©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Secondary Structure of Uracil-DNA Glycosylase Inhibitor Protein (*)

(Received for publication, August 24, 1994)

Suganthi Balasubramanian (1)(§) Richard D. Beger (1)(§) Samuel E. Bennett (2) Dale W. Mosbaugh (2) (3)(¶) Philip H. Bolton (1)(**)

From the  (1)Chemistry Department, Wesleyan University, Middletown, Connecticut 06450, and the (2)Departments of Agricultural Chemistry and Biochemistry and Biophysics and the (3)Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon 97331

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Bacillus subtilis bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) is an acidic protein of 84 amino acids that inactivates uracil-DNA glycosylase from diverse organisms (Wang, Z., and Mosbaugh, D. W.(1989) J. Biol. Chem. 264, 1163-1171). The secondary structure of Ugi has been determined by solution state multidimensional nuclear magnetic resonance. The protein adopts a single well defined structure consisting of five anti-parallel beta-strands and two alpha-helices. Six loop or turn regions were identified that contain approximately one half of the acidic amino acid residues and connect the beta-strands sequentially to one another. The secondary structure suggests which regions of Ugi may be involved in interactions with uracil-DNA glycosylase.


INTRODUCTION

Uracil residues may be introduced into DNA by the incorporation of dUMP during DNA synthesis or by the deamination of cytosine in DNA. In vivo cytosine deamination occurs at a genetically significant rate to produce a premutagenic UbulletG mispair(1, 2, 3) . If unrepaired, the uracil-DNA lesion will promote a C to T transition mutation after the next cycle of replication(3) .

Most organisms eliminate uracil residues from DNA by means of the uracil-excision DNA repair pathway. Uracil-DNA glycosylase initiates repair by cleaving the N-glycosylic bond between uracil and deoxyribose in DNA to yield free uracil and an abasic site(4) . The abasic sites induced by the action of uracil-DNA glycosylase cause changes in the structure, dynamics, and chemical stability of DNA (5, 6, 7, 8) . The chemistry of abasic sites has also been investigated (9, 10, 11) . The ubiquitous uracil-DNA glycosylase enzyme has been highly conserved throughout evolution as evidenced by the significant amino acid similarity, 55.7% identical residues, between human and bacterial forms of uracil-DNA glycosylase(12, 13, 14) . The functional similarity between Escherichia coli uracil-DNA glycosylase (Ung) (^1)and its human counterpart are underscored by the observation that human uracil-DNA glycosylase complements E. coli ung mutants(15) . Further, extracts of both E. coli and human cell lines have been shown to generate one nucleotide repair patches following excision of uracil from DNA, suggesting that the entire excision repair pathway itself may be highly conserved(16) .

Interestingly, the genomes of several mammalian viruses have been reported to encode uracil-DNA glycosylase(12, 17, 18, 19, 20) . The function of these viral uracil-DNA glycosylases remains to be fully understood. However, recent evidence suggests that the viral enzyme plays a key role in viral replication and reactivation since viral uracil-DNA glycosylase activity is rapidly induced following both herpes simplex virus and pox virus infection(21, 22, 23) . In adult neurons mammalian uracil-DNA glycosylase activity was reportedly undetectable; thus, uracil residues may accumulate either by the oxidative deamination of cytosine or by the misincorporation of dUMP in place of dTMP during DNA repair synthesis(24) . When a nerve cell is attacked by herpes simplex, pox, vaccinia, or pseudorabies virus, there is often a long latency period. During this period the genome of the virus can acquire uracil residues if host uracil-DNA repair is absent(24) . Further, uracil residues located in the herpes simplex 1 origin of replication (Ori(s)) hamper specific recognition by the origin binding protein(24) , and inactivation of the vaccinia and pox virus-encoded uracil-DNA glycosylase gene eliminates viral viability(20, 25) . Thus, uracil-DNA glycosylase plays an important role in the DNA metabolism of most biological systems, including virus.

