Site-directed Mutagenesis and Characterization of Uracil-DNA Glycosylase Inhibitor Protein
ROLE OF SPECIFIC CARBOXYLIC AMINO ACIDS IN COMPLEX FORMATION WITH ESCHERICHIA COLI URACIL-DNA GLYCOSYLASE*

(Received for publication, December 18, 1996, and in revised form, May 19, 1997)

Amy J. Lundquist Dagger , Richard D. Beger §, Samuel E. Bennett Dagger , Philip H. Bolton §** and Dale W. Mosbaugh Dagger Dagger Dagger §§

From the Dagger  Departments of Agricultural Chemistry and Biochemistry and Biophysics and the Dagger Dagger  Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon 97331 and the § Chemistry Department, Wesleyan University, Middleton, Connecticut 06459

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein inactivates uracil-DNA glycosylase (Ung) by acting as a DNA mimic to bind Ung in an irreversible complex. Seven mutant Ugi proteins (E20I, E27A, E28L, E30L, E31L, D61G, and E78V) were created to assess the role of various negatively charged residues in the binding mechanism. Each mutant Ugi protein was purified and characterized with respect to inhibitor activity and Ung binding properties relative to the wild type Ugi. Analysis of the Ugi protein solution structures by nuclear magnetic resonance indicated that the mutant Ugi proteins were folded into the same general conformation as wild type Ugi. All seven of the Ugi proteins were capable of forming a Ung·Ugi complex but varied considerably in their individual ability to inhibit Ung activity. Like the wild type Ugi, five of the mutants formed an irreversible complex with Ung; however, the binding of Ugi E20I and E28L to Ung was shown to be reversible. The tertiary structure of [13C,15N]Ugi in complex with Ung was determined by solution state multi-dimensional nuclear magnetic resonance and compared with the unbound Ugi structure. Structural and functional analysis of these proteins have elucidated the two-step mechanism involved in Ung·Ugi association and irreversible complex formation.


INTRODUCTION

The Bacillus subtilis bacteriophage PBS1 and -2 exhibit a unique genetic system that naturally contains uracil in place of thymine in a double-stranded DNA genome (1, 2). Stable incorporation of uracil residues into the phage DNA is achieved by the substitution of dUTP for dTTP as precursor in DNA synthesis and the concomitant inactivation of the host uracil-mediated base excision DNA repair pathway (2-4). To block uracil-DNA repair and protect the uracil-containing phage DNA from degradation, an early phage gene (ugi)1 is expressed that inhibits the B. subtilis uracil-DNA glycosylase. The amino acid sequences of the PBS1 and -2 Ugi proteins appear to be identical (5-7).

The PBS2 ugi gene encodes a small (9,474 dalton), monomeric, heat stable protein of 84 amino acids that inactivates uracil-DNA glycosylases from diverse biological sources (5, 8, 9). The ugi gene product is an unusually acidic protein (12 Glu, 6 Asp) with a pI = 4.2 that migrates anomalously during SDS-polyacrylamide gel electrophoresis (5, 10, 11). Ugi inactivates Ung by forming a tightly bound noncovalent complex with 1:1 stoichiometry that is essentially irreversible under physiological conditions (10, 12). Stopped-flow kinetic studies of the Ugi interaction with Escherichia coli Ung indicate that complex formation is accomplished through a two-step binding reaction (12). In the initial step, the association between free Ugi and Ung is characterized by a rapid pre-equilibrium reaction with a dissociation constant (Kd) of 1.3 µM; the second step, the formation of an irreversible complex, is characterized by the rate constant k = 195 s-1. Thus, Ung·Ugi complex formation initiates with a "docking" interaction that facilitates optimal alignment between the two proteins. If correct alignment between Ung and Ugi does not occur, a reversible association will transpire. If, however, proper alignment is achieved, then a "locked" complex quickly follows.

The secondary and tertiary structures of free Ugi were recently determined by solution state multi-dimensional NMR techniques and found to include two alpha -helices and five anti-parallel beta -strands as illustrated in Fig. 1 (13, 14). The five contiguous beta -strands are connected by short loop regions to form an anti-parallel beta -sheet. Analysis of the electrostatic potential of Ugi revealed several striking features (14). Seven of the 18 acidic amino acid residues (Glu-20, Asp-48, Glu-49, Asp-52, Glu-53, Asp-74, and Glu-78) come together to form a region of high negative potential on one face of the protein. Each of the residues that form this electrostatic region or "knob" are located immediately adjacent to or terminate a beta -strand. Two other acidic amino acid residues (Asp-40 and Asp-61) are also in juxtaposition to the end of beta -strands; Glu-78 and Glu-64 reside in the loop regions (14). The remaining seven negatively charged residues are located in the alpha 1-helix (Asp-6, Glu-9, and Glu-11) and alpha 2-helix (Glu-27, Glu-28, Glu-30, and Glu-31). Both the alpha 1- and alpha 2-helix elements project away from the beta -sheet and are located on potentially flexible arms of the polypeptide (14). Furthermore, the alpha 2-helix is longitudinally segmented into a hydrophobic face and a negatively charged face where the four glutamic acid residues protrude.


Fig. 1. Tertiary structure of the uracil-DNA glycosylase inhibitor protein and location of Glu and Asp residues. The tertiary structure of Ugi determined by solution state NMR techniques (14) is shown with the 12 Glu residues in red and 6 Asp residues in yellow. Secondary structural elements include the alpha 1-helix (Ser-5 to Lys-14), alpha 2-helix (Glu-27 to Asn-35), beta 1-strand (Glu-20 to Met-24), beta 2-strand (Ile-41 to Asp-48), beta 3-strand (Glu-53 to Ser-60), beta 4-strand (Ala-69 to Asp-74), and beta 5-strand (Asn-79 to Leu-84).
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Several lines of evidence suggest that some of the negatively charged amino acid residues of Ugi may act as a DNA mimic and mediate the interaction with Ung. First, UV-catalyzed cross-linking of oligonucleotide (dT)20 to the DNA-binding site of Ung blocked Ugi from forming a Ung·Ugi complex (15). Second, the x-ray crystallographic structure of Ugi in complex with human (16) and HSV-1 (17) uracil-DNA glycosylase reveals that the interfacing surface of Ugi shares shape and electrostatic complementarity to the DNA-binding groove of the enzyme (16, 17). Third, the negative electrostatic knob of Ugi exhibits an electrostatic potential of >6.6 kcal, which is similar to that generated by the negatively charged phosphate backbone of DNA (14). Fourth, the recent x-ray structure of human uracil-DNA glycosylase complexed with a 10-bp oligonucleotide containing a target G·U mispair reveals the DNA complexed at the same site as Ugi (18). Fifth, charge neutralization by carbodiimide-mediated adduction of Ugi carboxylic acid residues caused a decrease in inhibitor protein activity (19). Finally, chemical adduction of specific glutamic acid residues (Glu-28 and Glu-31) of Ugi located in the alpha 2-helix correlated with the formation of an unstable Ung·Ugi complex (19).

Bennett et al. (12) suggested that after the Ung/Ugi association, the transition to the locked configuration may involve a conformational change in either one or both proteins. Subsequently, Sanderson and Mosbaugh (19) proposed that the locking reaction is caused predominantly by a change in Ugi structure. This position is supported by a comparison of the crystal structures of free human and HSV-1 uracil-DNA glycosylase with the structures of each enzyme in complex with Ugi (16, 17, 20, 21). In both cases, the tertiary structure of the enzyme shows only minor structural changes. In contrast, a comparison of the heteronuclear multiple quantum correlation spectra of free and bound [15N]Ugi indicates that many residues of Ugi undergo conformational change upon binding to Ung (14). At present, the tertiary structure of the unbound Ugi protein in solution was determined solely by solution state NMR techniques (14). Also, a comparison of the solution structure of free Ugi with the crystal structure of Ugi complexed with either the human or HSV-1 uracil-DNA glycosylase demonstrates significant structural changes occur in Ugi (14, 16, 17). A more complete understanding of the docking and locking reactions may well be gained by determining the solution state structure of Ugi in complex with Ung.

