Structure of the Subunit of Escherichia

coli DNA Polymerase III in Complex with

the Subunit

Max A. Keniry, Ah Young Park, Elisabeth A. Owen, Samir M.

Hamdan, Guido Pintacuda, Gottfried Otting and Nicholas E.

Dixon

J. Bacteriol. 2006, 188(12):4464. DOI: 10.1128/JB.01992-05.

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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.

Subunit of Escherichia coli DNA Polymerase III

Structure of the

in Complex with the Subunit

Max A. Keniry,1* Ah Young Park,1 Elisabeth A. Owen,1 Samir M. Hamdan,1 Guido Pintacuda,1,2

Gottfried Otting,1 and Nicholas E. Dixon1

Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia,1 and Department of

Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden2

Received 29 December 2005/Accepted 23 March 2006

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

Downloaded from http://jb.asm.org/ on February 12, 2013 by guest

of in a complex with the unlabeled N-terminal domain of the subunit, 186, was determined by multidi-

mensional nuclear magnetic resonance spectroscopy. The structure was re ned 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 satis es

the pseudocontact shifts better than either the structure of in methanol-water buffer or the structure of HOT.

satis es 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 A or farther from the lanthanide ion in the active site of 186, in agreement with the extensive

biochemical data for the - system.

The holoenzyme DNA polymerase III (Pol III) is the main structure of the subunit in aqueous buffer contains three

replicative polymerase in Escherichia coli (37). It is a remark- helices, a short stretch of structure, and large segments of

able multisubunit enzyme that is capable of extraordinary exible polypeptide chains (26). Preliminary spectral data sug-

speed and delity of action. Pol III is composed of 10 different gested that some of the poorly structured regions attain struc-

ture on binding to 186. The re ned structure of in a mixed

subunits, 7 of which act as accessory subunits for a catalytic

core composed of 3 tightly bound subunits. The polymerase alcohol-water buffer solution showed that this subunit is a

active site is in the large subunit (130 kDa) of the core (33, three-helix bundle (39). The bacteriophage P1 homolog of,

34). The 3 -5 proofreading exonuclease activity is located in HOT, has the same three-helix bundle fold with minor struc-

the subunit (28 kDa) (48), speci cally, in the N-terminal tural variations (7). No atomic resolution structure for the

domain ( 186) (21 kDa), which also contains the binding site of subunit or any related polymerase belonging to the PolC family

the subunit (9 kDa). The subunit has no unambiguously is known.

designated function. The three subunits are arranged linearly The role of in Pol III function beyond stabilizing the

such that the subunit occupies the central position, binding to structure of is unclear. is not required for the polymerase or

both and (51). exonuclease activities of the core or the holoenzyme (35, 50).

The possibility of understanding the detailed function of the It does, however, stimulate the exonuclease activity of two- to

Pol III holoenzyme has provoked interest in the structure of

fourfold (44, 51), enhances the interaction of with (52), and

this enzyme since its discovery more than 25 years ago. Unfor-

stabilizes 186 under thermal inactivation (19) and chemical

tunately, neither the complete holoenzyme complex nor the

denaturation (17) conditions. The biochemical evidence for a

catalytic core has been crystallized yet. Instead, there has been

direct interaction of with the DNA substrate or template

considerable effort to solve the structures of individual sub-

strand is ambiguous. HOT plays a similar role when it is sub-

units by both X-ray crystallographic and nuclear magnetic res-

stituted for, and there has been a report that it has an even

onance (NMR) methods, and there has been substantial suc-

greater stabilizing effect on (3). The structure of 186 changes

cess recently for two of the three elements of the core. The

little when it binds to (6), whereas some of the poorly struc-

structure of 186 has been determined by X-ray crystallogra-

tured regions of become more ordered (26).

phy (20) and has been modeled from NMR data (8). This

The -binding epitope on 186 was de ned using NMR

structure led to a detailed understanding of the mechanism of

chemical shift mapping experiments and was localized to a

the exonuclease activity (19, 20). NMR studies showed that the

mainly hydrophobic surface encompassing strands 2 and 3,

helices 1 and 2, and the N terminus of helix 7 (6). This

places the -binding site on a face of 186 far (approximately

* Corresponding author. Mailing address: Research School of Chem-

15 to 20 A) from the active site. Identi cation of the 186-

istry, Australian National University, Canberra, ACT 0200, Australia.

binding epitope on by similar experiments is more ambiguous

Phone: 61-2-61252863. Fax: 61-2-61250750. E-mail: abqnds@r.postjobfree.com.