Bacillus subtilis bacteriophages PBS 1 and 2 are unusual biological systems in that their double-stranded DNA genome contains uracil in place of thymine(26) . Upon infection of the host, phage-induced activities cause depletion of the dTTP pool concomitant with dramatic elevation of the dUTP pool, thereby facilitating the incorporation of dUMP into phage DNA during replication(27, 28, 29) . However, uracil-containing DNA synthesized under conditions of PBS infection must be protected from the B. subtilis uracil-DNA glycosylase to avoid uracil-DNA degradation. This is accomplished by expression of the PBS 2 ugi gene product which directly inactivates the host uracil-DNA glycosylase(29, 30) .

The ugi gene from bacteriophage PBS 2 has been cloned, sequenced, and overexpressed in E. coli, and the purified Ugi protein has been characterized(31, 32) . Ugi is an acidic protein with a pI of 4.2(33) . It is a heat stable, nonglobular monomeric protein of 84 amino acid residues that exhibits anomalous migration during SDS-polyacrylamide electrophoresis(32, 33, 34) . The Ugi protein has been shown to inactivate Ung by forming an extremely stable UngbulletUgi complex with 1:1 stoichiometry(33) . In addition, Ugi inactivates other uracil-DNA glycosylase including those isolated from Micrococcus luteus, Saccharomyces cerevisiae, rat liver, and human cells(30, 32, 34) . Inhibition of Ung occurs in a noncompetitive manner with respect to the uracil-DNA substrate(35) .

Kinetic studies have shown that the interaction of Ugi with Ung occurs by way of a two-stage mechanism involving a rapid pre-equilibrium ``docking'' step, followed by a rearrangement or ``locking'' step that leads irreversibly to the final complex(36) . Several lines of evidence suggest that Ugi binds Ung at or near the DNA binding site of the enzyme. Ugi binding to Ung prevents enzyme association with DNA (33) . Ung that has been UV-cross-linked to the DNA oligonucleotide dT at the enzyme DNA binding site fails to form a complex with Ugi; and Ung complexed with Ugi does not UV-cross-link to dT(37) . The high percentage of negatively charged amino acids, 12 Glu + 6 Asp out of 84 residues, further suggests that Ugi may act as a DNA mimic in binding to Ung. Such mimicry may provide an explanation as to how the Ugi protein from PBS is capable of inactivating uracil-DNA glycosylase from diverse biological systems that are under no selective pressure to maintain a Ugi binding site.

Additional understanding of the UngbulletUgi complex requires elucidation of the individual protein structures as well as that of the complex. In this article we report on the determination of the secondary structure of Ugi by multidimensional nuclear magnetic resonance (NMR) utilizing isotopically labeled protein. The Ugi secondary structure contains five antiparallel beta sheets, that form a contiguous structure with one another, and two helices. The information gained from the secondary structure allows assessment of the regions of Ugi likely to be involved in binding to Ung.


EXPERIMENTAL PROCEDURES

Preparation of [N]Uracil-DNA Glycosylase Inhibitor

E. coli JM105 transformed with pZWtac1 was grown at 37 °C in 10 liters of M9 medium in which [N]ammonium chloride (Cambridge Isotope Laboratories) was the sole nitrogen source. The [N]M9 medium was supplemented with 10 µg/ml thiamine and 0.01% ampicillin. When the culture density reached 7.5 times 10^8 cells/ml, isopropyl-1-thio-beta-D-galactopyranoside was added to 1 mM to induce ugi gene expression. Following 3 h of additional growth, cells were harvested by centrifugation, and the cell pellets were stored at -80 °C. A cellular extract was obtained subsequent to sonification and centrifugation; purification of [N]Ugi was performed essentially as described by Wang et al.(35) . The DEAE-cellulose chromatography step was carried out using a 19.6 cm^2 times 12-cm column. Purified Ugi was concentrated 12-fold under 55 p.s.i. of prepurified N(2) in an Amicon stirred cell equipped with a YM10 25-mm diameter DIAFLO membrane (Amicon). Diafiltration was then conducted with 200 ml of NMR buffer (see below). The sample was further concentrated to 3 mM [N]Ugi under a stream of nitrogen and contained 25 mM deuterated Tris, 0.2 mM EDTA, 0.2 mM EGTA, and 100 mM NaCl at pH 7.0. The Ugi preparation was >95% N-labeled based on the NMR results.

