Solution structure, mutagenesis, and NH exchange studies of the MutT enzyme-Mg2+-8-oxo-dGMP complex

Article abstract of DOI:10.1016/j.molstruc.2003.12.060

The MutT pyrophosphohydrolase from E. coli (129 residues) catalyzes the hydrolysis of nucleoside triphosphates (NTP), including 8-oxo-dGTP, by substitution at Pβ, to yield NMP and pyrophosphate. The product, 8-oxo-dGMP is an unusually tight binding, slowly exchanging inhibitor with a K _D=52nM, (ΔG°=-9.8kcal/mol) which is 6.1kcal/mol tighter than the binding of dGMP (ΔG°=-3.7kcal/mol). The higher affinity for 8-oxo-dGMP results from a more favorable ΔH_binding (-32kcal/mol) despite an unfavorable -TΔS°_binding (+22kcal/mol). The solution structure of the MutT-Mg²⁺-8-oxo-dGMP complex shows a narrowed, hydrophobic nucleotide-binding cleft with Asn-119 and Arg-78 among the few polar residues. The N119A, N119D, R78K and R78A single mutations, and the R78K+N119A double mutant all showed largely intact active sites, on the basis of small changes in the kinetic parameters of dGTP hydrolysis and in ¹H-¹⁵N HSQC spectra. However, the N119A mutation profoundly weakened the active site binding of 8-oxo-dGMP by 4.3kcal/mol (1650-fold). The N119D mutation also weakened 8-oxo-dGMP binding but only by 2.1kcal/mol (37-fold), suggesting that Asn-119 functioned both as a hydrogen bond donor to C8O, and a hydrogen bond acceptor from N7H of 8-oxo-dGMP, while aspartate at position -119 functioned as an acceptor of a single hydrogen bond. Much smaller weakening effects (0.3-0.4kcal/mol) on the binding of dGMP and dAMP were found, indicating specific hydrogen bonding of Asn-119 to 8-oxo-dGMP. While formation of the wild type MutT-Mg²⁺-8-oxo-dGMP complex slowed the backbone NH exchange rates of 45 residues distributed throughout the protein, the same complex of the N119A mutant slowed the exchange rates of only 11 residues at or near the active site, indicating an increase in conformational flexibility of the N119A mutant. The R78K and R78A mutations weakened the binding of 8-oxo-dGMP by 1.7 and 1.1kcal/mol, respectively, indicating a lesser role of Arg-78 than of Asn-119 in the selective binding of 8-oxo-dGMP, likely donating a single hydrogen bond to its C6O. The R78K+N119A double mutant weakened the binding of 8-oxo-dGMP (K_I ^slope=3.1mM) by 6.5±0.2kcal/mol which overlaps, within error with the sum of the effects of the two single mutants (6.0±0.3kcal/mol). Such additive effects of the two single mutants in the double mutant are most simply explained by the independent functioning of Asn-119 and Arg-78 in the binding of 8-oxo-dGMP. Independent functioning of these two residues in nucleotide binding is consistent with their locations in the MutT-Mg ²⁺-8-oxo-dGMP complex, on opposite sides of the active site cleft, with a distance of 8.4±0.5A? between their side chain nitrogens.