4464

IN - COMPLEX

VOL. 188, 2006 STRUCTURE OF 4465

because chemical shift changes in occur for most of the (12), and the ratio of the intensities from these data sets, ( 300/ 300)/( 0/ 0),

molecule when it binds to 186 (26). 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

One of the goals of structural genomics is to assign functions was taken to indicate that a side chain was not at the interface.

based on studies of the three-dimensional structures of pro- C/15N-labeled 186 with a single lanthanide ion (Dy3, Er3, or Ho3 ) in

13

the active site of 186 was prepared as described previously (45). For these

teins. With this in mind, and knowing that has a modest effect

on the activity of, we have performed structural studies with experiments, the buffer was exchanged with 20 mM Tris and 100 mM NaCl (pH

the - 186 complex to clarify the source of the effect on . This 7.0) by ultra ltration because of the limited solubility of lanthanide phosphates.

Pseudocontact shifts induced when the lanthanide ion bound to the - 186

study is the latest in a series of studies directed at elucidating complex were measured and evaluated by using 15N and 13C heteronuclear single

the interactions between,, and the DNA substrate. Here, we quantum correlation (HSQC) spectra. Well-resolved shifted cross-peaks in the

N-HSQC spectra of lanthanide-labeled - 186 were initially assigned by in-

15

report the three-dimensional structure of when it is com-

plexed with 186; the results were re ned using distance and spection since they were displaced from their diamagnetic counterparts along

approximately diagonal lines. The remaining cross-peaks were assigned using

angular restraints supplemented with restraints derived from predicted PCS calculated from a preliminary model of that was derived from

pseudocontact shifts (PCS) that were induced by inserting a NOE and angular restraints. The assignments were veri ed by an iterative pro-

single lanthanide ion into the metal-binding site of 186 (45). cess that involved comparing the calculated PCS following structure re nement

We compared the structure of in complex with 186 with the with the measured PCS derived from inspection of cross-peak positions in the

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diamagnetic and paramagnetic two-dimensional HSQC spectra. In addition,

structure of in mixed alcohol-water buffer and with the struc-

diamagnetic and paramagnetic 3D HNCO spectra were recorded for the - 186-

ture of HOT in aqueous buffer. Below we also report the Dy3 complex, and the PCS were observed in all three dimensions. This facili-

results of nuclear Overhauser enhancement (NOE) difference tated assignment of PCS that were larger in this complex than in the complexes

spectroscopy experiments that de ned the contact surface of with other lanthanide ions.

on 186 and compare the results with a model of the - Spectral analysis and experimental restraints. Spectral weighting followed by

multidimensional Fourier transformation employing linear prediction was car-

complex that was assembled using only the PCS data (46). ried 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

MATERIALS AND METHODS SPARKY (12) format. Peak picking, spectral analysis, and semiautomated res-

Proteins. Methods for overproduction and puri cation of and 186 and for onance assignment were performed with XEASY, SPARKY, and CANDID

production and puri cation of the - 186 complex have been described in detail (22). Three-dimensional NOESY cross-peaks were integrated using the program

SPSCAN v1.0.53 (R. W. Glaser and K. Wuthrich, http://www.mol.biol.ethz.ch

previously (19, 20). 13C/15N-labeled and 15N-labeled were produced using

/wuthrich/software/spscan/). CALIBA (part of the CYANA suite of programs)

minimal media supplemented with L- -[6-13C]glucose and 15NH4Cl (Cam-

(16) was used to convert NOESY cross-peak volumes into upper distance

bridge Isotope Laboratories) as described previously (26). Concentrations of

- 186 were determined spectrophotometrically using an 280 value of 14,650 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