Nuclear Magnetic Resonance Analysis

All of the NMR spectra were obtained using a Varian Unityplus 400 spectrometer equipped with a Nalorac ID400 probe. The HSQC spectrum of the labeled Ugi was obtained in the usual fashion and a typical result is shown in Fig. 1. The data were collected with 256 increments of the evolution time and processed with a shifted Gaussian in both dimensions. Three-dimensional NOESY-HMQC and TOCSY-HSQC spectra were also obtained for Ugi. The TOCSY-HSQC data was used to group the resonances into spin systems with typical data shown in Fig. 2. The data were collected with eight transients per increment and obtained by the States-Haberkorn method along each indirectly detected dimension. There were 128 increments of t(1) and 24 increments of t(2) for each of the complex data sets. The data was linear predicted to 256 points along t(1) and 48 points along t(2). The data were Fourier transformed into 512 times 128 times 1024 points using shifted Gaussians along each dimension. The TOCSY-HSQC data was obtained using the sensitivity enhancement feature(38) . Fig. 3contains some of the NOESY-HMQC data used to make sequential assignments in one of the helices and one of the beta-sheet regions of the protein. The data was collected with 16 transients per increment and obtained by the States-Haberkorn method along each indirectly detection dimension. There were 128 increments of t(1) and 20 increments of t(2) for each of the complex data sets. The data were linear predicted to 256 points along t(1) and 40 points along t(2). The data were Fourier transformed into 512 times 128 times 1024 points using shifted Gaussians along each dimension. Conventional DQCOSY, NOESY, and TOCSY spectra were also obtained on the sample. The chemical shift differences of the CH protons relative to the random coil, unshifted positions were also determined and reported in Fig. 4.


Figure 1: The HSQC spectrum was obtained on a N-labeled sample of Ugi. The cross-peaks are at the chemical shifts of the nitrogen along axis F(1) and the chemical shift of the proton along axis F(2). The cross-peaks of the amide nitrogen-amide proton pairs are labeled by residue number and residue type and the cross-peak of the ring nitrogen of the tryptophan is also labeled. The chemical shifts of the amide proton and nitrogen resonances are given in Table 1. The unlabeled cross-peaks arise from glutamine and asparagine NH(2) groups and some of the assignments of these cross-peaks are given in Table 1.




Figure 2: The spectra shown are taken from a three-dimensional TOCSY-HSQC data set obtained on Ugi. The two spectra show the connectivities made from amide proton to side chain protons. The bottom spectrum is from the most congested region of the three-dimensional data.




Figure 3: The spectra shown are taken from a three-dimensional, NOESY-HMQC data set obtained on Ugi. The spectra at the top are arranged as ``strips'' and the sequential NOE connectivities used to assign the resonances of the alpha-helix from residue 27 to 33 are shown. All of the strips have the same range of chemical shifts along F(3), and the width of the chemical shift range along F(2) is indicated. The spectra at the bottom are also arranged as ``strips'' and show the sequential NOEs used to assign the resonances of the beta-strand residues from 68 to 77.




Figure 4: The sequential NOEs between the residues of Ugi are depicted with the NOEs characterized as small, medium, or large. The sequential NOEs are depicted as well as those between residue i and i + 2, and i and i + 3. The residues with slow amide exchange are indicated by a filled circle. The chemical shift of the CH proton of each assigned residue relative to the chemical shift of the random coil, unshifted position is given as DeltaH.





