From the
The Bacillus subtilis bacteriophage PBS2 uracil-DNA
glycosylase inhibitor (Ugi) is an acidic protein of 84 amino acids that
inactivates uracil-DNA glycosylase from diverse organisms. The
secondary structure of Ugi consists of five anti-parallel
The Bacillus subtilis bacteriophage PBS2 uracil-DNA
glycosylase inhibitor protein (Ugi)
The ugi gene has been
cloned, sequenced, and overexpressed in E. coli. It encodes a
small (M
Ugi inactivates E. coli uracil-DNA glycosylase noncompetitively; the K
Several lines of evidence
suggest that Ugi binds Ung at or near the DNA-binding site of the
enzyme. Ung bound to Ugi no longer associates with DNA; Ung
UV-cross-linked to single-stranded oligonucleotide fails to form
complex with Ugi, and Ung complexed with Ugi does not UV-cross-link to
dT
The secondary structure of Ugi was
recently determined by multidimensional NMR methods and found to
consist of two helices and five anti-parallel
In this paper, we describe the
tertiary structure of Ugi as determined by solution-state
multidimensional NMR and calculate the electrostatic potential of the
protein. The tertiary structure brings together a large number of
negatively charged residues to form an ``electrostatic knob''
that extends over about 18 Å on one face of the protein. The
electrostatic potential of this region is similar to that generated by
the phosphates in DNA. This large area of negative charge is likely to
be the portion of Ugi that interacts with the DNA binding site of Ung.
Preliminary results indicate that Ugi undergoes some conformational
change upon binding to Ung.
NOESY-HMQC data were
obtained using a mixing time of 200 ms at 17, 30, and 37 °C with 16
transients per increment and obtained by the Stakes-Haberkorn method
along each indirectly detected dimension. There were 128 increments in t
A series
of eight J-modulated HSQC experiments were obtained at 25 °C to
determine the
A series of seven XT1 HSQC (17) experiments was
obtained at 25 °C to determine the
A total of 67
An initial linear Ugi structure was
built using Biosym Insight II by piecing together the primary structure
sequence with the residue append command. This structure was used as a
template structure for the X-PLOR simulated
annealing(16, 18) . The energy function used during all
of the simulated annealing and energy minimization routines was
confined to covalent geometry terms (bonds, angles, torsion, and
chirality), distance and dihedral angle constraints, and a non-bonded,
van der Waals term.
A soft square potential was used during the
initial simulated annealing protocol for all NOE distance constraints
within a specified ``switching'' region, and outside the
switching region a soft asymptote was used:
On-line formulae not verified for accuracy
for R > d + d
On-line formulae not verified for accuracy
for R < d - d
Refined
simulated annealing consisted of molecular dynamics at 1000 K, and the
temperature was lowered to 100 K in 50-K increments. At each
temperature, 667 steps of a 2-fs timestep (1.34 ps total) molecular
dynamics were done. A hard square potential of 400 kcal/mol was used
for the NOE constraints:
On-line formulae not verified for accuracy
where ceil is 1000 kcal/mol, S is 400 kcal/mol, T is 300 K, K
The secondary structure of Ugi was recently determined by
multidimensional NMR methods and is shown in Fig. 1. The
secondary structure has two helices and five
Of the three aromatic residues, the two not
present in secondary structural elements are Tyr-65 and Trp-68. Both
Tyr-65 and Trp-68 are close to the large negative potential surface and
could also be involved in the interaction of Ugi with Ung. Tyr-65 is
close to His-44 in the tertiary structure of free Ugi, but this may not
be the case when Ugi is bound to Ung. Tyr-47 is also in this region and
may also be involved in the Ugi-Ung interactions.
Thus, the tertiary
structure of Ugi brings together a large number of negatively charged
residues to form an electrostatic knob. This unusual feature is the
result not only of the negatively charged residues at the ends of all
five of the
The Ung
These results indicate that the Ugi
protein folds in such a manner that a large region of highly negative
electrostatic potential is generated. The acidic residues that comprise
this region are the terminal residues of all five
The assistance of Dr. Igor Goljer in performing some
of the NMR experiments is appreciated.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-strands
and two
-helices (Balasubramanian, S., Beger, R. D., Bennett, S.
E., Mosbaugh, D. W., and Bolton, P. H.(1995) J. Biol. Chem. 270, 296-303). The tertiary structure of Ugi has been
determined by solution state multidimensional nuclear magnetic
resonance. The Ugi structure contains an area of highly negative
electrostatic potential produced by the close proximity of a number of
acidic residues. The unfavorable interactions between these acidic
residues are apparently accommodated by the stability of the
-strands. This negatively charged region is likely to play an
important role in the binding of Ugi to uracil-DNA glycosylase.