Full text of DOI:10.1016/j.molstruc.2003.12.060

Journal of Molecular Structure 700 (2004) 247–254
www.elsevier.com/locate/molstruc
Solution structure, mutagenesis, and NH exchange studies of the MutT
enzyme–Mg2þ-8-oxo-dGMP complex
*
M.A. Massiah, V. Saraswat, H.F. Azurmendi, A.S. Mildvan
Department of Biological Chemistry, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA
Received 7 November 2003; accepted 2 December 2003
Abstract
The MutT pyrophosphohydrolase from E. coli (129 residues) catalyzes the hydrolysis of nucleoside triphosphates (NTP), including
8-oxo-dGTP, by substitution at Pb, to yield NMP and pyrophosphate. The product, 8-oxo-dGMP is an unusually tight binding, slowly
exchanging inhibitor with a KD ¼ 52 nM, (DG8 ¼ 29:8 kcal/mol) which is 6.1 kcal/mol tighter than the binding of dGMP
(DG8 ¼ 23:7 kcal/mol). The higher affinity for 8-oxo-dGMP results from a more favorable DHbinding (232 kcal/mol) despite an
unfavorable 2TDS8binding (þ22 kcal/mol). The solution structure of the MutT–Mg2þ-8-oxo-dGMP complex shows a narrowed, hydrophobic
nucleotide-binding cleft with Asn-119 and Arg-78 among the few polar residues. The N119A, N119D, R78K and R78A single mutations, and
the R78K þ N119A double mutant all showed largely intact active sites, on the basis of small changes in the kinetic parameters of dGTP
hydrolysis and in 1H–15N HSQC spectra. However, the N119A mutation profoundly weakened the active site binding of 8-oxo-dGMP by
4.3 kcal/mol (1650-fold). The N119D mutation also weakened 8-oxo-dGMP binding but only by 2.1 kcal/mol (37-fold), suggesting that
Asn-119 functioned both as a hydrogen bond donor to C8vO, and a hydrogen bond acceptor from N7H of 8-oxo-dGMP, while aspartate at
position 2119 functioned as an acceptor of a single hydrogen bond. Much smaller weakening effects (0.3–0.4 kcal/mol) on the binding
of dGMP and dAMP were found, indicating specific hydrogen bonding of Asn-119 to 8-oxo-dGMP. While formation of the wild type
MutT–Mg2þ-8-oxo-dGMP complex slowed the backbone NH exchange rates of 45 residues distributed throughout the protein, the same
complex of the N119A mutant slowed the exchange rates of only 11 residues at or near the active site, indicating an increase in
conformational flexibility of the N119A mutant. The R78K and R78A mutations weakened the binding of 8-oxo-dGMP by 1.7 and
1.1 kcal/mol, respectively, indicating a lesser role of Arg-78 than of Asn-119 in the selective binding of 8-oxo-dGMP, likely donating a
single hydrogen bond to its C6vO. The R78K þ N119A double mutant weakened the binding of 8-oxo-dGMP (KIslope ¼ 3:1 mM) by
6.5 ^ 0.2 kcal/mol which overlaps, within error with the sum of the effects of the two single mutants (6.0 ^ 0.3 kcal/mol). Such additive
effects of the two single mutants in the double mutant are most simply explained by the independent functioning of Asn-119 and Arg-78 in
the binding of 8-oxo-dGMP. Independent functioning of these two residues in nucleotide binding is consistent with their locations in the
MutT–Mg2þ-8-oxo-dGMP complex, on opposite sides of the active site cleft, with a distance of 8.4 ^ 0.5 A between their side chain
˚
nitrogens.
q 2004 Elsevier B.V. All rights reserved.
Keywords: Nucleoside triphosphate; MutT enzyme; Hydrogen bonds; Double mutant; Additive effects
1. Introduction
The solution structure [2,3], together with product
inhibition and mutational studies have led to kinetic [4]
and chemical mechanisms [5] for this prototypical Nudix
hydrolase [6]. The best substrate for MutT is the oxidatively
damaged, mutagenic nucleotide, 8-oxo-dGTP [7]
(Scheme 1), which can mispair with template adenine
during DNA replication [8,9]. Genetic inactivation of the
MutT enzyme in E. coli results in a 104-fold increase in
mutations, all of which are AT ! CG transversions [10,11].
Inactivation of a homologous 8-oxo-dGTPase in mice
The MutT enzyme, a nucleoside triphosphate (NTP)
pyrophosphohydrolase, catalyzes the hydrolysis of NTPs to
yield NMPs and inorganic pyrophosphate by nucleophilic
substitution at the rarely attacked Pb [1].
NTP þ H2O ! NMP þ PPi þ Hþ
*
Corresponding author. Tel.: þ1-410-955-2038; fax: þ1-410-955-5759.
E-mail address: mildvan@jhmi.edu (A.S. Mildvan).
0022-2860/$ - see front matter q 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2003.12.060

248
M.A. Massiah et al. / Journal of Molecular Structure 700 (2004) 247–254
Scheme 1.
results in major increases in the occurrence of tumors [12].