Three-dimensional NOESY-HMQC and TOCSY-HSQC spectra were obtained at 15, 30, and 37 °C so as to resolve the residual water signal from some of the C protons. The NMR properties of the protein did not change significantly over this temperature range. The NMR properties of the protein did not change significantly over the pH range of 5.5 to 8. The amide protons exchange sufficiently slowly with water at pH 7 to allow the NMR experiments to be carried out a this pH.

The exchange rates of the amide protons were determined at pH 7.0 and 20 °C by observing the rate of loss of intensity of cross-peaks in the two-dimensional HMQC spectrum as a function of time after the Ugi has been transferred from a H(2)O solution to a ^2H(2)O solution. The amide protons with the slowest exchange rates are indicated in Fig. 4.


RESULTS AND DISCUSSION

The characterization of Ugi began with the determination that this protein adopts a single well defined structure in solution. The HSQC spectrum in Fig. 1contains cross-peaks whose coordinates are the chemical shifts of the amide nitrogen and amide proton of the non-proline residues. (Chemical shifts of all proton and nitrogen resonances are given in Table 1.) The cross-peaks arising from the NH(2) of glutamine and asparagine also appear in this region but are much broader than the cross-peaks from amides. The examination of this spectrum shows that the protein adopts a defined structure since there is a wide dispersion of the chemical shifts along both the nitrogen and proton dimensions. The presence of a number of resonances with proton chemical shifts between 8 and 10 ppm suggests that the protein has a significant percentage of the residues in beta-strands.

Many proteins are investigated by NMR at low pH since the crucial amide protons undergo rapid base-catalyzed exchange with the bulk water at neutral pH. It was found that the amide protons of Ugi exchange sufficiently slowly with water at pH 7.0 to allow the NMR experiments to be carried out successfully. This slow rate of amide exchange is also indicative of a highly structured protein. Examination of the spectrum indicated that only one structural form of the protein was present in solution, as there is one cross-peak for the amide of each non-proline residue in the protein. These NMR results indicated that Ugi had a well defined, single structure in solution and that it was well suited for NMR structural studies based on multidimensional NMR and sequential assignment strategies.

The next step in the structural characterization was the determination of the chemical shifts of the CH protons. The resonances of these protons were ascertained from the information present in a DQCOSY spectrum of the proton. The DQCOSY data, not shown, contains cross-peaks between the amide and alpha protons, and makes it possible to distinguish resonances in the HSQC spectrum of amide protons from those of the amino protons of glutamine and asparagine residues.

Once determined, the resonances of the amide and alpha protons were combined with the results of three-dimensional TOCSY-HSQC data to identify the spin systems(39, 40, 41) . These experiments allow the connection of the alpha protons to the other protons of the same residue via spin-spin and scalar couplings, and hence allow the classification of groups of resonances to a residue of a particular spin system type. Typical TOCSY-HSQC data is shown in Fig. 2.

The sequential assignments of the individual spin systems were based primarily on the information present in the three-dimensional NOESY-HMQC data. From this information the sequential connectivities from amide proton of residue i to amide proton of residue i - 1, the amide proton of residue i + 1 as well as from the amide proton of residue i to CH proton of residue i - 1 were determined(39, 40, 41) . The spectra containing the sequential connectivities used to assign the resonances of residues in the long helix are shown in Fig. 3. Fig. 3also contains the set of spectra used to make the sequential assignments in one of the beta-strand regions. The sequential NOE connectivities of all of the residues are indicated in Fig. 4. All of the non-proline residues were assigned except for the terminal residues 1 through 4.