(
)has been
shown to inactivate Escherichia coli uracil-DNA glycosylase
(Ung) by forming a stable Ung
Ugi protein complex in 1:1
stoichiometry(1) . Interaction with Ugi prevents the enzyme from
binding to DNA and dissociates a preformed Ung
DNA
complex(1, 2) . Ugi bound in a preformed complex does
not exchange with free Ugi(2) . Ugi also inactivates uracil-DNA
glycosylase isolated from other sources, including Microccocus
luteus, Mycoplasma lactucae, Saccharomyces
cerevisiae, rat liver (nuclear and mitochondrial), herpes simplex
virus types 1 and 2, human placenta, and KB
cells(3, 4, 5, 6, 7) . In
vivo expression of the ugi gene in E. coli leads
to inactivation of Ung and to bacteria with phenotypic properties
consistent with other ung mutants(8) . In vitro studies have indicated that Ugi is specific for uracil-DNA
glycosylase, as it had no inhibitory effect on other DNA-metabolizing
enzymes that were tested (4, 6).
= 9,477) heat-stable, non-globular
polypeptide of 84 amino acid residues (1, 6). Ugi is an acidic protein
(12 Glu, 6 Asp) with a pI of 4.2 that migrates anomalously during
SDS-polyacrylamide electrophoresis(1, 4, 5) .
Whereas the native molecular weight as determined by gel filtration
chromatography was
18,000, sedimentation equilibrium
centrifugation established that Ugi existed as a monomer in
solution(1, 8) .
(0.14 µM) is more than 10-fold lower than the K
(1.7 µM) of the enzyme
(9). Stopped-flow kinetic analysis suggests that the Ung/Ugi
association is described by a two-step mechanism(2) . The first
step involves a rapid pre-equilibrium or ``docking,''
characterized by the dissociation constant K (1.3
µM), in which Ugi and Ung associate to form a pre-complex.
The second step entails ``locking,'' characterized by the
rate constant k (195 s
), in which optimal
alignment is obtained. Two experiments indicate that the individual
components of the Ung
Ugi complex are not apparently altered by
the interaction. First, the dissociation of the complex by
SDS-polyacrylamide gel electrophoresis revealed no detectable
modification in the molecular weight of either protein subsequent to
binding(1) . Second, when the Ung
Ugi complex was treated
with urea and the individual proteins resolved by urea-polyacrylamide
gel electrophoresis, both uracil-DNA glycosylase and inhibitor
activities were recovered(1) .
(10) . Interestingly, the Ung peptides that
photocross-link with dT
are highly conserved in amino acid
sequence when compared with nine other uracil-DNA
glycosylases(11) . The fact that the Ugi protein from
bacteriophage PBS2 is capable of inactivating uracil-DNA glycosylase
from diverse biological systems, which are under no selective pressure
to maintain an Ugi binding site, lends support to the notion that Ugi
binds to the same site as DNA.
-strands(12) . Six loop or turn regions were identified
that connect the
-strands sequentially to one another to form a
contiguous anti-parallel sheet. All five of the
-strands have
terminal acidic amino acid residues on the same side of the
anti-parallel sheet(12) .
Preparation of [
[N]Uracil-DNA
Glycosylase Inhibitor, E. coli Uracil-DNA Glycosylase, and the
Ung
[
N]Ugi
Complex
N]Ugi was purified as previously
described(12) . Briefly, E. coli JM105 transformed with
pZWtac1 was grown to
7.5
10
cells/ml at 37
°C in 10 liters of M9 medium, in which
[
N]ammonium chloride (0.1%) provided the sole
nitrogen source. Expression of the ugi gene was induced by the
addition of isopropyl-1-thio-
-D-galactopyranoside to 1
mM, and after 3 h of additional growth, cells were harvested
by centrifugation and stored at -80 °C. Purification was
carried out essentially as described by Wang et
al.(9) . After purification to apparent homogeneity, Ugi
was diafiltered against NMR buffer (25 mM deuterated Tris, 0.2
mM EDTA, 0.2 EGTA, and 100 mM NaCl, pH 7.0) and
concentrated to
2.2 mM. E. coli uracil-DNA
glycosylase was purified to apparent homogeneity as previously
described (1) and concentrated under 55 p.s.i. of prepurified
N
in an Amicon stirred cell equipped with a 62-mm diameter
YM10 DIAFLO membrane (Amicon) to 24.5 µM. A portion (625
µl) of [
N]Ugi was combined with 54.3 ml of
Ung and incubated at 4 °C for 1 h. The protein mixture was then
concentrated under prepurified N
in an Amicon 10-ml stirred
cell (25-mm diameter, YM10 membrane) and diafiltered against 125 ml of
NMR buffer to a final volume of 1.8 ml. The concentration of the
complex was determined by absorbance spectroscopy using the extinction
coefficient
= 5.4
10
,
which represents the sum of the extinction coefficients of the protein
components (4.2
10
for Ung and 1.2
10
for Ugi) and found to be 739 µM. Samples (9 µg)
of the complex reaction taken before and after concentration and
diafiltration were analyzed by nondenaturing 12.5% polyacrylamide gel
electrophoresis as previously described(1) ; greater than 95% of
the protein was in complex as judged by Coomassie Blue staining.