In the present report we (i) conduct site-directed mutagenesis of seven acidic residues of Ugi; (ii) purify each mutant Ugi protein to apparent homogeneity; (iii) characterize each mutant Ugi with regard to specific activity and Ung·Ugi complex stability and reversibility; (iv) determine the structural similarity between wild type Ugi and the Ugi mutant proteins using NMR methods; (v) compare the free and complexed Ugi solution structures; and (vi) model the interactions in the wild type and mutant Ung·Ugi complexes.


EXPERIMENTAL PROCEDURES

Materials

Restriction endonucleases (EcoRI, EcoRV, HindIII, PstI, SacI, and XmnI), T4 polynucleotide kinase, T4 DNA polymerase, and T4 DNA ligase were purchased from New England Biolabs. Isopropyl-1-thio-beta -D-galactopyranoside and ScaI were obtained from Life Technologies, Inc. and AcyI came from Promega. [3H]Leucine and [35S]methionine were obtained from NEN Life Science Products; [3H]dUTP was from Amersham Corp., and [13C]glucose and [15N]ammonium chloride were from Cambridge Isotope Laboratories.

E. coli JM105 was provided by W. Ream (Oregon State University), and E. coli CJ236 was obtained from T. A. Kunkel (NIEHS). Epicurian coli XL2-Blue ultracompetent cells, phagemid pBluescript II SK(-), and VCS-M13 helper phage were supplied by Stratagene. Plasmid pKK223-3 was obtained from Pharmacia Biotech Inc.; pZWtacl (22) and pSB1051 (12) were constructed as described previously. Oligonucleotides were synthesized using an Applied Biosystems 380B DNA Synthesizer by the Center for Gene Research and Biotechnology (Oregon State University).

Site-directed Mutagenesis of the Uracil-DNA Glycosylase Inhibitor Gene

The first step in the site-directed mutagenesis procedure involved subcloning of the ugi gene into a pBluescript-based phagemid to produce single-stranded DNA. The pZWtac1 EcoRI-HindIII restriction fragment (726 bp) containing the ugi gene (Fig. 2) was inserted into the corresponding EcoRI and HindIII sites of pBluescript II SK(-) using T4 DNA ligase. The resulting phagemid (pAL) was transformed into E. coli JM105, plated on LB plates containing 100 µg/ml ampicillin, 40 mM isopropyl-1-thio-beta -D-galactopyranoside, and 40 µg/ml 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside, after which pAL DNA was purified from white colonies. E. coli CJ236 (dut, ung) was then transformed with phagemid pAL and grown at 37 °C in 1.0 liter of 2 × YT medium supplemented with 34 µg/ml chloramphenicol and 100 µg/ml ampicillin. Upon reaching a cell density of 108 cells/ml (1 A600 nm = 8 × 108 cells/ml), uridine was added to a final concentration of 0.25 µg/ml; VCS-M13 helper phage was added at a multiplicity of infection equal to 1.0, and incubation was continued at 37 °C for 1.5 h. Kanamycin (26 µg/ml final concentration) was added to select for infected E. coli cells and growth continued for an additional 5.5 h. The culture was centrifuged at 7000 rpm for 15 min at 4 °C in a GSA (Sorvall) rotor, and the supernatant fraction was processed to precipitate pAL phage with the addition of 0.25 volume of a 15% PEG-8000 and 2.5 M NaCl solution. Phage DNA was isolated from the supernatant fraction following extractions with phenol and chloroform:isoamyl alcohol (24:1) and precipitation with ethanol (23). The precipitated DNA was centrifuged at 9500 rpm for 20 min at 4 °C in a SA600 (Sorvall) rotor, air dried, and resuspended in 500 µl of TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). This single-stranded uracil-substituted DNA that contained the antisense ugi gene sequence was termed pALU(ss) DNA.


Fig. 2. Strategy for oligonucleotide-directed mutagenesis of the ugi gene. The EcoRI/HindIII DNA fragment (726 bp) from pZWtac1 contains the ugi structural gene with nucleotide position +1 starting the ATG codon and position +255 terminating the TAA stop codon. Seven oligonucleotides were synthesized as shown that are partially complementary to the antisense sequence of the ugi structural gene over the region depicted. The beginning and ending nucleotide positions are indicated. Noncomplementary bases were engineered at the positions (*) to introduce site-specific mutations that eliminated Glu or Asp residues by amino acid substitution and introduced endonuclease recognition sites into the ugi gene. Endonuclease recognition sites are underlined, and the cleavage sites are indicated (and ;2). Each oligonucleotide was separately hybridized to uracil-containing pALU(ss) DNA and served to initiate in vitro primer extension. Oligonucleotidedirected mutagenesis was conducted as described by Kunkel et al. (23) with modifications indicated under the "Experimental Procedures." The specific amino acid location and substitution produced by each oligonucleotide is indicated in the inlaid table along with the novel restriction endonuclease sites and cleavage location within the 726-bp DNA fragment.
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The second step involved in vitro primer extension of various oligonucleotides containing site-directed mutations in the ugi structural gene. Deblocked/deprotected oligonucleotides were purified by Sephadex G-25 chromatography and phosphorylated at the 5' end using ATP and T4 polynucleotide kinase as described previously (23). Defined DNA primer/templates were constructed by annealing various oligonucleotides (20 pmol) to pALU(ss) DNA at a 3:1 (primer:template) ratio in a 100-µl volume as described (24, 25). Primer extension reaction mixtures (100 µl) contained 20 mM Hepes-KOH (pH 8.0), 2 mM dithiothreitol, 10 mM MgCl2, 2 mM ATP, 500 mM each of dATP, dCTP, dGTP, and dTTP, 0.5 mg/ml BSA, 6 units of T4 DNA polymerase, 200 units of T4 DNA ligase, and 1.8 pmol of the heteroduplex pALU DNA primer/template. Following incubation for 5 min at 25 °C and then 2 h at 37 °C, a sample (10 µl) terminated with the addition of 1.5 µl of 0.1 M EDTA was analyzed by 1% agarose gel electrophoresis to determine the extent of primer extension. Transformation of E. coli JM105 CCMB80 competent cells was performed with 10 µl of the primer extension reaction mixture (26). Transformed bacterial colonies were grown on LB plates containing 100 µg/ml ampicillin, and isolated colonies were subsequently grown to saturation in 2 ml of 2 × YT medium supplemented with ampicillin. Plasmid DNA (pSugi) was isolated using the Wizard Miniprep DNA purification technique (Promega). Isolated plasmids (pSugi) were analyzed by 1% agarose gel electrophoresis after three independent restriction endonuclease digestions as follows: (i) HindIII, (ii) HindIII plus EcoRI, and (iii) the appropriate novel restriction endonuclease for the site introduced into the pSugi DNA by site-directed mutagenesis (Fig. 2).

The third step of the procedure involved subcloning the ugi genes containing site-directed mutations from pSugi to the pKK223-3 derived overexpression vector pZWtac1, replacing the wild type ugi gene. Both pSugi and pZWtac1 DNA (1 µg) were separately digested with excess HindIII and EcoRI, and the products were resolved by 0.8% agarose (low melting point) gel electrophoresis. Bands corresponding to the ugi gene containing DNA fragment (726 bp) from pSugi and the 4.6-kb fragment from pZWtac1 were excised, DNA extracted, and purified using glass milk (27). Samples containing the 726-bp and 4.6-kb EcoRI/HindIII DNA fragments were mixed, and the complementary ends were joined using T4 DNA ligase. The ligation reaction mixture (10 µl) contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP, 25 µg/ml BSA, 0.75 pmol of the 726-bp fragment, and 0.25 pmol of the 4.6-kb DNA fragment. Following incubation for ~16 h at 16 °C, the ligation mixture was used to transform either E. coli JM105 or XL2-Blue competent cells (26). Transformed colonies were isolated after growth on LB plates supplemented with 100 µg/ml ampicillin and grown overnight in 2 ml of 2 × YT medium containing ampicillin. Plasmid DNA was then isolated using the Wizard Miniprep procedure and analyzed using restriction endonuclease digestion for HindIII, HindIII plus EcoRI, and the appropriate introduced restriction sites (Fig. 2) as described above. In the resulting plasmids (pKugi), the mutant ugi gene was expressed under the control of the isopropyl-1-thio-beta -D-galactopyranoside-inducible tac promoter.