Characterization of the secondary structural elements of Ugi was based on several pieces of information. The helices were identified primarily on the basis of strong NOE connectivities between the NH of residue i and the NH of residue i + 1 and residue i - 1. The helices were also characterized by the presence of weak to medium connectivities from the NH proton of residue i to the NH proton of residue i + 3 and weak NOE connectivities between the CH of residue i and the NH proton of residues i + 2 and i + 3. These NOE connectivities are depicted in Fig. 4and allowed the identification of the presence of two alpha-helices in the secondary structure. One of these helices is from residue 5 to residue 14 and the other helix is from residue 27 to residue 35. In general, downfield shifts, positive differences, correlate with beta-sheets and upfield shifts, negative differences, correlate with alpha helices(42) . The chemical shift differences of the CH protons relative to the random coil, unshifted positions are also given in Fig. 4. In the two regions of Ugi assigned to alpha-helices the expected negative differences between the observed chemical shifts and the random coil chemical shifts are found.

The presence of five beta-strand regions in the secondary structure of Ugi was determined primarily on the basis of the presence of strong NOE connectivities between the CH proton of residue i and the amide proton of residue i + 1, strong connectivities between the CH proton of residue i and the amide proton of residue i + 1, and by the fact that the amide protons of residues in beta-strands tend to exchange more slowly with water. The beta-strands are from residues 20-24, 41-48, 53-60, 69-74, and 79-84. The characteristic NOE connectivities of the beta-strand regions suggested that they are all anti-parallel(40, 41, 43) . This information is summarized in Fig. 4. The chemical shift differences of the CH protons of the beta-strands are expected to exhibit positive differences relative to the random coil, unshifted positions. The experimental results, summarized in Fig. 4, are in agreement with this expectation. Indeed, grouping resonances to spin system type, sequential assignments, NOE connectivities, amide exchange rates, and differences in chemical shift relative to the random coil, unshifted positions are all consistent with each other and the secondary structure assignments.

The determination of the arrangements of the beta-strands relative to one other was based on the information present in NOE connectivities between CH and CH protons on separate beta-strands. These NOEs appear at every other site along a beta-strand, and are indicated in the depiction of the beta-strands in Fig. 5and in the depiction of the entire secondary structure in Fig. 6. These connections allowed the arrangement of the five beta-strands in a connected anti-parallel manner that is shown in Fig. 5and Fig. 6.


Figure 5: A depiction of the topology of the beta-strands of Ugi. The protons connected by NOEs are indicated by the double-headed arrows.




Figure 6: A depiction of the overall secondary structure of Ugi. The beta-strand regions are indicated by the large arrows and the alpha-helices by the helices.



The presence of five beta-strands in Ugi is consistent with prior data concerning the high stability of the protein(30, 31, 32, 33) . Other proteins of similar size with five beta-strands include ubiquitin (44) and the RAS binding domain of human RAF-1(45) . Both of these proteins contain five anti-parallel beta-strands and a single helix, and they are very stable proteins, presumably because of the high beta-content of the structures(44, 45) . In ubiquitin and the RAS binding domain, the helix is between the first and fourth beta-strands; whereas in Ugi the intervening helix is between the first and second beta-strands. The somewhat unusual feature of the beta-strands in Ugi is that they are connected sequentially.

The secondary structure has six loop or turn regions in which one half of the 18 acidic amino acid residues of the protein are found. The regions from residues 36-40, 49-52, and 61-68 contain six acidic groups, two each, whereas the region 75-78 contains one acidic group; other regions constituting loops or turns are residues 15-19, 25, and 26. The anomalous electrophoretic mobility of Ugi may arise, in part, from the presence of these acidic groups in regions of the protein not involved in the secondary structure.

The biological function of Ugi is to inhibit uracil-DNA glycosylase, a DNA repair enzyme that binds single- and double-stranded DNA, recognizes uracil residues, and then catalyzes the hydrolysis of the N-glycosylic bond between the uracil base and the deoxyribose-phosphate ``backbone'' of DNA. Ugi and Ung react to form a stable protein-protein complex that does not interact with DNA. Thus, Ung may recognize Ugi because the inhibitor protein mimics in some fashion the general properties of its DNA substrate. The secondary structure of Ugi can reveal which regions and residues may be critical to the interaction with Ung.