Nuclear Magnetic Resonance
All of the NMR
spectra were obtained using a Varian Unityplus 400 spectrometer
equipped with a Nalorac ID400 probe. Three-dimensional NOESY-HMQC and
TOCSY-HSQC were obtained using uniformly N-labeled Ugi
(13-16). Sensitivity enhanced TOCSY-HSQC data obtained using a
60-ms mixing time were applied to group the spin systems of the
N amide-labeled residues. TOCSY-HSQC data were collected
at 17, 30, and 37 °C with eight transients per increment and
obtained by the States-Haberkorn method along each indirectly detected
dimension. Each of the complex data sets was analyzed using 128
increments of t
and 24 increments of t
. The data were linearly predicted to 256 points
in t
and 48 points in t
prior
to Fourier transformation into 512
128
1024 points using
shifted Gaussians along each dimension.
and 20 increments in t
. The
data were linearly predicted to 256 points in t
and 40 points in t
before Fourier
transformation into 512
128
1024 points using shifted
Gaussians along each dimension. Representative planes from the
NOESY-HMQC data have been previously presented(6) .
J
dihedral angle-coupling
constants(13, 14, 15, 16) . Mixing times
of 10, 30, 50, 60, 70, 80, 100, and 140 ms were used. Data were
collected with 256 points along t
and 2048 points
during the acquisition time. The data were zero filled to 512 points
along t
and Gaussian shifted before Fourier
transformation into 512
2048 points. The modulation of the
volume, V, of each HSQC signal was linearly approximated to V(
) =
constant
cos(
J
)
with respect to the mixing time,
, to produce 67
NH-C
H dihedral angle-coupling constants. The linearly predicted
time of the change in sign of a peaks amplitude was within 2% error of
the exact non-linear expression for the J-modulated evolution of a HSQC
signals.
N T
times. Delays of 0, 35, 80, 150, 300, 600, and 1200 ms were used
with 256 points along t
and 2048 points during the
acquisition time. The data were zero filled and Gaussian shifted along
both dimensions prior to Fourier transformation into 1024
4096
points. The volume of each HSQC peak was fitted with respect to mixing
time and least square fitted to V(t) = V(t =
0)
exp(-t/T
), with t the delay time, to produce a best fit T
time
for each
N-labeled atom that could be accurately found in
at least four of the seven XT1 HSQC experiments.
Method of Structure Calculation
A total
of 968 interproton restraints were acquired from 200-ms
three-dimensional HMQC NOESY data obtained at 17, 30, and 37 °C
experiments. Of these NOESY constraints, 270 were intraresidue and 636
were interresidue. Of the 636 interresidue NOESY constraints, 329 were
sequential. The 908 NOESY restraints were divided into three categories
corresponding to strong, medium, and weak. The respective ranges of the
categories were 1.8-5.0, 2.1-5.0, and 2.4-5.0
Å.
dihedral constraints was obtained from
a series of six J-modulated HMQC experiments. The 67 dihedral angle
constraints were divided into three categories. When
J
had a coupling constant greater than
8 Hz,
was constrained to -140 ± 40°. When the
coupling constant was below 6 Hz and the residue was part of a helical
secondary structure element,
was constrained to -80
± 40°. Coupling constants below 6 Hz of residues not in a
helical secondary structure element were not given any dihedral
constraints. The rest of the coupling constants were constrained to
-120 ± 40°.