M 1 cm 1.

restraints, 48 hydrogen bond restraints (rNH-O 1.5 to 2.4 A, rN-O 2.4 to 3.4

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 A) were included based on the CSI and other indicators of -helical structure

and where a single hydrogen bond acceptor was identi ed in preliminary struc-

600-MHz INOVA spectrometer equipped with a 5-mm PENTA probe, a Varian

ture calculations. A total of 162 pseudocontact shifts of amide protons derived

800-MHz INOVA spectrometer equipped with a 5-mm triple-resonance probe,

from separate experiments with 13C/15N-labeled - 186-Dy3, 13C/15N-labeled

and a Bruker Avance 800 spectrometer equipped with a 5-mm TXI probe. Each

- 186-Er3, and 13C/15N-labeled - 186-Ho3 were used in the nal structural

probe was equipped with triple-axis actively shielded gradients. Each protein

re nement with the program XPLOR-NIH (49).

sample was extensively dialyzed and then concentrated by ultra ltration to obtain

Structure calculations. Initial structures were calculated using CYANA (16)

a concentration of 1 mM in 20 mM sodium phosphate (pH 7.0) buffer con-

and CANDID (22) with distance and dihedral angle restraints. Typically, each

taining 100 mM NaCl and 0.1 mM dithiothreitol, and D2O was added to a

CYANA run was composed of 100 steps of minimization, followed by 4,000

concentration of 10% (vol/vol) before NMR spectra were acquired. Sequential

molecular dynamics steps and then by 1,000 energy minimization steps. An

backbone and C resonances were assigned by combined analysis of the results

iterative process involving several rounds of calculations was employed to assign

of the following experiments: HNCA (55), HN(CO)CA (55), C -decoupled

previously ambiguous NOE cross-peaks using the program module CANDID

HNCA (36), C -decoupled HN(CO)CA (36), HNCO (41), HCACO (15),

(22). During nal structure re nement with the program XPLOR-NIH (49), the

HN(CA)CO (11), HNCACB (55), CBCA(CO)NH (14), three-dimensional (3D)

1

H-1H NOEs and angular restraints were supplemented with 162 PCS estimated

sensitivity-enhanced 15N-separated total correlation spectroscopy (TOCSY) (56)

from the chemical shifts of backbone resonances in 15N-HSQC experiments with

with a mixing time of 70 ms, and 3D sensitivity-enhanced 15N-separated nuclear

C/15N-labeled - 186 recorded with and without a lanthanide ion (Dy3, Er3,

13

Overhauser enhancement spectroscopy (NOESY) (56) and 13C-separated

or Ho3 ). The parameters of the magnetic susceptibility anisotropy tensor NOESY (40) with mixing times of 80 ms. Side chain 13C and 1H resonances were

were determined using the PCS values obtained for samples of - 186 complexes

assigned based on the results of HCCH-TOCSY (25), H(CCO)NH (13), and

when each leucine and phenylalanine in 186 had been selectively 15N labeled as

C(CO)NH (13) experiments. The results of all the experiments mentioned above

described previously (45). The values for the tensor anisotropy parameter

were recorded at a 1H frequency of 600 MHz. The results of an additional 3D ax

for Dy3, Er3, and Ho3 used in the calculations were 40.3, 10.9, and 14.7 M3

sensitivity-enhanced 15N-separated NOESY experiment and a 13C-separated

32

10, respectively, and the values for the tensor anisotropy parameter

NOESY experiment, each with a mixing time of 80 ms, were recorded at a 1H rh

used in the calculations were 4.5, 5.1, and 3.2 m3 10 32, respectively. Each

frequency of 800 MHz. The results of 13C/15N-half- ltered and double-half-

XPLOR-NIH run was composed of 18,000 molecular dynamics steps, followed by

ltered NOESY experiments (43) were recorded (at a 1H frequency of 800 MHz)

5,000 energy minimization steps. The quality of the structures was evaluated with

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 PROCHECK-NMR (28). Experimental restraints were analyzed using the programs

XPLOR-NIH and AQUA (28). Figures 2, 3, and 6 were prepared with MOLMOL

described previously (26).