These observations suggest that the biological role of MutT
is to prevent errors in DNA replication by hydrolyzing
oxidatively damaged, mutagenic nucleotides such as 8-oxo-
dGTP [7,12]. While the in vivo substrate of the MutT
enzyme has not been unequivocally identified [13], 8-oxo-
guanine nucleotides show unusually high affinities for
MutT, as indicated by the low Km of 8-oxo-dGTP
(0.48 mM), which is 2300-fold (4.5 kcal/mol) lower
than that of dGTP (1100 mM) [7], and by the 35,000-fold
(6.1 kcal/mol) tighter binding of the reaction product, 8-oxo-
dGMP (KD ¼ 52 nM), than of dGMP (KD ¼ 1:8 mM) to the
enzyme–Mg2þ complex [4].
NOESY HSQC spectra [14]. Further structural refinement
was facilitated by the use of residual dipolar couplings of
backbone 15N–H groups [15], calculated from in phase/
anti-phase 1H–15N HSQC spectra, with and without
orienting bicelles [16], as described [14]. Restraints used
in computing the final structures were 1746 NOEs (13.5 per
residue), 41 backbone hydrogen bonds, 186 f and c values
determined with the program TALOS on the basis of 15N,
Ha, Ca, and Cb chemical shifts [17], and the residual
dipolar couplings of 53 backbone 15N–H vectors [18,19].
The structures were computed as described [14] with the
program CNS 1.1 [20] on a Silicon Graphics R10000
computer and a Pentium–Linux work station.
The tight binding of 8-oxo-dGMP (DG8binding
¼
Exchange rates of backbone 15NH protons with deuter-
1
29:8 kcal/mol) results from a favorable enthalpy change
(DHbinding ¼ 232 kcal/mol) which exceeds an unfavorable
entropy change (2TDS8binding ¼ þ22 kcal/mol), the latter
indicating strong ordering in the enzyme–Mg2þ-8-oxo-
dGMP complex [4]. Here, we review the structural basis for
the unusually high affinity of the MutT enzyme for 8-oxo-
dGMP, in comparison with dGMP and dAMP, and show
that local and remote hydrogen bonding play major roles.
ons in D2O were measured by monitoring H–15N HSQC
spectra of MutT as a function of time after having replaced
the solvent with 99.9% D2O by freeze drying, and the data
were analyzed as described [14,21,22].
Selective 1H–15N HSQC spectra of MutT focusing
on the Arg–N1H region were obtained from uniformly
15N-labeled samples, as described [14,23].
2.2. Kinetic methods
2. Experimental
(g-32P)dGTP was prepared by transphosphorylation of
dGDP with (g-32P)ATP as previously reported [5]. Steady-
state kinetic experiments with Mg2þ-activated MutT were
performed by measuring the amount of (g-32P) pyrophos-
phate released from (g-32P)dGTP in Tris–HCl buffer
(50 mM, pH 7.5) at 23 8C [4]. Linear and nonlinear
regression analysis of the kinetic data were performed
using the program Grafit (Erithacus Software Ltd, Staines,
UK).
2.1. General NMR methods
Heteronuclear NMR spectra were collected at 23 8C on a
Varian Unity Plus 600 MHz spectrometer using a Varian
5 mm triple resonance probe. NMR samples (0.4 ml)
typically contained 0.5–1.0 mM MutT, 15 mM MgCl2,
4 mM d11-Tris–HCl, pH 7.5, 21 mM NaCl, 0.34 mM NaN3,
and 10% D2O. Solution structures of free MutT and of the
quaternary MutT–Mg2þ(H2O)AMPCPP–Mg2þ complex
were determined as previously described [2,3]. Because of
2.3. Preparation of mutants of Asn-119 and Arg-78
the extensive chemical shift changes in the H–15N HSQC
The construction of the plasmid pETMutT, containing
the MutT gene under control of the T7 promoter has been
described previously [24]. Appropriate primers, 33 nucleo-
tides in length, containing the desired mutation, were
provided by the Johns Hopkins Medical Institutions
Synthesis and Sequencing Facility. The plasmid isolation
and purification was done, as described [5,22] following the
instruction manuals of the QIAGEN plasmid isolation kit
1
spectra of the MutT–Mg2þ-8-oxo-dGMP complex [4],
reassignment of all resonances was necessary [14]. This
was accomplished by 3D HNCA, 3D HNCACB, 3D
1H–15N NOESY-HSQC, 2D H–1H NOESY, 2D H–1H
TOCSY, and 3D 1H–15N TOCSY HSQC experiments [14].