If Ugi is to mimic DNA, then it should have a number of acidic groups which can mimic the electrostatic potential of the phosphate groups in DNA. The secondary structure has eight acidic groups that may be available for this role. The regions 36-40, 49-52, and 61-68 are prime candidates for this activity since these regions are not involved in the secondary structure and contain six of the 18 acidic residues present. While the tertiary structure of the beta-strands is not yet known, it may be the case that a barrel type structure is formed in which these negative changes are distributed in a manner similar to the arrangement of the negative changes of DNA, and thus may act as the primary binding site of Ugi to uracil-DNA glycosylase. It is noted that amino acids 49-52 contains the sequence Glu-Ser-Thr-Asp and that the serine and threonine residues may serve as hydrogen bond donors in complex formation. It is also possible that residues 75-78 are involved in complex formation as this region contains the sequence Ser-Gln-Gly-Glu. The serine and glutamine residues offer hydrogen bonding opportunities and the glutamic acid a negative charge.

The helix from residues 27 to 35 has a distinctly charged side and a hydrophobic side; however, the helix from 5 to 13 does not appear to have as significant a difference between its two sides. While either or both of these helices could be involved in complex formation, the secondary structure does not make a clear prediction as to what that role(s) might be.

The beta-strand regions of proteins are typically not involved directly in the activity of proteins. Hence, the beta-strand regions of Ugi are likely to be important structurally but are not expected to play significant roles in the interaction with uracil-DNA glycosylase. Based on the secondary structure of the beta-strands shown in Fig. 5, no specific pattern in the arrangement of the acidic or other types of residues can be inferred.

Ugi contains only three aromatic residues and of these Tyr and Trp are not present in a secondary structural feature. If Ugi binds to the uracil binding site of uracil-DNA glycosylase, then it is possible that an aromatic residue may occupy that site. The Tyr and Trp are the only residues apparently available for this role.

The tertiary structure of Ugi in solution is now being determined in order to gain information concerning the arrangement of negative charges and other features of Ugi which may be important to the interaction with uracil-DNA glycosylase. Additional studies are planned which will examine the changes in the amide exchange rates and chemical shifts of the amide nitrogen and protons, as well changes in the mobility of the amide nitrogens which may occur when Ugi is complexed with uracil-DNA glycosylase. These studies should offer information about the regions of Ugi which directly interact with uracil-DNA glycosylase. Preliminary results on the complex indicate that the structure of Ugi undergoes considerable change upon complex formation. This is consistent with kinetic results which indicated the presence of a slow, locking step in complex formation(36) .


FOOTNOTES

*
This work was supported by the National Institutes of Health Grants GM 32823 and ES 00210 (to D. W. M.) and National Science Foundation Grant DMB 91-05003 (to P. H. B.). The NMR spectrometer was purchased with support from National Science Foundation BIR 93-03077 (to P. H. B.). This is Technical Report 10,551 from the Oregon Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Contributed equally to the structure determination.

To whom correspondence should be addressed: Dept. of Agricultural Chemistry, Oregon State University, Agricultural and Life Sciences Bldg. 1007, Corvallis, OR 97331-7301. Tel.: 503-737-1797; Fax: 503-737-0497.

**
To whom correspondence should be addressed. Tel.: 203-685-2668; Fax: 203-685-2211.

(^1)
The abbreviations used are: Ung, uracil-DNA glycosylase; Ugi, uracil-DNA glycosylase inhibitor; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect correlation spectroscopy; TOCSY, total correlation spectroscopy; HMQC, heteronuclear multiple quantum correlation spectroscopy; HSQC, heteronuclear single quantum correlation spectroscopy; DQCOSY, double quantum filtered correlation spectroscopy.


ACKNOWLEDGEMENTS

We appreciate the assistance of Dr. Igor Goljer in performing some of the 400 MHz NMR experiments.


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