The 186 epitope on was determined by NOE difference spectroscopy by (27).

using the method of Eichmuller et al. (10), except that the experiment was not

performed in the constant-time mode. The experiment created parallel ( mode)

RESULTS

and antiparallel ( mode) alignments of the longitudinal magnetization of un-

labeled 186 and 13C/15N-labeled . Two separate experiments were performed Resonance assignment and NMR spectroscopy. The opti-

at a 1H frequency of 600 MHz, and in each experiment the and modes were

mum temperature range for acquisition of the spectra of the

acquired in an interleaved fashion as described previously (10), using mixing

- 186 complex was 25 to 30 C. At temperatures above 30 C,

times of 300 and 0 ms. The cross-peak intensities of the two-dimensional maps

the sample aggregated, and at temperatures below 25 C, spec-

for the four data sets ( 300, 300, 0, and 0) were quanti ed using SPARKY

4466 KENIRY ET AL. J. BACTERIOL.

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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.

tral resolution was not suf cient for analysis. Figure 1 shows the resonances for free . In contrast, the resonances of S67 to

L69 were broad for the - 186 complex, although they were

superposition of the 15N-HSQC spectra recorded with Dy3

and in the absence of a paramagnetic ion, from which the PCS narrow for free, which may be explained by close proximity to

186. Interestingly, DeRose et al. (7) reported low-intensity

were estimated. The 15N-HSQC and 15N-separated NOESY

spectra of the 13C/15N-labeled subunit bound to 186 showed amide peaks for A22 to S24 for HOT, but the equivalent peaks

for 186-bound, A21 to A23, were intense. Backbone non-

a chemical shift dispersion characteristic of a folded, mostly

exchangeable 1H and 13C resonances were assigned by stan-

helical protein. Narrow linewidths observed for the amide pro-

tons of the residues following P70 indicated that there were dard procedures using HNCA, HN(CO)CA, and HNCO spec-

mobile residues at the C terminus. Nearly complete assignment tra, which resulted in assignment of 73 H (96%), 70 C (92%),

of the backbone resonances (N, HN, C, C ) was achieved for and 61 C (80%) resonances. Most of the unassigned nonex-

the residues from D9 to K76, and partial assignments (N, HN) changeable resonances were from residues near the N termi-

were obtained for backbone resonances for L2 to L8. The nus. The absence of any intense exchange peaks between the

water and the amide protons of these residues in the 15N-

amide cross-peaks for residues L2 to L8 were broad, and their

intensities in the 15N-HSQC and 15N-separated NOESY and separated NOESY spectrum indicated that the broadening of

TOCSY spectra were low. All the observable backbone amide the amide signals was due to conformational exchange rather

cross-peaks (70 of an expected 71 cross-peaks) in the 15N- than H exchange with the water.

HSQC spectrum were assigned. Only the amide resonance of Secondary structure. The NOE data were obtained from

80-ms-mixing-time 15N-separated NOESY and 13C-separated

H47 could not be assigned. In contrast to the corresponding

cross-peaks in the 15N-HSQC spectrum of uncomplexed, in NOESY spectra at 600 and 800 MHz. The presence of medium

which the amide resonances in the N-terminal section of helix to strong dNN and weak d N(i,i 1) and d N(i,i 3) NOEs

1 (L8 to V18) were narrow (26), more uniform signal intensi- and the consensus CSI (53, 54) identi ed three regions of

ties were observed for these resonances in the - 186 complex, helicity between Q10 and Y31 (helix 1), between A37 and R42

indicating that there was reduced mobility. Similarly, amide (helix 2), and between L48 and L66 (helix 3). Analysis using

resonances from the C-terminal part of helix 1 (D19 to K28) the programs TALOS (4) (H, C, C, and C chemical shifts)

and SHIFTY (42) (H, C, C, C, N, and HN chemical shifts)

and the C-terminal section of helix 3 (E54 to A62), which were

either broad or not observed in uncomplexed (26), were placed the helices at Q10 to Y31, A37 to R42, and R49 to R68.

