of the electrostatic surface of the folded core of (residues 10 to 66) in the complex with 186. Figure 2C has the same orientation as the stereo views shown in Fig. 2A and D, and Fig. 2B shows the opposing face of the surface shown in Fig. 2C. The surface shown in Fig. 2B comprising mostly residues from helices 2 and 3 is highly positively charged with several arginine and lysine side chains in close proximity. The functional significance of the cluster of arginine residues oriented outward on helix 3 is unknown. There are also small pockets of hydrophobic residues at the C terminus of helix 3 and the turn structure between helices 1 and 2. Hydrophobic residues from helix 1 and helix 3 dominate the opposing face, which can be segregated into distinct regions (Fig. 2C). The surface of helix 1 is hydrophobic, resulting from the predominance of hydrophobic residues in the central part of the helix (V16 to F27). This hydrophobic surface extends to the base of the turn structure encompassing N32, M33, and P34. On either side of the central hydrophobic spine are positively and negatively charged residues.

- interface. A conventional method for defining the interface between two proteins is to map the perturbation of chemical shifts and NMR signal linewidths of one protein component caused by complexation of the other. Clustering of large changes in chemical shifts in a particular region is indicative of a potential binding surface. The epitope on 186 was identified in this way (6). Figure 4A shows the changes in the chemical shift of the amide proton, amide nitrogen, backbone carbonyl, and carbons of when the complex is formed. Most of the residues show significant changes in chemical shifts; the exception is the region between M33 and W51. These results suggest that the N-terminal segment of helix 1 and the C terminus of helix 3 are involved in contacts with 186. The previously identified (26) sections of uncomplexed that are affected by conformational or chemical exchange phenomena include the C-terminal sections of helices 1 and 3 (D19 to Y31 and R53 to S67). Thus, it is difficult to distinguish the effects of the direct contact with 186 from indirect effects due to a more stable secondary structure.

View larger version (19K): [in this window] [in a new window]   FIG. 4. Changes in chemical shifts of upon binding to 186 and 1HN pseudocontact shifts of . (A) Changes in chemical shifts of 1HN () and 15N[amide] () and 13C () and 13C' (+) spins. There are significant changes in regions where contact between and 186 is also suggested by the results shown in Fig. 5. Little or no change is observed in the region from V35 to R53, suggesting that this region is not in contact with 186. The resonance assignments in free were obtained from reference 9. (B) Plot of pseudocontact shifts of the backbone HN induced by Dy3+ (), Er3+ (), and Ho3+ (x) versus residue number.

View larger version (19K): [in this window] [in a new window]   FIG. 5. 1H-13C correlation maps obtained by the technique of Eichmüller et al. (10), showing the differential decay of 1H magnetization after preparation of the and spin modes of the 13C-HSQC spectra of 186-bound 13C/15N-labeled . The two spectra were recorded with an NOE mixing period of 300 ms. A reference pair of spectra obtained by using a 0-ms NOESY mixing period (not shown) was also recorded to account for differences in one-bond heteronuclear scalar coupling constants.

Notably, ¹⁵N-HSQC cross-peaks could be assigned for residues 4 to 9 preceding helix 1 in the samples with Er³⁺ and Ho³⁺. Some of these cross-peaks could also be observed for the -186-Dy³⁺ complex, but they were more difficult to assign. The fact that cross-peaks could be observed at all for these residues in the paramagnetic samples indicates that their amide protons must be located more than 14 Å from the paramagnetic ion (2). Cross-peaks of residues 2 and 3 were already weak in the diamagnetic complex and were therefore less likely to be observed in the paramagnetic complexes. The magnitude of the PCS observed for these residues thus indicates that they are closer to the active site of 186 than any other residue of is but may not reach all the way to this site.