DNA Sequence Analysis

Double-stranded pKugi DNA containing site-directed mutations were used as templates for nucleotide sequencing using the dideoxynucleotide chain termination method originally described by Sanger et al. (28). Either primer FP/PKK that was complementary to the ugi sense strand at position -143 to -122 or primer IRPUGI that hybridized to the antisense strand at position +379 to +401 was used to initiate DNA polymerase-mediated synthesis. DNA sequencing was conducted using an Applied Biosystems model 373A sequencer by the Center for Gene Research and Biotechnology (Oregon State University).

Purification of Uracil-DNA Glycosylase and Inhibitor Protein

Purification of fraction V [leucine-3H]Ung (13.5 cpm/pmol) was carried out similarly to that described by Bennett et al. (15). Nonradioactive Ung (fraction V) was purified from E. coli JM105/pSB1051 grown in LB medium supplemented with 100 µg/ml ampicillin (19). Fraction IV [35S]methionine-labeled Ugi (40 cpm/pmol) and [13C,15N]Ugi were purified as described by Sanderson and Mosbaugh (19) from E. coli JM105/pZWtac1 cultures (1-1.5 liters) grown in M9 minimal medium supplemented with 100 µg/ml ampicillin, 10 µg/ml thiamine, and either 4.2 nmol of [35S]methionine or 0.2% [13C]glucose and 0.17% [15N]ammonium chloride, respectively. Nonradioactive Ugi and the various site-directed mutants of Ugi were purified following the same procedure, except that bacterial growth occurred in LB medium containing 100 µg/ml ampicillin.

Purification of [3H]Ung·Ugi Complexes

Wild type Ugi or various site-directed mutants of Ugi protein were mixed with [3H]Ung in buffer A (30 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, 5% (w/v) glycerol) containing 50 mM NaCl and incubated at 25 °C for 10 min and then at 4 °C for 20 min. Following complex formation, each sample was applied to a DE52 cellulose column equilibrated in buffer A containing 50 mM NaCl, washed with equilibration buffer, and step-eluted, as described previously (19). Fractions (1 ml) were collected and samples were analyzed for 3H radioactivity. The [3H]Ung·Ugi complex was detected by 18% nondenaturing polyacrylamide gel electrophoresis, and fractions containing complex were pooled and concentrated using a Centriplus-10 (Amicon) concentrator.

Enzyme Assays

Uracil-DNA glycosylase inhibitor activity was measured under previously described conditions (10). When appropriate, Ugi was diluted with IDB buffer (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl). One unit of uracil-DNA glycosylase inhibitor inactivates 1 unit of uracil-DNA glycosylase in the standard reaction. Uracil-DNA glycosylase activity was similarly measured except that Ugi was omitted (10). One unit of uracil-DNA glycosylase is defined as the amount that releases 1 nmol of uracil/h under standard reaction conditions.

Protein Measurements

Protein concentrations were determined by absorbance spectroscopy using the molar extinction coefficients epsilon 280 nm = 4.2 × 104 liter/mol cm (Ung) and epsilon 280 nm = 1.2 × 104 liter/mol cm (Ugi). The concentration of [3H]Ung·Ugi complex was determined from the specific radioactivity of [3H]Ung (10).

Electrophoresis

Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis was performed essentially as described by Laemmli (29) and modified by Bennett et al. (12).

Nondenaturing polyacrylamide slab gel electrophoresis was performed at 4 °C essentially as described by Sanderson and Mosbaugh (19) with resolving gels containing 18% acrylamide and 0.39% N,N'-methylenebis (acrylamide). The gel was immediately stained using the rapid stain procedure described by Reisner (30) and modified by Sanderson and Mosbaugh (19).

Nondenaturing polyacrylamide tube gel electrophoresis was conducted using the same components in the resolving gel (9 × 0.6 cm diameter) and stacking gel (1 cm) as described above. Following electrophoresis the resolving gel was either stained with Coomassie Brilliant Blue G-250 or sliced horizontally into 3.1-mm sections, placed into scintillation vials, dehydrated overnight, and solubilized in 500 µl of H2O2 at 55 °C for 24-36 h as described by Sanderson and Mosbaugh (19). After complete solubilization, 5 ml of Formula 989 fluor was added and 3H and 35S radioactivity was measured by scintillation spectrometry.

Nuclear Magnetic Resonance Analysis

All of the wild type and mutant Ugi protein samples were concentrated to 7-18 mg/ml and dialyzed against NMR buffer containing 25 mM deuterated Tris, 0.2 mM EDTA, 0.2 mM EGTA, and 100 mM NaCl, at pH 7.0. NOESY experiments were carried out using a 150-ms mixing time at 25 °C on a Varian INOVA 500 MHz NMR spectrometer. The spectral width in each dimension was 7500 Hz. The final pulse in the NOESY experiment was replaced with a watergate gradient pulse sequence and also a gradient pulse before each equilibration delay. The watergate sequence used a 1-ms z-direction gradient followed by a 2.2-ms selective shaped pulse for water and a 180° pulse followed by another 2.2-ms selective shaped pulse and another 1-ms z-direction gradient. A weak z-direction gradient was applied in the first half of t1 and its negative applied in the second half of t1. Each NOESY experiment had 512 increments in t1 with an acquisition time of 137 ms. Each NOESY spectrum was transferred into 1024 × 4096 points and weighted using shifted Gaussians along each dimension.

Protein Structure Determination of [13C,15N]Ugi Complexed to Ung in Solution

Samples of [13C,15N]Ugi were complexed with unlabeled Ung as described previously (14). All of the NMR spectra were obtained with the sample at 30 °C using a Varian Unityplus 400 spectrometer equipped with a Nalorac ID400 probe (31-34). Data were acquired using States-Haberkorn for the indirectly detected dimension and using shifted Gaussians in the data processing along each dimension. The results of a 60-ms mixing time 15N TOCSY-HSQC spectrum were used to identify spin systems. The experiment had an acquisition time of 0.108 s and there were 128 increments of t1 and 24 increments of t2 for each data set. The data were linearly predicted to 256 points in t1 and 48 points in t2 before being Fourier-transformed into 512 × 128 × 1024 points. The spectral widths were 5000 Hz for F1, 1500 Hz for F2, and 5000 Hz for F3.

A 15N/1H NOESY-HMQC spectrum was recorded with a mixing time of 200 ms and 16 transients per increment. There were 1024 points in F3 and an acquisition time of 0.108 s was used. There were 128 increments in t1 and 20 increments in t2. The data were linearly predicted to 256 points in t1 and 40 points in t2 before being Fourier-transformed into 512 × 128 × 1024 points. The 15N NOESY-HMQC data was used along with the 15N TOCSY-HSQC to make the chemical shift identification with the 15N and HN chemical shifts of Ugi. These assignments led to 707 NOE constraints for structure determination. The spectral widths were 5000 Hz for F1, 1500 Hz for F2, and 5000 Hz for F3.

A 13C TOCSY-HSQC-SE spectrum with a 40-ms mixing time was used to group the spin systems for 13C-labeled atoms. The data were collected with 16 transients per increment. The acquisition time was 0.108 s. There were 256 increments of t1 for each of the complex data sets. The data were linearly predicted to 512 points in t1 before being Fourier-transformed into 512 × 1 × 1024 points. The spectral widths were 5000 Hz in F1 and 12000 Hz in F2.

A 13C NOESY-HMQC with a mixing time of 150 ms was collected with 16 transients per increment. The acquisition time was 0.0832 s. There were 128 increments in t1 and 48 in t2. The data were linearly predicted to 228 points in t1 and 96 points in t2 before being Fourier-transformed into 512 × 256 × 1024 points. The spectral widths were 5000 Hz in F1, 12000 Hz in F2, and 5000 Hz for F3. These assignments led to 325 NOE constraints. A two-dimensional 13C NOESY-HMQC with a mixing time of 160 ms was collected with 16 transients per increment. There were 330 increments in t1 with the offset set in the middle of the aromatic region. Analysis of this NOE spectrum gave 35 NOE constraints. The normalized Z4-score analysis of 1HN, 1Halpha , 13Cbeta , and 15N chemical shifts for Ugi produced 106 phi  and psi  dihedral constraints (35).