easily detected and well resolved for the - 186 complex, indi- Helical wheel representations showed that helix 1 is mostly

cating that complex formation with 186 abolished a chemical hydrophobic but helix 2 is amphipathic, with a predominance

exchange process on the millisecond timescale that broadened of acidic residues on one of its surfaces. On the other hand,

IN - COMPLEX

VOL. 188, 2006 STRUCTURE OF 4467

helix 3 is amphipathic with mostly basic residues covering one TABLE 1. Structural statistics and root mean square deviations for

the 12 NMR structure conformers representing the subunit

face. The helical regions are broken by a sharp turn between

of DNA polymerase III in complex with 186

N32 and P34, a short section of extended structure from V35 to

Parameter Value

A37, and a break between helices 2 and 3 that occurs near P45.

The segment from P34 to I36 is characterized by weak dNN Structural restraints

Distance restraints

and strong d N(i,i 1) NOEs and an inversion of the CSI,

Meaningful NOEs 584

indicating the presence of an extended conformation. Simi-

Intraresidue 152

larly, the pattern of strong dNN NOEs in the segment from Medium range, i-j 5 343

E38 to Q44 is broken at P45 but resumes again at L48. The CSI Long range, i-j 5 41

Dihedral angle restraints 111

again inverts at E46, but values characteristic of a helix resume

Hydrogen bond restraints 48

at L48. Strong sequential d (i,i 1) NOEs observed for all

Pseudocontact shifts 162

proline H resonances indicated that they are all in the trans

conformation. Glycine is frequently a helix breaker (47), but all Statistics for structure calculations

Average restraint violations

the markers for helical structure continue past G24 in the

NOE violation (A 0.19 0.05

middle of helix 1. This may be due to a sequence of three

Downloaded from http://jb.asm.org/ on February 12, 2013 by guest

Largest violation 12

alanine residues that precede G24; alanine strongly favors Largest violation 10

helix formation (47). The presence of d N(i,i 2) NOEs and Atomic RMS deviations, residues 10 to 66

the lack of d N(i,i 4) NOEs suggest that helix 1 terminates Backbone (A) 1.03 0.30

All heavy atoms (A) 2.11 0.35

with a short stretch of a 310 helix.

Ramanchandran analysis, residues 10 to 66

Structure calculations. The input data for structure calcu- Residues in the most favored regions 92.8

lations included 1H NOEs from 15N- and 13C-separated Residues in additionally allowed regions 7.2

NOESY spectra, angular data from chemical shifts, and PCS as Residues in generously allowed regions 0

Residues in disallowed regions 0

described in Materials and Methods. The PCS data were par-

ticularly valuable as the number of long-range NOEs was rel- Energetics

atively small (Table 1), in part because most long-range con- CHARMm energy (kcal/mol 881

tacts involved poorly resolved side chain resonances and most

of the resonances from the aromatic rings of F27, Y31, and F52

were broad.

meaningful NOE data to restrict the protein backbone to any

Several rounds of structure calculations with CYANA (16)

region of conformational space.

and CANDID (22) were used to re ne the structure and assign

Description of the solution structure and comparison with

cross-peaks. The nal re nement was performed with the pro-

uncomplexed and HOT. The NMR-derived structure of

gram XPLOR-NIH, with which the structure was re ned

bound to 186 is a three-helix bundle (Fig. 2 and 3). Helix 1 is

against the complete set of distance, angular, and PCS (Dy3,

approximately antiparallel to helix 2 and parallel to helix 3,

Er3, and Ho3 ) restraints. After the nal round of calcula-

making angles of 170 and 50, respectively. Helix 2 makes an

tions, the 12 structures with the lowest energies were retained angle of about 130 with helix 3. A hydrophobic core between

from the 200 structures calculated (Fig. 2A). The backbone the helices is populated mostly by short- and long-chain ali-

conformation is well de ned except for nine residues at the N phatic residues. Several side chain resonances of K15 are

terminus and 10 residues at the C terminus. The structural shifted up eld, in agreement with the location of K15 between

statistics and residual violations of the experimental restraints helices 1 and 3 and packed above W51. D19 and R55 are

are shown in Table 1. Inclusion of the PCS restraints reduced adjacent and partially protected from the solvent, and they may

the root mean square (RMS) deviation values and slightly form a salt bridge. Several basic residues in helices 2 and 3 are

changed the relative orientations of the helices. Re nement oriented away from the protein core into the solution and are

against the distance and angular restraints yielded backbone clustered on one side of . These residues are arginine residues

RMS deviations of about 1.5 A (residues 10 to 66), whereas the at positions 42, 49, 53, 60, and 68.