	DISCUSSION

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The small subunit of DNA polymerase III forms an isolable complex with the catalytic N-terminal domain (186) of the proofreading 3'-5' exonuclease subunit (19, 44). The high-resolution crystal structure of 186 (20) has provided impetus to modeling the structure of the -186 complex, both to explain its stability and to obtain insight into the roles of in DNA replication. Although is not necessary for the exonuclease activity of , it has been shown to protect 186 against thermal inactivation, as measured by the activity remaining after treatment for 10 min at an elevated temperature; the temperature at which 50% of the activity remained was about 14°C higher for -186 than for free 186 (19). Free 186 was also observed to precipitate irreversibly in a few hours under conditions used here for extensive NMR studies of the complex (at 25 or 30°C); NMR spectra of free 186 could be obtained only at 10°C (18). Furthermore, as judged from the stability of the intact Pol III core (-- complex) during 28,000-fold purification from plasmid-free E. coli (38), the - complex is probably extremely stable to dissociation. Direct measurement of the dissociation constant of the complex has not been reported, but an upper limit for the dissociation constant of 20 nM has been obtained by electrospray ionization-mass spectrometry (17).

Our determination of the three-dimensional structure of complexed with 186 is the next step for understanding the basis for this intimate and stable protein-protein interaction, and our findings elucidated the binding interface. Remarkably, the NMR spectra obtained for in the 30-kDa complex with 186 were easier to interpret than the NMR data obtained for free , yet the number of long-range NOEs that could be observed between the helices in was still small for the complex. In this situation the global PCS restraints made a significant contribution to the quality of the structure.

Overall, the structure obtained for 186-bound is similar to the structure of free in mixed alcohol-water solvent (39) and to the structure of the close homolog HOT in aqueous buffer (7). The differences among these structures are either due to structural changes in the free and complexed proteins or due to limitations of the structure determination protocols. Since PCS restraints were used in the structure calculation, the present NMR structure of satisfies the PCS restraints better than the structure of either free (39) or HOT (7). We noted that the side chain of L21 is barely solvent exposed in the structure of HOT (7), so it would not be expected to engage in intermolecular NOEs with 186. In contrast, the corresponding residue in , L20, is much more solvent exposed in the complexed and free forms of (39). Since the methyl groups of this leucine have intermolecular NOEs with 186 and experience strong ring currents in the complex with 186 but not in free , the two structures of provide plausible models of the structure in the complex. The small chemical shift changes observed for helix 2 and most of helix 3 in suggest that any changes in the global fold of upon complexation with 186 are small.

The intermolecular NOEs observed for L20 and the NOE difference experiments (10) resulted in identification of a hydrophobic surface along helix 1, as shown in Fig. 2C, as an important part of the interaction surface with . The result is in agreement with the chemical shift mapping of the binding epitope of on 186 that indicated that the interface between and is primarily hydrophobic (6). Electrospray ionization-mass spectrometry studies of the dissociation of -186 also suggested that the - interface is hydrophobic (17). Furthermore, preliminary chemical shift mapping experiments with the HOT-186 complex placed the interaction site near the N terminus of helix 1 and the C terminus of helix 3 (7), in agreement with our results.

The intermolecular NOEs and reduced mobility observed for A6 show that the N-terminal segment of is involved in specific contacts with 186, whereas it is highly mobile in uncomplexed in aqueous (26) and mixed-solvent buffers (39). Thus, it is clear that the N-terminal segment of folds upon complex formation with 186, although most interactions of this segment may be intermolecular rather than intramolecular. Although the absence of intraprotein long-range NOEs precluded structure determination for the first nine residues of -bound , PCS values and paramagnetic relaxation enhancements indicated that the N-terminal residues of are closer to the metal ion at the active site of 186 than are any of the other residues of .