The constraints were grouped into strong, medium, and weak. A strong NOE peak was constrained to 1.8 <r <4.0 Å, a medium NOE peak was constrained to 2.1 < r < 4.5 Å, and a weak NOE peak was constrained to 2.4 < r < 5.0 Å during simulated annealing and refined simulated annealing protein structure determinations protocols. Once the secondary structure of Ugi in the complex was determined there were 24 sets of hydrogen bonds that were used for a total of 48 constraints. The hydrogen bond constrained the oxygen to amide proton to be 1.8 < r < 2.5 Å and the oxygen to nitrogen distance to be 2.5 < r < 3.3 Å. The normalized Z4 score analysis of chemical shifts for Ugi produced 106 phi  and psi  dihedral constraints (35).

The simulated annealing and refinements protocols followed the same procedures as described for the structure of the free uracil-DNA glycosylase inhibitor protein (14) as were previously reported (36). The simulated annealing and refinement protocols were run on an IBM 3CT running X-PLOR 3.1 (37).

Protein Modeling

Starting with the HSV-1 uracil-DNA glycosylase·Ugi complex co-crystal coordinates described by Savva and Pearl (6, 17), mutant Ugi forms in complex were generated by exchanging an individual wild type Ugi amino acid with a mutant residue using the residue replacement command in INSIGHT (BIOSYM). This is thermodynamically reasonable as all the mutant Ugi structures are similar to that of the free Ugi structure as evidenced by their NOESY spectra. Complete free energy analysis of the transition from the free to the bound form of Ugi is not computationally feasible. Therefore, rigid body energy minimizations were performed to determine a reasonable estimate of the Delta Delta E involved between mutant forms of Ugi and wild type Ugi when bound to Ung (38). These calculations do not take into account the energy differences involved in the structural conformation changes that occur during binding to Ung. Rigid energy minimizations were then executed using an IBM 3CT running X-PLOR 3.1 (36). The rigid energy minimization procedure utilized all residues within 5 Å of the alpha 2-helix and beta 1-strand of Ugi which included 40 residues of Ugi and 38 residues of uracil-DNA glycosylase. A dielectric constant of 6.0 was used to compensate for not using water with a cut-on distance of 6.0 Å and a cutoff distance of 6.5 Å. Energy minimizations were conducted using a two-step method. The first step involved 1000 iterations of rigid energy minimization with a large van der Waals radius but without considering electrostatic forces. In the second step, 2000 iterations were conducted with both electrostatic interactions and normal van der Waals radius influencing the structure. After the rigid energy minimizations converged, the minimized structure of the uracil-DNA glycosylase·Ugi complex emerged. Each individual unbound wild type and mutant Ugi structure was similarly generated. Interaction energies were calculated by combining the van der Waals, electrostatic, and hydrogen bond energies of the enzyme-inhibitor complex and unbound Ugi. Changes in interaction energies, Delta Eint, are defined as the difference in the interaction energies of the uracil-DNA glycosylase·Ugi wild type and mutant complex. The differences in the change in the interactive energies Delta Delta Eint are defined by subtracting the difference of the Delta Eint of the wild type and mutant Ugi from the Delta Eint of the mutant Ugi-containing complex.


RESULTS

Site-directed Mutagenesis of the Uracil-DNA Glycosylase Inhibitor Gene

To investigate the role of specific negatively charged amino acid residues in the Ung/Ugi interaction, site-directed mutagenesis producing single amino acid substitutions was performed on the ugi gene. The specific sites and substitutions selected were based on knowledge of the 1.9-Å crystal structure of Ugi complexed with human uracil-DNA glycosylase (16). Significant similarity exists between the human and E. coli enzyme around the proposed sites of Ung/Ugi interaction (Table I). Oligonucleotides were synthesized that introduced a codon change at seven Glu or Asp sites and a new restriction endonuclease cleavage site into the ugi gene as indicated in Fig. 2. To overproduce the mutant Ugi proteins, the EcoRI/HindIII DNA fragment containing the ugi structural gene was subcloned into the overexpression vector pKK223-3 producing a set of pKugi plasmids. Two methods were used to verify that the engineered mutations had been introduced into each pKugi DNA. First, restriction endonuclease digestions were conducted to establish the presence of the newly introduced recognition site within the EcoRI/HindIII DNA (726 bp) fragment. Second, dideoxynucleotide chain termination DNA sequencing of double-stranded pKugi DNAs was performed (data not shown). For all mutants, the entire ugi gene was bidirectionally sequenced and the results confirmed the designed nucleotide changes, exclusively.

Table I. Interactions of Ugi amino acid residues with various uracil-DNA glycosylases


Ugi, amino acid Uracil-DNA glycosylasea
HSV-1b
Humanb
E. coli Amino Acidc
Interaction Amino acid Interaction Amino acid

 beta 1-Strand
  Glu-20 H2 Ser-202 H2 Ser-169 Ser-88
WH1 Ser-302 Ser-270 Ser-189
 alpha 2-Helix
  Glu-27
  Glu-28 H1 Thr-280 H2 Ser-247 Ser-166
His-300 His-268 His-187
WH2
Ser-305 Ser-273 Ser-192
  Glu-30
  Glu-31 Lys-306 SB Arg-276 Arg-195
Loop regions
  Asp-61 H1 Arg-252 SB Lys-218 Ala-137
  Glu-78 Pro-303 Pro-271 Pro-190
Leu-304 Leu-272 Leu-191
Ser-305 WHN Ser-273 Ser-192
Lys-306 Val-274 Ala-193
Val-307 Tyr-275 His-194

a Amino acid sequence alignment and position numbers of uracil-DNA glycosylase correspond to those described by Caradonna et al. (9). The original reference and GenBank accession number for each enzyme corresponding to HSV-1, herpes simplex virus type 1 (40) is X14112; UDG1, human (39) is X15653; and E. coli (41) is J03725.
b Interactions between PBS1 Ugi and HSV-1 uracil-DNA glycosylase have been described based on a 2.7-Å crystal structure of the complex (17), that involving PBS2 Ugi and human UDG1 were described from a 1.9-Å crystal structure (16).
c E. coli Ung amino acid residues corresponding to the aligned HSV-1 and human uracil-DNA glycosylase polypeptides are indicated (9).
d Chemical interactions listed include the following: H1, a hydrogen bond between carboxylate and Thr backbone amide or Arg side chains; H2, a pair of hydrogen bonds between the carboxylate and Ser backbone amide and side chain Ogamma ; WH1, water-mediated hydrogen bond between the carboxylate and Ser-Ogamma ; WH2, water-mediated hydrogen bonds with His backbone amide and Ser-Ogamma ; WHN, hydrogen-bonded network with ordered solvent molecules and backbone atoms of UDG1 residues; and SB, salt bridge.

Purification and Specific Activity of the Mutant Ugi Proteins

To facilitate characterization of the inhibitor activity exhibited by wild type Ugi and seven mutant Ugi proteins, each protein was overproduced using the appropriate pKugi vector and purified according to Sanderson and Mosbaugh (19). The purity of Ugi from the final purification step (fraction IV) was analyzed using 20% SDS-polyacrylamide gel electrophoresis (Fig. 3A). As previously observed the electrophoretic mobility of wild type Ugi was greater than that predicted for a 9474-dalton protein (10). Each mutant Ugi protein migrated with a unique slower mobility with respect to wild type Ugi, consistent with the elimination of a negatively charged residue by site-directed mutagenesis. However, these observations also imply that the mutant Ugi proteins exhibit different propensities to bind SDS or adopt to different protein conformations during electrophoresis, since each mutant protein carries the same charge. The specific activity of each purified Ugi protein was determined under standard conditions (Fig. 3B). Ugi E20I was essentially void of inhibitory activity, displaying ~1% of the wild type specific activity, whereas Ugi E78V was virtually unaffected, displaying ~105% activity. The four mutations in Glu residues located in the alpha 2-helix (E27A, E28L, E30L, and E31L) showed progressively decreased levels of activity with 95, 88, 70, and 53% of control activity, respectively. Significant inactivation was also observed with the Ugi D61G protein which showed ~25% of wild type Ugi activity.