RMS deviations decreased to about 1.0 A after inclusion of the The overall fold of the structure (Fig. 3A) is different from

PCS restraints. No well-de ned structure could be calculated the fold described previously for free in aqueous buffer (26)

for residues at the N terminus (M1 to L8) and the C terminus and is more similar to the fold of free in a mixed methanol-

(P70 to K76) because of the lack of meaningful intraprotein H2O buffer (39) and the fold of HOT in aqueous buffer (7).

medium- and long-range NOEs in these regions of . Narrow Although the number of long-range NOEs for complexed

with 186 is fairly small, the NOEs are more uniformly distrib-

cross-peaks for residues in the region from P70 to K76 indicate

that the C terminus of is highly mobile, whereas intermolec- uted over the sequence than they were for the calculation of

ular NOEs between A6 of and residues of 186 and confor- free in aqueous buffer (26). Since several of the characteris-

mational exchange broadening of resonances indicate that tically ring current shifted resonances and the patterns of

there may be some structure at the N terminus. NOEs are very similar in complexed and free in aqueous

The quality of the structure was supported by the appear- buffer, we concluded that the structure described here and the

ance of the Ramanchandran plot, generated by PROCHECK- structure of in the mixed solvent (39) re ect the structure of

NMR (28), which showed that all the residues in the gener- free in aqueous buffer more closely than the previously pub-

ously allowed and disallowed regions are in disordered parts of lished structure (26). The most signi cant difference between

free and 186-bound appears to be in different mobilities that

the protein (mostly the C terminus), for which there were no

4468 KENIRY ET AL. J. BACTERIOL.

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FIG. 2. NMR-determined structures of the subunit in complex with 186. (A) Stereo view of superposition of the backbone heavy atoms from

the nal ensemble of the 12 lowest-energy structures re ned 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).

IN - COMPLEX

VOL. 188, 2006 STRUCTURE OF 4469

inance 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 de ning the inter-

face between two proteins is to map the perturbation of chem-

ical shifts and NMR signal linewidths of one protein compo-

nent 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 iden-

ti ed in this way (6). Figure 4A shows the changes in the

FIG. 3. Comparison of the structures of the lowest-energy structure chemical shift of the amide proton, amide nitrogen, backbone

of 186-bound (A), the lowest-energy uncomplexed structure (B),

carbonyl, and carbons of when the complex is formed. Most

and the lowest-energy HOT structure (C). All structures have the same

of the residues show signi cant changes in chemical shifts; the

overall fold, but the angles between helices 1 and 3 are different. The

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ribbons trace the backbones of 186-bound, free (PDB code 2AE9), exception is the region between M33 and W51. These results

and HOT (PDB code 1SE7), respectively. suggest that the N-terminal segment of helix 1 and the C

terminus of helix 3 are involved in contacts with 186. The

previously identi ed (26) sections of uncomplexed that are

affected by conformational or chemical exchange phenomena

cause line broadening for different segments of the polypeptide

include the C-terminal sections of helices 1 and 3 (D19 to Y31

chain, particularly at the interface between helix 1 and helix 3.

Binding to 186 stabilizes the structure rather than inducing and R53 to S67). Thus, it is dif cult to distinguish the effects of

the direct contact with 186 from indirect effects due to a more

any large changes.

stable secondary structure.