Tertiary structural information was obtained from the 3D
1H–13C NOESY, 2D 1H–1H NOESY and 3D 1H–15N
1
1

M.A. Massiah et al. / Journal of Molecular Structure 700 (2004) 247–254
249
and the QIAquick gel extraction kit, respectively. The wild-
3. Results and discussion
type MutT gene in the pET11b vector, [pETMutT], was
used as the template for the polymerase chain reaction in
order to generate the single mutants of MutT, following the
instruction manual of the QuickChange site-directed
mutagenesis kit. After confirming the sequence of each
mutant of MutT, the E. coli BL21 (DE3) competent cells
were transformed with the plasmid having the mutant gene.
The plasmid containing the mutant gene was isolated from
E. coli BL21 (DE3) and the sequence of the gene was
reconfirmed by DNA sequencing. With the Asn-119
mutants, the mutation was also confirmed by the disap-
pearance of the Asn-119-15NdH2 cross peaks from the
1H–15N HSQC spectrum [22].
3.1. Nucleotide binding site
The solution structure of the MutT–Mg2þ(H2O)
AMPCPP-Mg2þ complex showed the AMP moiety to be
57% buried in a cleft, making direct contact with five
residues, and showing second sphere interactions with nine
additional residues. Of these 14 residues which constitute
the nucleotide binding site, 10 were hydrophobic (Leu, Ile,
Phe), the exceptions being Lys-3, Lys-39 near the
phosphate, and Arg-78 and Asn-119 near the purine ring
[3,14]. Calorimetric studies of the binding of 8-oxo-dGMP
to the Mg2þ complex of MutT showed a negative change
in heat capacity (DCp ¼ 2680 cal mol21 K21), consistent
with the burial of hydrophobic groups on nucleotide
binding [4].
2.4. Preparation of MutT
The recombinant E coli strain HMS174(DE3)[pET-
MutT] was used for wild-type MutT, whereas the strains
BL21(DE3)[pETMutT-R78A], BL21(DE3)[pETMutT-
R78K], BL21(DE3)[pETMutT-N119A], BL21(DE3)[pET-
MutT-N119D], and BL21(DE3)[pETMutT-R78K-N119A]
were used for preparing uniformly 15N-labeled mutants of
MutT, as described [5,22]. The enzymes were purified to
.95% homogeneity on the basis of SDS-PAGE, as
described [5,22].
3.2. Conformation change in MutT on binding 8-oxo-dGMP
1
As detected by H–15N HSQC experiments, the weak
binding of dGMP (KD ¼ 1:8 mM) to a single site on MutT
altered the backbone 15N and NH chemical shifts of 22
residues, mainly surrounding the nucleotide binding site [4].
In contrast, the tight binding of 8-oxo-dGMP (KD ¼ 52 nM)
to a single site on MutT altered the chemical shifts of 62
residues widely distributed throughout the protein, and by
much greater amounts, indicating diffuse structural changes
throughout MutT [4].
2.5. Measurement of enzyme-Mg2þ-nucleotide dissociation
constants
Independent evidence for widespread conformational
changes in MutT induced by the binding of 8-oxo-dGMP
was provided by measurements of backbone NH exchange
rates with D2O. After 51 h in D2O, dGMP was found to
The dissociation constant of 8-oxo-dGMP from the
ternary MutT–Mg2þ-8-oxo-dGMP complex was deter-
mined kinetically from the KIslope of the nucleotide, and
the DHbinding was determined by van’t Hoff analysis of the
temperature dependence of the KIslope; as described [4]. Both
the dissociation constant and the DHbinding of 8-oxo-dGMP
were independently checked by isothermal titration calori-
metry, as described [4]. Samples containing 6 mM enzyme
and 15 mM MgCl2 in 50 mM Tris–HCl or 50 mM
NaHEPES, pH 7.5, were titrated with small 0.6 mM
increments of 8-oxo-dGMP at 18, 23, and 28 8C [4]. The
release of heat at constant temperature was monitored using
an Omega calorimeter manufactured by Microcal Inc.,
Northampton, MA. Appropriate corrections were made for
the separately measured heat of dilution of the nucleotide.