Fig. 3. Purity and specific activity of site-directed mutant Ugi proteins. A, SDS-polyacrylamide gel electrophoresis of purified mutant Ugi preparations. Nine samples (50 µl) each containing 3.6 µg of fraction IV wild type or mutant Ugi protein were applied to a 20% polyacrylamide gel containing 0.1% SDS, and electrophoresis was conducted as described under "Experimental Procedures." Protein bands were visualized after staining with Coomassie Brilliant Blue G-250 (Bio-Rad). The molecular weight standards for BSA, ovalbumin, glyceraldehyde-3-phosphate dehydrogenase, carbonic anhydrase, trypsinogen, and trypsin inhibitor are indicated by arrows from top to bottom, respectively. The location of the tracking dye (TD) is indicated by an arrow. Lanes 1 and 9 contain wild type Ugi; lane 2, Ugi E20I; lane 3, Ugi E27A; lane 4, Ugi E28L; lane 5, Ugi E30L; lane 6, Ugi E31L; lane 7, Ugi D61G; and lane 8, Ugi E78V. B, determination of wild type and mutant Ugi specific activity. Standard uracil-DNA glycosylase inhibitor assays were performed on fraction IV Ugi preparations as described under "Experimental Procedures." Relative specific activity was determined by comparing each mutant Ugi activity to the control (wild type Ugi) that equaled 1.1 × 106 units/mg.
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Ability of Mutant Ugi Proteins to Form a Complex with Ung

To determine whether the mutant Ugi proteins were able to form a Ung·Ugi complex, a 3-fold molar excess of Ung was incubated with each Ugi protein under standard binding conditions. The resultant Ung·Ugi complexes were resolved from the component proteins by nondenaturing polyacrylamide gel electrophoresis (Fig. 4). As controls, free Ung, wild type Ugi, and a 3:1 ratio of Ung·Ugi were analyzed for comparison with mutant forms of free Ugi and Ung·Ugi complexes. Each mutant Ugi protein migrated as a single band with a mobility similar to but slightly slower than that of wild type Ugi. In each case, the mutant Ugi proteins formed a Ugi·Ung complex that also migrated slightly slower than the wild type complex. With the exception of Ugi E20I, it appeared that each mutant Ugi protein formed a stable and complete complex with Ung since no free Ugi was detected. In contrast, the appearance of some free Ugi E20I, less Ung·Ugi E20I complex, and a smear of protein between the Ung and Ung·Ugi E20I complex bands suggested that Ugi E20I formed an unstable complex (Fig. 4, lane 5).


Fig. 4. Ability of mutant Ugi proteins to form complex with E. coli Ung. Wild type or various mutant Ugi proteins (96 pmol) were mixed with or without 288 pmol of Ung and the sample (45 µl) incubated at 25 °C for 10 min followed by 20 min at 4 °C to form complex. Each sample was then mixed with loading buffer, applied to a nondenaturing 18% polyacrylamide gel, electrophoresis was carried out, and the gel was stained with Coomassie Brilliant Blue G-250 as described under "Experimental Procedures." Lanes 1 and 20 contained 288 pmol of Ung; lanes 2 and 19 contained 96 pmol of wild type Ugi; lanes 3 and 18, wild type Ugi plus Ung; lanes 4, 6, 8, 10, 12, 14, and 16, various mutant Ugi proteins; lanes 5, 7, 9, 11, 13, 15, and 17, contained mutant Ugi plus Ung as indicated. The arrows indicate the location of Ung, Ung·Ugi, Ugi, and the tracking dye (TD).
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Relative Ability of Mutant and Wild Type Ugi Proteins to Complex with Ung

Competition experiments were conducted to determine the relative ability of each mutant Ugi protein to form a complex with Ung in the presence of wild type Ugi. Ung was incubated with a 2-fold molar excess of a mixture of Ugi and/or Ugi E27A at various ratios. The proteins were then resolved by nondenaturing polyacrylamide gel electrophoresis and detected by Coomassie Blue staining (Fig. 5A, lanes 1-6). Under these conditions, the Ung·Ugi E27A complexes were only partially resolved, whereas free Ugi and Ugi E27A were separated as independent bands. To quantitatively analyze the ability of mutant Ugi proteins to compete with the wild type inhibitor protein, similar experiments were conducted after mixing each mutant Ugi with wild type [35S]Ugi and incubating the mixtures with Ung. Following electrophoresis, 35S radioactivity was detected in two bands that corresponded to [35S]Ugi free and in complex. Thus, the amount of [35S]Ugi detected in the complex band reflected the competitive ability of the mutant inhibitor protein to stably associate with Ung while in the presence of wild type Ugi. As a control, various ratios of [35S]Ugi to Ugi (both wild type proteins) were mixed and analyzed by electrophoresis (Fig. 5B, black bars). The amount of [35S]Ugi detected in the complex approximately equaled the amount expected based on equal competition between the two inhibitor proteins and on the various ratios between [35S]Ugi and Ugi. Thus, this result confirms the identical nature of the two wild type inhibitor protein preparations and validates the experimental design. After examining each of the mutant Ugi proteins for their ability to compete with [35S]Ugi for complex formation, it was observed that two mutant Ugi proteins (Ugi E20I and Ugi E28L) consistently showed an increased amount of [35S]Ugi in complex with Ung over that predicted by equal competition. Hence, Ugi E20I and Ugi E28L demonstrated a decreased ability to form complex with Ung in the presence of wild type [35S]Ugi. The results also suggested that Ugi E20I competes very poorly with wild type Ugi since the maximum amount of [35S]Ugi based on the molar amount of Ung was found in complex for all ratios utilizing Ugi E20I.


Fig. 5. Ability of mutant Ugi proteins to compete with wild type [35S]Ugi for complex formation with E. coli Ung. A, six competition reaction mixtures (70 µl) containing 314 pmol of Ung and 628 pmol (total) of Ugi and/or Ugi E27A were prepared at molar ratios of 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100 ([35S]Ugi to Ugi E27A) for lanes 1-6, respectively. After the addition of Ung, the samples were mixed, incubated under standard complexing conditions, tracking dye was added, and the samples were loaded onto nondenaturing 18% polyacrylamide tube gels as described under "Experimental Procedures." Following electrophoresis at 4 °C, each gel was stained with Coomassie Brilliant Blue G-250. The direction of migration was from top to bottom, and the tracking dye (TD) is located by an arrow. The location of the Ung·Ugi and Ung·Ugi E27A complex, free Ugi E27A, and Ugi are indicated by arrows. B, eight sets of gels were prepared as described in A except that [35S]Ugi (wild type) was mixed at various molar ratios, excluding the 0:100 ([35S]Ugi to Ugi) sample, with either wild type Ugi (control) or various mutant Ugi proteins, as indicated. After electrophoresis, each gel was horizontally sliced (3.1 mm), dried, solubilized, and analyzed for 35S radioactivity. The average amount of [35S]Ugi (wild type) that formed complex with Ung in duplicate competition reactions was determined from the amount of 35S radioactivity detected in the Ung·Ugi complex band. The expected amount of [35S]Ugi to form complex when competing with wild type Ugi (control) is indicated (bullet ).
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Reversibility of the Ung·Ugi Complex with Various Mutant Ugi Proteins

To characterize further the nature of the Ung·Ugi complexes containing mutant Ugi proteins, we assessed the ability of wild type Ugi to displace mutant Ugi from a preformed complex. [3H]Ung·Ugi complexes were formed by individually incubating either wild type or mutant Ugi with a 3-fold molar excess of [3H]Ung and the complex species isolated by DEAE-cellulose chromatography. Purification of [3H]Ung·Ugi E27A is illustrated in Fig. 6A. Analysis of fractions across the peak by nondenaturing polyacrylamide gel electrophoresis verified that >95% of [3H]Ung formed complex and that no detectable free [3H]Ung or Ugi was observed in these fractions (Fig. 6A, inset). Stable preformed [3H]Ung·Ugi complexes were isolated using this procedure for each mutant Ugi protein (Fig. 6B) except Ugi E20I, which was unable to form a stable complex that could be purified (data not shown).