HOT, which exhibits 53% sequence identity with (29), also

folds into a three-helix bundle (7). Ribbon diagrams of 186- NOE difference spectroscopy (10) is an alternative method

for mapping the binding epitope on a protein when the target

bound, uncomplexed in mixed buffer (39), and uncom-

protein is uniformly 13C/15N labeled and the other protein

plexed HOT are shown in Fig. 3. Each structure has helices

that are approximately the same length, and each structure has component is not labeled. We used this method to identify the

side chains of methyl-bearing residues of 13C/15N-labeled

loop regions that reverse the direction of the peptide chain.

that are at the interface with 186. Figure 5 shows the and

The orientations of helices 1 and 3 differ by about 10 for

modes of part of the 1H-13C correlation map of the complex

complexed and uncomplexed (Fig. 3A and B) and by 20 for

complexed and HOT (Fig. 3A and C). A DALI (23) search recorded with a mixing time of 300 ms. Substantial differential

of the Protein Data Bank (PDB) revealed that of the proteins relaxation ( 50%) was observed for the side chain resonances

in the PDB, HOT is the protein most closely related to, with of A6, M13, V16, L20, A21, A23, A26, and M33 (not shown),

a Z score of 5.8 for the structured regions. which reside in or just precede helix 1. A62 and L66, which

The structures of complexed and uncomplexed in the reside in helix 3 but face helix 1, also showed substantial dif-

mixed-solvent buffer are very similar, and the greatest differ- ferential relaxation. Figure 2D shows the locations of all of

ences are the orientation of helix 1 with respect to the other these residues in the 3D structure. Very little differential re-

two helices and the conformation of loop L1. These differences laxation was observed for V35, I36, A37, A39, I57, A58, and

may be due to structural changes on complexation with or to V64, which reside either in the turn structure or in helix 2 or 3.

the use of long-range PCS restraints. The latter better de ne A6 and L20 displayed several NOEs to the protons of unla-

beled 186 in half- ltered and double-half- ltered NOESY

the orientation of helices and loops, especially where there are

few long-range distance restraints. Mueller et al. (39) com- experiments (46). Furthermore, the methyl resonances of L20

mented on the paucity of long-range restraints in the vicinity of were shifted up eld, indicating the presence of a nearby aro-

matic residue, which could only come from 186 since there are

helix 2 and loop L2 of uncomplexed .

Electrostatic surface of . Figure 2B and C show opposing no nearby aromatic rings in the structure. In the structure of

faces of the electrostatic surface of the folded core of (resi- HOT, L21 (the corresponding residue) forms part of the hy-

dues 10 to 66) in the complex with 186. Figure 2C has the drophobic core (7), but in in the mixed-solvent buffer L20 is

same orientation as the stereo views shown in Fig. 2A and D, fully exposed to the solvent. The interaction interface of with

186 clearly involves the hydrophobic surface of helix 1. The

and Fig. 2B shows the opposing face of the surface shown in

Fig. 2C. The surface shown in Fig. 2B comprising mostly res- data are consistent with the chemical shift perturbation data

idues from helices 2 and 3 is highly positively charged with (Fig. 4A), in that the residues with the smallest chemical

several arginine and lysine side chains in close proximity. The shift perturbation show the smallest net differential relax-

functional signi cance of the cluster of arginine residues ori- ation effects.

An interface aligning helix 1 of with 186 is also qualita-

ented outward on helix 3 is unknown. There are also small

pockets of hydrophobic residues at the C terminus of helix 3 tively consistent with the results of experiments with lantha-

nide ions at the 186 active site, since residues in the N-

and the turn structure between helices 1 and 2. Hydrophobic

residues from helix 1 and helix 3 dominate the opposing face, terminal half of helix 1 had larger average PCS than residues in

which can be segregated into distinct regions (Fig. 2C). The helices 2 and 3 (Fig. 4B). This indicates that helix 1 is closer to

the active site of 186 than are other regions of . The position

surface of helix 1 is hydrophobic, resulting from the predom-

4470 KENIRY ET AL. J. BACTERIOL.

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FIG. 4. Changes in chemical shifts of upon binding to 186 and

1

HN pseudocontact shifts of . (A) Changes in chemical shifts of 1HN

(E) and 15N[amide] and 13C and 13C spins. There are

signi cant 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 (E), and Ho3 versus residue number.