The data were analyzed as described [4] with the program
Origin 2.9, provided by the manufacturer, to yield KD and
DHexperimental: Measurements of DHexperimental in two buffers
with different values of DHionization permitted the determi-
nation of DHbinding of 8-oxo-dGMP. Measurements of
DHbinding of 8-oxo-dGMP at three temperatures permitted
evaluation of the change in heat capacity DCp on nucleotide
binding [4].
protect 20 residues against exchange in the MutT–Mg2þ
-
dGMP complex. These protected residues were mainly near
the nucleotide binding site. In contrast, after 62 h in D2O,
8-oxo-dGMP protected 45 residues against exchange, and
by much greater factors [14]. These residues were widely
distributed throughout the protein. Since the nucleotide
binding site consists of approximately 14 residues (see
above), widespread tightening of the protein structure must
have occurred on the binding of 8-oxo-dGMP [4,14].
3.3. Solution structure of the MutT–Mg2þ-8-oxo-dGMP
complex
Because of the extensive chemical shift changes in the
1H–15N HSQC spectra of the MutT–Mg2þ-8-oxo-dGMP
complex, reassignment of all resonances and determination
of the solution structure of this complex were carried out as
described in Section 2. The secondary structure of the
enzyme in the MutT–Mg2þ-8-oxo-dGMP complex was
identical to that of free MutT and to MutT in the enzyme–
Mg2þ(H2O)AMPCPP-Mg2þ complex (Fig. 1) [14]. How-
ever, the tertiary structure of the MutT–Mg2þ-8-oxo-dGMP
complex showed a significant change in the position of
The dissociation constants of other nucleotides were also
determined kinetically from the KIslope; and in several cases
1
checked by H–15N HSQC titrations, as described [4,22].

250
M.A. Massiah et al. / Journal of Molecular Structure 700 (2004) 247–254
Fig. 1. (A) Stereo pair showing the solution structure of MutT in the MutT–Mg2þ-8-oxo-dGMP complex. Superposition of backbone Ca, C, and N atoms of 20
converged structures computed with the restraints given in the text. (B) Superposition of the lowest energy structure of MutT in the MutT–Mg2þ-8-oxo-dGMP
complex (green) onto the lowest energy structure of free MutT (white). (C) Superposition of the lowest energy structure of MutT in the MutT–
Mg2þ(H2O)AMPCPP-Mg2þ complex (red) onto the lowest energy structure of free MutT (white). The nucleotides are omitted for clarity [14]. Structures (B)
and (C) were made with Molscript [30].
˚
helix I, which moved 4.5 A toward the active site from its
˚
8.0 ^ 1.7 A from the enzyme-bound divalent cation, and
one of its phosphate oxygens was restrained to be
˚
3.3 ^ 0.7 A from Nz of the catalytic residue, Lys-39,
distances very similar to those found in the AMPCPP
complex [3].
position in the free enzyme. Also, helix II together with loop
4, which precedes helix II, moved toward the cleft, resulting
in an overall closing of the active site (Fig. 1B) [14]. In
contrast, AMPCPP which binds to MutT with a KD which is
103.7-fold (5.0 kcal/mol) weaker than that of 8-oxo-dGMP,
induced much smaller changes in the tertiary structure, in
comparison with that of free MutT (Fig. 1C) [3,14].
NMR evidence suggested hydrogen bonding of the
purine rings of bound nucleotides by Asn-119 and an
arginine residue, likely Arg-78, the only arg in the
nucleotide binding cleft. Thus, the binding of AMPCPP
resulted in a small upfield shift (20.05 ppm) of the
downfield of the two NdH2 resonances of Asn-119, while
the binding of dGTP resulted in a larger downfield shift of
this resonance (þ0.54 ppm), suggesting an interaction of the
guanine base with Asn-119-NH2 [3]. The binding of 8-oxo-
dGMP resulted in the disappearance of both side chain-
NdH2 resonances of Asn-119 in the 15N HSQC spectrum of
the MutT–Mg2þ-8-oxo-dGMP complex, likely due to
exchange broadening. No downfield shifted proton
resonances (.11 ppm) appeared at 5 or 258 which would
indicate unusually strong hydrogen bonds to the purine ring
of 8-oxo-dGMP [14].