Fig. 6. Ability of free [35S]Ugi to exchange with various mutant Ugi proteins in a preformed [3H]Ung·Ugi complex. A, formation and purification of the [3H]Ung·Ugi E27A complex. A sample (300 µl) containing 12 nmol of [3H]Ung and 36 nmol of Ugi E27A was incubated under standard complexing conditions and applied to a DE52 cellulose column (0.8 cm2 × 3 cm). The column was eluted with buffer A containing 150 mM and 250 mM NaCl (arrows) as described under "Experimental Procedures." Fractions (1 ml) were collected, and samples (100 µ1) were analyzed for 3H radioactivity (bullet ). Samples (40 µl) from fraction numbers 26-40 were analyzed on a nondenaturing 18% polyacrylamide slab gel (lanes 5-17, respectively). Electrophoresis was carried out at 4 °C, and protein bands were visualized after staining with Coomassie Brilliant Blue G-250 as shown in the inset. Lane 1 contained 260 pmol of [3H]Ung; lane 2, 640 pmol of Ugi E27A; lane 3, 136 pmol of [3H]Ung·Ugi; and lane 4 contained a sample (4 µl) of the [3H]Ung/Ugi E27A mixture that was loaded onto the DE52 column. The location of the tracking dye (TD) is indicated by an arrow. Peak fractions containing the [3H]Ung·Ugi E27A complex were pooled (bar) and concentrated using a Centriplus-10 (Amicon) concentrator. B, purity of the various [3H]Ung·Ugi complexes. Samples (40 µl) containing 100 pmol of [3H]Ung (lane E), [3H]Ung·Ugi (lanes 1 and 8), [3H]Ung·Ugi E27A (lane 2), [3H]Ung·Ugi E28L (lane 3), [3H]Ung·Ugi E30L (lane 4), [3H]Ung·Ugi E31L (lane 5), [3H]Ung·Ugi D61G (lane 6), and [3H]Ung·Ugi E78V (lane 7) were applied to a nondenaturing 18% polyacrylamide slab gel, and electrophoresis was conducted at 4 °C. The location of free Ung (arrow) and Ung·Ugi complexes (arrow) are indicated. C, ability of free [35S]Ugi to exchange with various mutant Ugi proteins in a preformed complex. Seven competition reactions (150 µl) containing 0.6 nmol of [3H]Ung·Ugi (wild type or mutant) and 6.0 nmol of [35S]Ugi (wild type) were incubated under standard complexing conditions and applied to a nondenaturing 18% polyacrylamide tube gel; electrophoresis was conducted,and gels were horizontally sliced (~3 mm), dried, solubilized as indicated under "Experimental Procedures," and analyzed for 3H and 35S radioactivity using a double isotope detection technique. The amount of [3H]Ung (square ) and [35S]Ugi (black-square) found in the Ung·Ugi complex band is plotted for the seven competition reactions as indicated. The background level of 35S radioactivity detected in the wild type [3H]Ung·[35S]Ugi complex was not subtracted from the mutant complexes. Each reaction was carried out in duplicate and represents the average values.
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Competition experiments were conducted to determine if wild type [35S]Ugi could exchange with mutant Ugi contained in the preformed [3H]Ung·Ugi complexes. Each complex preparation was incubated with a 10-fold molar excess of [35S]Ugi, and nondenaturing polyacrylamide gel electrophoresis was performed, as described above. If a mutant [3H]Ung·Ugi association was reversible, then wild type [35S]Ugi would exchange with the mutant Ugi in complex; the amount of [35S]Ugi in the complex would reflect the amount of mutant Ugi exchanged. As a control, wild type [35S]Ugi was incubated with preformed [3H]Ung·Ugi (wild type) complex; 6.8% of the [3H]Ung was found associated with [35S]Ugi in complex (Fig. 6C). This value represents a background level when comparing results with the mutant preformed complexes. Of the six mutant Ugi contained in preformed complexes, only Ugi E28L was significantly displaced by wild type [35S]Ugi (Fig. 6C). In this case, 50% of Ugi E28L was replaced by [35S]Ugi, demonstrating that the Ung·Ugi E28L complex was reversible. The other preformed complexes containing mutant Ugi proteins showed slightly above background levels of wild type [35S]Ugi exchange (1.9% E27A, 0.5% E30L, 3.4% E31L, 1.4% D61G, and 1.8% E78V). Thus, in contrast to Ung·Ugi E28L the other mutant complexes were irreversible, as is the wild type Ung·Ugi complex.

Rate and Extent of Wild Type Ugi Exchange with the Ung·Ugi E28L Complex

The reversible nature of the [3H]Ung·Ugi E28L complex was exploited to determine the rate of exchange with wild type [35S]Ugi. Competition reaction mixtures containing the preformed [3H]Ung·Ugi E28L complex (0.6 nmol) and [35S]Ugi (6 nmol) were incubated at 25 °C for various times to allow exchange before determining the amount of [35S]Ugi that resided with [3H]Ung in complex (Fig. 7, closed circles). The results indicated that rapid exchange occurred since 28% of the complex contained [35S]Ugi without incubation. The amount of exchange increased with incubation time and reached a plateau after 120 min with ~75% of the Ugi E28L exchanging with wild type [35S]Ugi. In contrast, the [3H]Ung·Ugi (wild type) complex showed no significant exchange with [35S]Ugi after 240 min confirming the irreversible nature of this association (Fig. 7, open circles). While the Ugi E28L mutant was capable of forming a stable complex with Ung, an irreversible complex was not achieved.


Fig. 7. Time course of the [35S]Ugi-induced displacement reaction of Ugi E28L from the preformed [3H]Ung·Ugi E28L complex. Six competition reaction mixtures (150 µl) containing 0.6 nmol of [3H]Ung·Ugi E28L and 6 nmol of [35S]Ugi were prepared in duplicate as described in Fig. 6. Two control reactions containing 0.54 nmol of [3H]Ung·Ugi (wild type) and 5.4 nmol of [35S]Ugi were prepared. Each reaction was incubated at 25 °C for the times indicated and then loaded onto nondenaturing 18% polyacrylamide tube gels. The unincubated sample (0 min) was mixed on ice and electrophoresis initiated as rapidly as possible. Following electrophoresis at 4 °C, the gels were sliced and processed as described under "Experimental Procedures." The 3H and 35S radioactivities were measured in each gel slice, and the percentage of the once [3H]Ung·Ugi E28L (bullet ) or [3H]Ung·Ugi (open circle ) complex that contained [35S]Ugi was determined by dividing the amount (nmol) of [35S]Ugi by the amount (nmol) of [3H]Ung found in the Ung·Ugi complex band and multiplying by 100.
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Solution State Structure of Mutant and Wild Type [15N]Ugi Proteins

NMR structural determinations were made to analyze and compare the polypeptide structures of the wild type and seven mutant Ugi proteins. Each Ugi protein showed one-dimensional proton spectra consistent with a well ordered and folded structure (data not shown). The one-dimensional spectra obtained on the samples in 2H2O also indicated that all eight Ugi proteins exhibited about the same number of slowly exchanging amide protons. In addition, the distribution of the amide proton chemical shifts was consistent with each mutant Ugi containing a high percentage of beta -structure, as is the case for wild type Ugi (13, 14). Structural determinations were also made by comparing secondary structural NOE peaks from amide to amide and amide to alpha -NOESY spectra. The amide to amide NOESY spectra for wild type and mutant Ugi forms are shown in Fig. 8. Each mutant Ugi protein was found to contain two alpha -helices and five beta -strands identical to the secondary structural elements as exhibited by the unbound wild type Ugi protein. The NOESY spectra of each mutant Ugi was compared with that of the assigned wild type spectrum. Detailed examination showed that Ugi E20I, D61G, and E78V have structures that are very similar to wild type Ugi. However, the chemical shifts of many of the cross-peaks in the Ugi E20I spectrum are distinct from those of the wild type protein. Analysis of the NOESY spectra for Ugi E27A, E28L, E30L, and E31L likewise indicated close structural similarity to wild type Ugi, with the exception of the alpha 2-helix length. Ugi E27A and E28L contained an alpha 2-helix that was shorter at the N-terminal end, whereas Ugi E30L and E31L exhibited a shorter alpha 2-helix at the C-terminal end.