FIG. 5. 1H-13C correlation maps obtained by the technique of Eich-

muller et al. (10), showing the differential decay of 1H magnetization

of the lanthanide ion with respect to the structure of is shown after preparation of the and spin modes of the 13C-HSQC spectra

of 186-bound 13C/15N-labeled . The two spectra were recorded with

in Fig. 2D. The closest distance of approach of the lanthanide

an NOE mixing period of 300 ms. A reference pair of spectra obtained

and helix 1 of, 15 A, is consistent with the presence of an

interaction site near a hydrophobic patch on 186 encompass- by using a 0-ms NOESY mixing period (not shown) was also recorded

to account for differences in one-bond heteronuclear scalar coupling

ing helix 1, strands 2 and 3, and the N terminus of helix 7 constants.

(6). Chemical shift mapping of the binding epitope of on 186

IN - COMPLEX

VOL. 188, 2006 STRUCTURE OF 4471

demonstrated that the interface between and is primarily to the structure of the close homolog HOT in aqueous buffer

hydrophobic (6), which is in good agreement with the results of (7). The differences among these structures are either due to

the experiments described above. This places the 186 epitope structural changes in the free and complexed proteins or due to

on primarily along helix 1, as shown by the hydrophobic limitations of the structure determination protocols. Since PCS

surface in Fig. 2C. Preliminary chemical shift mapping exper- restraints were used in the structure calculation, the present

iments of the HOT- 186 complex placed the interaction site NMR structure of satis es the PCS restraints better than the

near the N terminus of helix 1 and the C terminus of helix 3 (7), structure of either free (39) or HOT (7). We noted that the

in good agreement with the results of the NOE difference side chain of L21 is barely solvent exposed in the structure of

experiments. HOT (7), so it would not be expected to engage in intermo-

lecular NOEs with 186. In contrast, the corresponding residue

Notably, 15N-HSQC cross-peaks could be assigned for resi-

dues 4 to 9 preceding helix 1 in the samples with Er3 and in, L20, is much more solvent exposed in the complexed and

Ho3 . Some of these cross-peaks could also be observed for free forms of (39). Since the methyl groups of this leucine

have intermolecular NOEs with 186 and experience strong

the - 186-Dy3 complex, but they were more dif cult to as-

ring currents in the complex with 186 but not in free, the two

sign. The fact that cross-peaks could be observed at all for

structures of provide plausible models of the structure in the

these residues in the paramagnetic samples indicates that their

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complex. The small chemical shift changes observed for helix 2

amide protons must be located more than 14 A from the

and most of helix 3 in suggest that any changes in the global

paramagnetic ion (2). Cross-peaks of residues 2 and 3 were

fold of upon complexation with 186 are small.

already weak in the diamagnetic complex and were therefore

The intermolecular NOEs observed for L20 and the NOE

less likely to be observed in the paramagnetic complexes. The

difference experiments (10) resulted in identi cation of a hy-

magnitude of the PCS observed for these residues thus indi-

cates that they are closer to the active site of 186 than any drophobic surface along helix 1, as shown in Fig. 2C, as an

important part of the interaction surface with . The result is in

other residue of is but may not reach all the way to this site.

agreement with the chemical shift mapping of the binding

epitope of on 186 that indicated that the interface between

DISCUSSION

and is primarily hydrophobic (6). Electrospray ionization-

mass spectrometry studies of the dissociation of - 186 also

The small subunit of DNA polymerase III forms an iso-

suggested that the - interface is hydrophobic (17). Further-

lable complex with the catalytic N-terminal domain ( 186) of

the proofreading 3 -5 exonuclease subunit (19, 44). The more, preliminary chemical shift mapping experiments with

the HOT- 186 complex placed the interaction site near the N

high-resolution crystal structure of 186 (20) has provided im-

petus to modeling the structure of the - 186 complex, both to terminus of helix 1 and the C terminus of helix 3 (7), in

agreement with our results.

explain its stability and to obtain insight into the roles of in

The intermolecular NOEs and reduced mobility observed

DNA replication. Although is not necessary for the exonu-

clease activity of, it has been shown to protect 186 against for A6 show that the N-terminal segment of is involved i

Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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