3.4. 8-oxo-dGMP Binding Site
While 12 intermolecular NOEs from assigned protons of
MutT to deoxyribose protons of bound 8-oxo-dGMP
defined the position of the bound nucleotide, additional
restraints were required to define its conformation and
orientation [14]. Because of the absence of nonexchange-
able protons on the 8-oxo-guanine ring, no intramolecular
NOEs between the base and deoxyribose were observed.
Hence, the conformation of bound 8-oxo-dGMP was
assumed to be the same as that found for MutT-bound
dGMP, with a high anti-glycosidic torsional angle ðx ¼
73 ^ 98Þ and a C10-endo sugar pucker ðd ¼ 118 ^ 118Þ [25].
These dihedral angles were very similar to those found for
bound AMPCPP [25]. To facilitate the orientation of bound
8-oxo-dGMP, its phosphorus was restrained to be
MutT contains seven arginine residues of which five
1
Arg–N1H resonances were detected in a H–15N HSQC
spectrum of the MutT–Mg2þ complex. The binding of
8-oxo-dGMP to the MutT–Mg2þ complex induced small

M.A. Massiah et al. / Journal of Molecular Structure 700 (2004) 247–254
251
Fig. 2. Comparison of the two alternative orientations for 8-oxo-dGMP bound to MutT [14]. (A) Lowest energy structure with bifunctional hydrogen bonding
from Asn-119 NdH and Od to the C8vO and N7H, respectively, of 8-oxo-dGMP, and monofunctional hydrogen bonding from Arg-78 NhH2 to the C6vO of
8-oxo-dGMP. Bound 8-oxo-dGMP is shown in red, hydrophobic regions are green, polar regions are yellow, and cationic regions are blue. (B) Details of
hydrogen bonding in (A). (C) Lowest energy structure with monofunctional hydrogen bonding from Arg-78 NhH2 to the C8vO of 8-oxo-dGMP, and
bifunctional hydrogen bonding from Asn-119 NdH and Od to the C6vO and N1H, respectively, of 8-oxo-dGMP. (D) Details of hydrogen bonding in (C).
Structures (A) and (C) were made with Molscript [30].
shifts in these signals and the appearance of two additional
Arg–N1H resonances, presumably due to slowed exchange
with the solvent, suggesting increased N1H hydrogen bond
donation by these two arginine residues [14].
3.5. Effects of mutations of Asn-119 and Arg-78 on kinetic
and structural properties of MutT
To resolve the orientational ambiguity of 8-oxo-dGMP
(Fig. 2), and to determine the quantitative contributions of
Asn-119 and Arg-78 to the high affinity and specificity of
MutT for 8-oxo-nucleotides, the effects of mutating these
residues on the nucleotide binding and structural properties
of MutT were determined [22]. In the standard assay of
MutT with Mg2þdGTP as substrate, mutations of Asn-119,
Arg-78, and the double mutant R78K þ N119A resulted in
small or no effects on kcat (#2-fold) and on Km (#4-fold),
indicating largely intact active sites. Constant specific
activities of these mutants after weeks of storage at
220 8C indicated stable protein structures. Intact protein
structures for N119A, N119D, R78K and R78A single
mutants, and for the R78K þ N119A double mutant were
Application of these hydrogen bonding restraints led to
two alternative orientations of enzyme-bound 8-oxo-dGMP
(Fig. 2). In the first case (Fig. 2A and B), bifunctional
hydrogen bonding occurred from Asn-119 NdH and Od to
the C8vO and N7H of 8-oxo-dGMP, respectively, and
monofunctional hydrogen bonding occurred from Arg-78
NhH2 (or N1H) to the C6vO of 8-oxo-dGMP [14]. This
structure buried 78% of the total surface area of the
nucleotide [14]. In the second case (Fig. 2C and D), the
opposite orientation of the purine ring was assumed, with
Asn-119 NdH and Od hydrogen bonded to the C6vO and
N1H, respectively, of 8-oxo-dGMP, and with Arg-78 NhH2
(or N1H) hydrogen bonded to the C8vO of 8-oxo-dGMP.