Fig. 8. The NOESY spectra of wild type Ugi and seven mutant Ugi proteins. The two-dimensional 500-MHz, 150-ms mixing time NOESY spectra of wild type Ugi (A) and Ugi E20I (B), E27A (C), E28L (D), E30L (E), E31L (F), D61G (G), and E78V (H) are shown. The region shown contains signals primarily from amide, aromatic, and amino protons and the NOE cross-peaks between them.
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Solution State Structure of [13C,15N]Ugi Complexed to Ung

To determine the structure of Ugi bound to Ung, a sample of [13C,15N]Ugi (820 nmol) was combined with an excess of unlabeled Ung (1220 nmol), and the Ung·[13C,15N]Ugi complex (1.27 mM) was prepared as described previously (14). NMR structural determinations were made using 15N-TOCSY-HSQC, 15N/1H-NOESY-HMQC, 13C-TOCSY-HSQC, and 13C-TOCSY-HSQC-SE spectra, and the solution state structure of [13C,15N]Ugi complexed to Ung is shown in Fig. 9. A comparison of free [15N]Ugi (Fig. 9A) to [13C,15N]Ugi bound to Ung (Fig. 9B) indicates that significant structural change of Ugi occurred as a consequence of complex formation. The electrostatic surfaces of the free (Fig. 9, C and E) and complexed Ugi protein (Fig. 9, D and E) were evaluated using the GRASP program (38).


Fig. 9. Tertiary structure of free Ugi and Ugi bound to Ung. The tertiary structure of the solution state [15N]Ugi (A) was previously determined by Beger et al. (14). The tertiary structure of [13C,15N]Ugi bound to E. coli Ung was determined by solution state multidimensional NMR techniques as described under "Experimental Procedures." Several secondary structural elements are highlighted in both free Ugi (A) and in complex (B) structures as follows: alpha 1-helix (light blue); alpha 2-helix (dark blue); beta l-strand (red); beta 2-beta 5-strands (salmon); and the loop between beta 3- and beta 4-strands (yellow). The location of the N-terminal (N), C-terminal (C), Glu-28 (28), and Glu-3l (31) residues are also indicated. The electrostatic surfaces of the free (C) and complexed (D) forms of Ugi were generated using the program GRASP (38) as described previously (14). Structures A and B of the free and bound Ugi correspond to the same view as indicated in C and D, respectively. The bottom panel depicts Ugi rotated 180°. The electrostatic potentials were calculated with a dielectic constant of 6.0 for the protein and 80.0 for the solvent. The ionic strength of the solution was set to 0. Only the charges of the side chains of Lys, Asp, Asn, Glu, and Gln residues were used. The electrostatic potential cutoff was set to 6.6 kcal/mol, and the regions with a negative potential of this magnitude are shown in red, and the regions with a positive potential of this magnitude are shown in blue.
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DISCUSSION

We have used site-directed mutagenesis to assess the role of specific negatively charged amino acids in Ugi activity. Three structural domains of Ugi were targeted that included the beta l-strand (E20I), the alpha 2-helix (E27A, E28L, E30L, and E31L), and the loop regions joining the anti-parallel beta -strands (D61G, E78V). To gain information about the structural changes induced by the specific amino acid replacements, NMR spectral analysis was performed for each Ugi protein. The one-dimensional proton spectra of the wild type and mutant Ugi proteins appeared to be quite similar indicating that each Ugi protein folded in much the same manner. The results of binding experiments indicated that each mutant Ugi protein remained capable of associating with Ung and forming a Ung·Ugi complex. However, complex stability and reversibility was found to be altered by some amino acid substitutions. These results suggest that none of the individual Ugi amino acids examined play an essential role in mediating Ung/Ugi binding. Rather, the negatively charged residues act collectively to facilitate stable complex formation.

The specific chemical interactions that stabilize the Ung·Ugi complex can be inferred from those in the x-ray crystallographic structures identified of HSV-1 (17) and human (16) uracil-DNA glycosylase·Ugi complexes. Such a comparison is justified since E. coli Ung shares extensive amino acid homology with its HSV-1 and human counterparts (39), and both co-crystal structures show significant structural similarity (16, 17). The structure of Ugi in complex with Ung has been determined by conventional solution state methods and found to be essentially the same as the crystal structure (16, 17). As indicated in Table I, the locations and types of interactions linking Ugi residues with either HSV-1 or human uracil-DNA glycosylase were found to be highly conserved. Amino acid sequence alignment of E. coli Ung to both the HSV-1 and human enzyme revealed identical or conservative substitutions at the sites of Ugi interaction. The ability of Ugi to perform DNA mimicry has apparently capitalized on the conservation of Ung residues located in the highly conserved DNA-binding pocket (16, 18, 21). This striking amino acid correspondence suggests that similar interactions most likely mediate the Ugi association with all three uracil-DNA glycosylases examined here and possibly others.

The Ugi E20I protein, although capable of forming a Ung·Ugi complex, did not completely block Ung activity, presumably due to an inability to form a stable complex with Ung. The instability of this association was evident from the dissociation detected during nondenaturing polyacrylamide gel electrophoresis, the inability to isolate a Ung·Ugi E20I complex by anion exchange chromatography, and the ineffectiveness of Ugi E20I to compete with wild type Ugi for Ung binding. The position of Glu-20 is apparently stabilized by a pair of hydrogen bonds between the carboxylate side chain of the conserved Ser-88 backbone amide and Ogamma of E. coli Ung (Table I). In addition, a water-mediated hydrogen bond may also form between Ugi Glu-20 and Ung Ser-189, as has been described for the complex involving the HSV-1 enzyme (17). The loss of Ugi E20I activity may be explained by a weakening of these interactions due to charge neutralization or peptide conformational change surrounding this key residue. Protein modeling indicated that the van der Waals energy dropped considerably (-17 kcal/mol); in this case, more than enough to stop the interaction. Taken together the results suggest that Ugi E20I forms a frail unlocked complex that fails to prevent Ung association with uracil-DNA.

The four mutations (E27A, E28L, E30L, and E31L) created within the alpha 2-helix had quite different effects on Ugi activity. While Ugi E27A retained near full inhibitor activity, the other three mutations caused a progressive reduction of Ugi-specific activity (E28L > E30L > E31L) with Ugi E31L maintaining ~50% wild type activity. The influence of these mutations was particularly interesting since a major structural difference between the free and complexed forms of Ugi involves the orientation of the alpha 2-helix (Fig. 9, A and B). Furthermore, x-ray crystallographic studies have indicated that when in complex the alpha 2-helix and beta 1-sheet resides over the DNA-binding groove and provides the majority of contacts between the enzyme and inhibitor (16, 17). Therefore, it was not surprising that Ugi E27A activity was unaffected, since Glu-27 has not been implicated in complex interaction (Table I). The results obtained for Ugi E28L and E31L support the inference drawn from chemical modification that Glu-28 and/or Glu-31 play an important role in promoting stable Ung·Ugi complex formation (19). The unique ability of Ugi E28L to form a stable but reversible complex when challenged with wild type Ugi indicates that this residue plays a critical role in forming the locked complex. Like Glu-20, Glu-28 appears to form hydrogen bonds with a conserved Ser (Ser-166 of E. coli Ung) amide and the side chain Ogamma (Table I). In addition, Glu-28 also forms water-mediated hydrogen bonds to a universally conserved active site His backbone amide and Ser Ogamma atom (His-187 and Ser-192 of E. coli Ung). We speculate that these contacts are responsible, at least in part, for creating the irreversible nature of the Ung·Ugi complex. Additionally, the observation that Ugi E31L, like wild type Ugi, formed an essentially irreversible complex with Ung argues that Glu-31 does not play a major role in the locking reaction.