+ r
or
- r
, where ceil is 1000 kcal/mol, S is a scale factor of 100 kcal/mol, T is the
temperature 300 K, and K
is the Boltzmann
constant.
is the distance from the edge of the square well NOE
constraints, and a and b are determined by X-PLOR
such that the function is a smooth function at the point R = d + d
+ r
. The slope from the asymptote is given by c, which was set to 0.1 during the initial high temperature
molecular dynamics; otherwise, it was set to 1.0. The switching
distance was set to 0.5 Å. The exponent softexp was set to 1, and
exp was set to 2. A square biharmonic potential of 100 was used for all
dihedral constraints. The simulated annealing protocol started with 500
steps of energy minimization, followed by 6000 steps of a 4-fs timestep
(24 ps total) molecular dynamics at 1000 K with a scaled energy
function such that angular energies were multiplied by 0.4, torsions
were multiplied by 0.1, and van der Waals terms were multiplied by
0.002. Another 3000 steps of 4-fs timestep (12 ps total) molecular
dynamics at 1000 K were carried out with an intact energy function
except for the van der Waals, which was scaled to 0.002 its normal
value. The system was cooled by dropping the temperature at 50-K
intervals to a final temperature of 200 K. At each temperature, 1000
steps of a 4-fs timestep (4 ps total) molecular dynamics were done
while slowly scaling the van der Waals radius up to a final value of
0.75 at 200 K. This was followed by 2000 steps of square well conjugate
energy minimization with the van der Waals energy function multiplied
by 4.0. The 40 calculated Ugi structures with the lowest total energy
and smallest number of violations were saved for 40 additional,
separate iterations of refined simulated annealing.
is the Boltzmann
constant, and
is the distance from the edge of the square well
NOE constraints. The NOE distance constraints were 1.8-4.0,
2.1-4.5, and 2.4-5.0 Å for strong, medium, and weak
NOE constraints, respectively. An additional 19 hydrogen bonds were put
in as NOE distance constraints; only those hydrogen bonds with long
amide exchange times that fit within the anti-parallel
-sheet
secondary and tertiary structure scheme were used. Hydrogen bonds were
constrained with a N-O distance of 2.5-3.3 Å and
H-O distance of 1.8-2.5 Å. After the simulated
annealing to 100 K, 500 steps of energy minimization with a van der
Waals energy function multiplied by 4.0 were executed. This was
followed by 100 steps of energy minimization with a hard square
potential of 200 kcal/mol for both NOE and dihedral angle constraints.
The 12 lowest energy structures with no NOE violations greater than 0.4
Å and no dihedral angle violations greater then 5° were
saved. These structures have a 1.0 Å root mean square deviation
for the heavy backbone atoms and 1.3 Å root mean square deviation
for all heavy atoms. Fig. 4contains the overlay of these 12
structures.
Figure 4:
The structures shown are 12 of the
structures found to be consistent with the NMR
data.
The electrostatic surfaces were generated using the
program GRASP(19) . The electrostatic potentials of Ugi were
calculated with a dielectric constant of 4.0 for the protein and 80.0
for the solvent. The ionic strength was set to zero. The charges of the
side chains of the Lys, Asp, and Glu residues were used. The
electrostatic potential of the duplex DNA was generated in the same
manner with charges on the phosphate backbone. The DNA duplex used is
the symmetric dimer of d(CGCGAATTCGCG) with an idealized B-70
structure.
-strands that are
arranged into a contiguous antiparallel sheet. Not present in secondary
structural features are seven acidic residues: Glu-38, Asp-40, Glu-49,
Asp-52, Asp-61, Glu-64, and Glu-78. In addition, all five of the
-strands have terminal acidic residues on the same side of the
antiparallel sheet as shown in Fig. 1.
Figure 1:
Secondary structure of Ugi. The Glu and
Asp residues not present in secondary structural features are
highlighted (blackovals). Also shown is the
-sheet topology of Ugi; arrows indicate which NOEs were
observed. The terminal Glu and Asp residues of the
-strands are
also highlighted (circledresidues).
The tertiary structure
of Ugi was determined using the combination of the NMR results and
simulated annealing. The relevant NMR information is summarized in Fig. 2and Fig. 3. The assignments of the resonances have
been previously presented(12) . The N T
values have been determined, and the relaxation
data are consistent with a well defined structure.
Figure 2:
Summary of the NMR data used to determine
the tertiary structure of Ugi. The sequential NOEs and the differences
in the chemical shifts of the H protons relative to their
unshifted positions are shown. The J
are in three
groups with ``+'' for couplings characteristic of
-strands, ``X'' for couplings characteristic of
helices, and ``
'' for average couplings. The slowing
exchanging amide protons are indicated by the blackdots.
Figure 3:
The number and types of NOEs for each
residue are indicated as are the N T
values of the amide nitrogens. Also shown are the root mean
square deviation for all heavy atoms of each residue as well as for the
heavy backbone atoms of each residue.