We used a novel NMR strategy (46) based on the PCS induced by inserting lanthanides at the 186 metal-binding site to dock the NMR-derived structure of described above with the X-ray-derived structure of 186 (20). The PCS-based routine for rigid docking of and 186 is described in detail elsewhere (46). Briefly, the PCS detected for the amide protons of [¹⁵N]Leu- and [¹⁵N]Phe-labeled 186 (with unlabeled ) were used to derive the tensor of Dy³⁺ with respect to the crystal structure of 186. The PCS of the amide protons of ¹⁵N-labeled (with unlabeled 186) were subsequently used to position and orient the tensor with respect to the solution structure of described here. The common coordinate system defined in this way by the principal axes of the tensor of the metal ion positioned and oriented the structures of 186 and with respect to each other. Er³⁺ data recorded in the same manner defined a second coordinate system that was equally centered about the metal ion but oriented somewhat differently. The combination of Dy³⁺ and Er³⁺ data provided a unique solution for the 186- complex (46). A stereo representation of the model is shown in Fig. 6. The overall structure agrees with the results of NOE difference spectroscopy, intermolecular NOEs, and chemical shift changes, placing the hydrophobic surface at the C terminus of helix 1 (Fig. 6) and part of the C terminus of helix 3 in contact with the short helix 2 in 186. The N terminus of helix 1 of is approximately parallel to one of the strands of the ß-sheet in 186. The closest approach of the backbone of is 15 Å from the active site of 186, and thus the backbone of the helices of is too far away to have a direct role in the proofreading process. Although there is no direct interaction with the catalytically important helix 7 of 186, may have a role in stabilizing the position of this helix by restraining the adjacent ß strand. This stabilizing role is consistent with biological evidence showing that is not required for the proofreading function of but rather has a modest effect on the activity of and the fidelity of replication (52).

Exonuclease I, a structural relative of , has an additional C-terminal helical domain that we speculated previously may be an analog of (20). However, when the location of this domain in exonuclease I was compared with the location of on 186, there was little overlap; the C-terminal domain of exonuclease I does not overlay the ß-sheet and only partially overlays helix 2. In addition, there is very little structural homology between and the C-terminal domain of exonuclease I, and its function is probably unrelated (46).

The interface between and 186 is large and relatively flat and is populated mostly by long-chain and aromatic side chains. The minimum contact surface between and 186 as shown in Fig. 6 was calculated to be 1,400 Ų per monomer by the program DSSP (24). The actual contact surface is almost certainly larger because the structure determination of did not take into account the interaction between 186 and the N terminus of shown by the intermolecular NOEs between A6 of and 186. This interface area corresponds to the standard-size protein interface as defined by Lo Conte et al. (30) and is typical of protein-protein complexes with nanomolar affinity (30), particularly those with hydrophobic interfaces.

Although there is no evidence that interacts with either single- or double-stranded DNA, it is intriguing that it has two motifs that figure prominently in DNA-binding proteins. The helix-loop-helix motif encompasses helix 1, loop 1, and helix 2 of , and the helix-hairpin-helix motif encompasses helix 2, loop 2, and helix 3 of . The helix-hairpin-helix motif has been proposed to be a nonspecific, single-stranded DNA-binding motif (9, 32), and the helix-loop-helix motif is also common in proteins that bind to DNA (21, 31). The solvent-facing surface of helix 3 of has several closely spaced arginine residues (Fig. 2B) that could also function as a DNA-binding region.

	ACKNOWLEDGMENTS

 
We are grateful to Varian Associates (Palo Alto, CA) for generously providing time on the INOVA 800 spectrometer. We thank Esther Bulloch and Tanya Ronson for assistance with sample preparation, Gabrielle Cavallaro for assistance with the PARA restraints module of XPLOR-NIH, and Robert London for making the coordinates of and HOT available prior to publication. We benefited from the use of NMR facilities at the Australian National University NMR Center.

This research was supported by grants from the Australian Research Council, including a Federation Fellowship to G.O.

	FOOTNOTES

 
* Corresponding author. Mailing address: Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia. Phone: 61-2-61252863. Fax: 61-2-61250750. E-mail: max{at}rsc.anu.edu.au.

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