(Received for publication, December 18, 1996, and in revised form, May 19, 1997)
From the Departments of Agricultural Chemistry and
Biochemistry and Biophysics and the
Environmental Health Sciences Center,
Oregon State University, Corvallis, Oregon 97331 and the
§ Chemistry Department, Wesleyan University,
Middleton, Connecticut 06459
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.
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 s1. 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 -helices and five anti-parallel
-strands as
illustrated in Fig. 1 (13, 14). The five
contiguous
-strands are connected by short loop regions to form an
anti-parallel
-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
-strand. Two other acidic amino acid residues (Asp-40
and Asp-61) are also in juxtaposition to the end of
-strands; Glu-78
and Glu-64 reside in the loop regions (14). The remaining seven negatively charged residues are located in the
1-helix (Asp-6, Glu-9, and Glu-11) and
2-helix (Glu-27, Glu-28, Glu-30, and Glu-31). Both the
1- and
2-helix elements project away from the
-sheet and are located on potentially flexible arms of the polypeptide (14).
Furthermore, the
2-helix is longitudinally segmented into a
hydrophobic face and a negatively charged face where the four glutamic
acid residues protrude.
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 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.
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--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).
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-
-D-galactopyranoside, and 40 µg/ml 5-bromo-4-chloro-3-indolyl
-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.
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--D-galactopyranoside-inducible tac promoter.
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 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 ComplexesWild 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 AssaysUracil-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 MeasurementsProtein concentrations were determined
by absorbance spectroscopy using the molar extinction coefficients
280 nm = 4.2 × 104 liter/mol cm (Ung)
and
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).
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 AnalysisAll 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 SolutionSamples 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,
1H, 13C
, and 15N chemical
shifts for Ugi produced 106
and
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 and
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 ModelingStarting 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 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
2-helix and
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,
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
Eint are defined by subtracting the difference of the
Eint of the
wild type and mutant Ugi from the
Eint of the
mutant Ugi-containing complex.
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.
|
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 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.
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).
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.
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).
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 ComplexThe 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.
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 -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
-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
-helices and five
-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
2-helix length. Ugi E27A
and E28L contained an
2-helix that was shorter at the N-terminal
end, whereas Ugi E30L and E31L exhibited a shorter
2-helix at the
C-terminal end.
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).
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 l-strand (E20I), the
2-helix (E27A, E28L, E30L, and E31L), and the loop regions joining the anti-parallel
-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 O 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
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
2-helix (Fig. 9, A and B). Furthermore, x-ray crystallographic
studies have indicated that when in complex the
2-helix and
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 O
(Table I). In
addition, Glu-28 also forms water-mediated hydrogen bonds to a
universally conserved active site His backbone amide and Ser O
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
-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 -helices and five
-strands). The
2-
3-
4-
5 portion of the anti-parallel
-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
1- and
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
1- and
2-helices. Upon binding to Ung, the flexible arms containing the
1- and
2-helix reorient to allow the positioning of
1 and
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
1-strand becomes twisted; (ii) the
1- and
2-helix move toward the core of Ugi; and (iii) the loop
between the
3- and
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 1- and
2-helices relative to the rest of the protein. The
1-helix is
positioned behind the
-sheet of the complex structure shown, and the
2-helix is positioned in front. There are smaller changes of the
-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
1-helix effectively covers
the electrostatic potential of the knob region that is exposed in the
free protein. The position of the
2-helix in combination with the
modest rearrangements of the
-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
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
1-strand and
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
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.
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.
We thank Dr. Reg McParland and Anne-Marie Girard for conducting the nucleic acid sequencing and Barbara Robbins for conducting the oligonucleotide synthesis.