The overlay of
the structure is shown in Fig. 4, and the average tertiary
structure is shown in two perspectives in Fig. 5. In this
structure, the two helices present in the protein are situated on the
same face of the -sheet. Neither of the helices appears to be in
close proximity to any other structural element. The loop formed by
residues 61-68 folds over the
-sheets. The
-strand and
helix regions of the Ugi structure are all quite regular.
Figure 5:
The top structures depict the electrostatic potential and tertiary
structure of Ugi with residue 5 in the upper right and residue
84 on the bottom right. In the electrostatic potential
structure, regions of negative electrostatic potential are indicated by red and positive electrostatic potential by blue. The
cutoff of the electrostatic potentials was set at 6.6 kcal. In the
tertiary structure, the Glu residues are purple, the Asp
residues red, the His residue blue, the Tyr residues green, and the Trp residue gold. In the bottomstructures, the views of the electrostatic potential and
tertiary structure of Ugi are rotated 180° about the vertical axis
so that residue 5 is at the upperleft and residue 84
at the bottomleft. The electrostatic potentials and
color coding are the same as described above.
The
tertiary structure of Ugi was used to calculate the electrostatic
potential that is shown in Fig. 5. The electrostatic potential of
Ugi is quite striking. The tertiary structure brings together Glu-20,
Asp-48, Glu-49, Asp-52, Glu-53, Asp-74, and Glu-78 to form a surface of
highly negative potential, indicated by the red surface, on one face of
the structure. The electrostatic potential of this region is greater
than 6.6 kcal and is thus similar to that generated by the phosphate
backbone of DNA. This large area of negative potential is likely to be
a region of Ugi that interacts with the residues of Ung that naturally
interact with the phosphates of DNA. The other side of Ugi is mostly
neutral as shown in Fig. 5. The electrostatic potential of Ugi is
compared with that of B-form DNA in Fig. 6. It is seen that both
the areas and magnitudes of the electostatic potentials in the two
instances are quite similar.
Figure 6:
The electrostatic potential of Ugi
compared with that of the duplex DNA. Regions of negative and positive
electrostatic potential are indicated by red and blue, respectively.
Other regions of Ugi with negative
potentials are found adjacent to the two helices. As the potentials in
these regions are greater than 6.6 kcal, they could contribute to the
binding to Ung; however, the areas of these potentials are not nearly
as large as that of the main negative potential patch. Moreover, the
negative potentials of these regions are not as strong as that of the
main negative potential region, due in part to charge neutralization by
adjacent Lys residues.
-strands but of those in adjacent loop regions as
well. The size of this knob as well as the magnitude of its
electrostatic potential strongly suggest that this region is important
in the interaction of Ugi with Ung. Since the electrostatic knob is
confined to one face of the Ugi structure, it is likely that most of
the interactions of Ugi with Ung will occur on this face. The
charge-charge repulsion generated by bringing together so many negative
charges is apparently offset by the stability of the
-sheet.
Ugi complex was prepared with
N-labeled
Ugi and the HSQC spectrum of the complex obtained. This spectrum is
compared with that of free Ugi in Fig. 7. The results indicate
that many residues of Ugi undergo considerable conformational change
upon binding to Ung. Preliminary assignments of the resonances and
preliminary analysis of NOE connectivities suggests that the secondary
structure of Ugi does not change upon complex formation. It appears,
rather, that the conformational changes are primarily confined to the
loop regions. The presence of a conformational change is also
consistent with prior kinetic studies of the Ung-Ugi interaction, which
indicated that Ugi underwent a slow, irreversible isomerization or
``locking'' step upon binding to Ung(2) . This
conformational change is most likely associated with this locking step.
Figure 7:
The HSQC spectra of N-labeled
Ugi in the presence and absence of Ung. The assignments of the spectra
are also presented.
The shape of the electostatic knob in free Ugi is determined in part
by the folding of the loop formed by residues 61-68 over the
-sheet. There is an apparent hydrogen bond between Tyr-65 and
His-44 that constitutes a part of the interaction between the loop and
the
-strands. Preliminary studies of the Ugi
Ung complex
suggest that this interaction is disrupted in the complex; hence, the
orientation of the 61-68 loop relative to the
-strands may
be different in the complex.
-strands and
those found in the loops. All four aromatic amino acid residues are
located next to this electrostatic knob, and we speculate at least one
of them may interact with the uracil binding site of Ung. Ugi undergoes
a considerable conformational change upon binding to Ung, which is
consistent with the kinetics of complex formation(2) . That this
conformational change occurs primarily in the loop regions of Ugi may
suggest that the electrostatic knob is present in the complex, although
its size and shape may change upon complex formation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.