The two mutations in the loop regions connecting the consecutive beta -strands of Ugi provided distinctly different results. Ugi D61G caused ~75% reduction of activity, whereas Ugi E78V showed a specific activity equivalent to wild type Ugi. The inability of the E78V mutation to affect activity may be explained since Glu-78 resides within the electrostatic knob region of Ugi that contains seven Glu or Asp residues (14). The results suggest that neutralizing the negative charge of Glu-78 may have little effect on the overall inhibitory action due to the relatively small individual contribution of Glu-78. The involvements of Glu-61 in Ugi/Ung binding remains to be determined; however, the reduced activity of Ugi D61G was not attributed to a defective locking reaction.

Several lines of evidence have led to a proposal that free Ugi undergoes a conformational change during formation of the Ung·Ugi complex (14, 16, 17, 19-21). A direct demonstration of this change is evident by comparing the NMR solution structure of free [15N]Ugi with that of [13C,15N]Ugi complexed to E. coli Ung (Fig. 9). Clearly, the tertiary structure of the free and bound Ugi are quite different; however, both forms of the protein contain similar secondary structural elements (i.e. two alpha -helices and five beta -strands). The beta 2-beta 3-beta 4-beta 5 portion of the anti-parallel beta -sheet remains generally unchanged in the two structures and provides a focal point for comparison. The major transition between these structures involves a collapse of the polypeptide segments containing the alpha 1- and alpha 2-helix. In the unbound state, both helices extend away from the core of Ugi (14). We speculate that this Y-shaped structure may arise from the negative charge repulsion between the negative electrostatic knob and both the negatively charged alpha 1- and alpha 2-helices. Upon binding to Ung, the flexible arms containing the alpha 1- and alpha 2-helix reorient to allow the positioning of beta 1 and alpha 2 over the positively charged DNA-binding pocket of uracil-DNA glycosylase (16, 17). As a consequence, several other structural changes occur as follows: (i) the beta 1-strand becomes twisted; (ii) the alpha 1- and alpha 2-helix move toward the core of Ugi; and (iii) the loop between the beta 3- and beta 4-strands becomes slightly reoriented. The orientation and negative charge of Glu-28 in the DNA-binding pocket mediates the formation of the locked complex and excludes DNA. The involvement of a Ugi structural change may explain the specificity exhibited by this inhibitor protein toward uracil-DNA glycosylases acting through a mechanism involving DNA mimicry.

The structural changes that occur during complex formation have a pronounced effect on the electrostatics of Ugi (Fig. 9). The primary changes appear to result from the positions of the alpha 1- and alpha 2-helices relative to the rest of the protein. The alpha 1-helix is positioned behind the beta -sheet of the complex structure shown, and the alpha 2-helix is positioned in front. There are smaller changes of the beta -strands. The helices appear to have relatively few interactions with the rest of the protein in the free form, and there may be no particularly large barriers between the free and bound conformations. The positioning in the bound state of the alpha 1-helix effectively covers the electrostatic potential of the knob region that is exposed in the free protein. The position of the alpha 2-helix in combination with the modest rearrangements of the beta -strands gives rise to a large negative electrostatic potential on the face that forms most of the contacts in the Ung complex. This suggests that electrostatic interactions will play a considerable role in the complex and that the alpha 2-helix appears to be involved in these interactions.

Molecular modeling studies were conducted using the co-crystal coordinates of the HSV-1 uracil-DNA glycosylase·Ugi complex and variations of the free and bound Ugi structure. The models only allowed differences in the position of the amino acid side chains corresponding to the mutant Ugi proteins. Information concerning the contributions to the energies of complex stability was assessed for the mutations in the beta 1-strand and alpha 2-helix. The modeling indicated that the complex containing Ugi E20I has by far the highest energy, consistent with the low activity of this protein. The modeling suggested that Ugi E20I forms the same unbound protein structure as wild type Ugi but that there are very unfavorable (~15 kcal) van der Waals interactions in the complex. In contrast, Ugi E27A and E30L were found to have energies that are essentially identical to that of wild type Ugi in the complex. This is consistent with neither Glu-27 nor Glu-30 residues participating in an interaction with the enzyme (Table I) and both Ugi E27A and E30L showing only partially reduced activity. Models of Ugi E28L and E31L were examined in an attempt to explain the reason that Ugi E28L was the only mutant protein capable of forming a stable but reversible complex. As shown in Fig. 10, the positions of the Leu side chains in both mutant Ugi polypeptides were quite similar to the wild type Glu, although they are shorter in length. Since both mutant proteins are structurally very similar to wild type, we infer that the absence of the carboxyl group precipitates the change in properties of each mutant. The Leu side chain should not be capable of mediating the hydrogen bond interactions that stabilize the enzyme-inhibitor complex (Table I). Under this condition Ugi E28L appears capable of conducting the docking reaction but not the locking reaction. Analysis of the energy terms showed that the electrostatics of the complexes with Ugi E28L and E31L are ~5 and ~2 kcal, respectively, less favorable than that of complex containing wild type Ugi. Both mutant proteins showed ~3 kcal less stability than wild type Ugi in complex based on van der Waals forces. Hence, Ugi E28L differed from the other mutations in the alpha 2-helix in that it not only had the highest energy but was unfavorable in both electrostatic and van der Waals energy relative to the wild type Ugi in complex. This suggests that the locking reaction may involve both the electrostatic potential and the hydrogen bond interactions of Glu-28.


Fig. 10. Molecular modeling of the Ugi E28L and E31L mutant proteins complexed with uracil-DNA glycosylase. The Ugi protein on the left represents a partial polypeptide structure of free Ugi as previously determined by Beger et al. (14). The alpha 2-helix and loop region joining the beta 3- and beta 4-strands are shown with the location of Glu-28 and Glu-31 indicated. A portion of the HSV-1 uracil-DNA glycosylase·Ugi complex derived from the co-crystal coordinates described by Savva and Pearl (6, 17) is illustrated on the right. Modeled structures of Ugi E28L and E31L (yellow) complexed with HSV-1 uracil-DNA glycosylase (light blue) were obtained as described under "Experimental Procedures" and are shown in the top and bottom middle, respectively. The changes in the location of the enzyme and inhibitor side chains in the complexes containing Ugi E28L and E31L are depicted (red).
[View Larger Version of this Image (119K GIF file)]

This study has demonstrated that Ugi exists in three different conformational states (free, unlocked, and locked Ugi) during the binding reaction with Ung. The involvement of a significant structural transformation and role of Glu-20 and Glu-28 in mediating the locking reaction has been demonstrated. However, several issues remain to be investigated concerning the structure and function of Ugi during Ung complex formation. First, do individual amino acid residues play an essential role in the docking reaction? Second, what is the effect of various mutations on the kinetics of the Ung-Ugi interaction? Third, what is the structure of E. coli Ung when complexed with Ugi? Additional protein structural and biochemical analysis will be required to elucidate these important issues.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants GM32823 and ES00210 (to D. W. M.) and NP-750 from the American Cancer Society (to P. H. B.). The NMR spectrometer was purchased with support from the National Science Foundation Grant BIR-95-12478 and by a grant from the Camille and Henry Dreyfus Foundation (to P. H. B.). This is Technical Report 11083 from the Oregon Agricultural Experimental Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   Present address: Laboratory of Molecular Genetics, NIEHS, Research Triangle Park, NC 27709.
**   To whom correspondence may be addressed: Dept. of Chemistry, Wesleyan University, Middleton, CT 06459. Tel.: 860-685-2668; Fax: 860-685-2211.
§§   To whom correspondence should be addressed: Dept. of Agricultural Chemistry, Oregon State University, 1007 Agricultural & Life Sciences Bldg., Corvallis, OR 97331-7301. Tel.: 541-737-1797; Fax: 541-737-0497.
1   The abbreviations used are: ugi and Ugi, bacteriophage PBS1 or 2 uracil-DNA glycosylase inhibitor gene and protein, respectively; Ung, E. coli uracil-DNA glycosylase; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect correlation spectroscopy; BSA, bovine serum albumin; HSV-1, herpes simplex virus type-1; bp, base pair(s); kb, kilobase pair(s); TOCSY-HSQC, total correlation spectroscopy-heteronuclear multiple quantum correlation spectroscopy.

ACKNOWLEDGEMENTS

We thank Dr. Reg McParland and Anne-Marie Girard for conducting the nucleic acid sequencing and Barbara Robbins for conducting the oligonucleotide synthesis.


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