This structure buried 71% of the total surface area of the
nucleotide [14].
1
confirmed by H–15N HSQC spectra which showed few
changes in backbone 15N and NH chemical shifts from those

252
M.A. Massiah et al. / Journal of Molecular Structure 700 (2004) 247–254
Table 1
Effects of mutations of Asn-119 and Arg-78 on the KIslope (mM) of NMP
Asn-119 to the selectivity of binding of 8-oxo-dGMP
compared to dGMP and dAMP is 4:3 2 0:4 ¼ 3:9 kcal/mol
(Table 2). Hence, 3.9 of the 6.1 kcal/mol tighter binding of
8-oxo-dGMP than of dGMP may be provided by Asn-119.
We have noted above that the tight binding of 8-oxo-
dGMP to wild type MutT is accompanied by a change in
tertiary structure which narrows the nucleotide binding cleft
[14], and by a general tightening of the protein structure, as
detected by the slowed H/D exchange of the backbone NH
resonances of 45 residues after 62 h in D2O [14]. The
weaker binding of dGMP to wild type MutT protected only
20 residues against NH exchange [14]. With the N119A
mutant, after 62 h of exchange in D2O under essentially
identical conditions to those used with the wild type
enzyme, 8-oxo-dGMP slowed the NH exchange of only
11 residues, indicating a much more flexible protein
structure in the N119A mutant. All of the 11 residues
strongly protected against exchange in the mutant were in or
near the active site suggesting that most of these residues
were directly protected by the bound nucleotide. Ten of
these 11 residues were among the 45 residues protected
against exchange in the 8-oxo-dGMP complex of the wild
type enzyme. Hence the N119A mutation accelerated the
NH exchange of 35 residues throughout the protein, by one
to four orders of magnitude, consistent with greater
conformational flexibility in this mutant [22].
products of the MutT reactiona
Enzyme
8-oxo-dGMP
dGMP
dAMP
Wild type
N119A
0.049 ^ 0.015
81.00 ^ 21.58b
1.80 ^ 0.16
0.32 ^ 0.03c
0.82 ^ 0.11
3100 ^ 500
1750 ^ 500
3200 ^ 650
6750 ^ 1050
5250 ^ 500
5500 ^ 1000
12400 ^ 1600
4750 ^ 250
8000 ^ 1950
11900 ^ 1500
6500 ^ 450
N119D
R78A
R78K
13950 ^ 500
22800 ^ 7800
R78K þ N119A
a
From Refs. [4,22].
b
c
KD from HSQC titration ¼ 240 ^ 130 mM [22].
KD from HSQC titration p0:5 mM [22].
of the wild type enzyme, and these were mainly near the
sites of mutation [22].
3.6. Effects of the N119A mutation on nucleotide binding
and NH exchange properties of MutT
Despite their largely intact protein structures, and nearly
wild type values for kcat and Km; several of the mutants
showed much weaker binding of the nucleotide product
8-oxo-dGMP. Other nucleotide products showed smaller
effects of the mutations, as accurately and conveniently
measured by their KIslope values (Tables 1 and 2). The
N119A mutation resulted in a major 1650-fold decrease in
the affinity of the enzyme for 8-oxo-dGMP, as measured by
the increase in KIslope; corresponding to a loss in binding free
energy ðDDG8bindingÞ of 4.3 kcal/mol (Table 2). Weakened
binding of 8-oxo-dGMP by the N119A mutant was
independently confirmed by 1H–15N HSQC titration,
which yielded a KD for 8-oxo-dGMP of 237 ^ 130 mM
that overlapped with its KIslope of 81 ^ 22 mM [22]. This
weakening effect of N119A was highly selective for 8-oxo-
dGMP, since dGMP and dAMP showed ,2-fold increases
in KIslope; corresponding to much smaller losses in binding
free energy ðDDG8bindingÞ of only 0.4 and 0.3 kcal/mol,
respectively. Hence, the quantitative contribution of
3.7. Properties of the N119D mutant
A 37-fold decrease in affinity for 8-oxo-dGMP, in
comparison with wild type MutT, was found with the
N119D mutant, as measured by the increase in KIslope
;
corresponding to a loss of binding free energy ðDDG8binding
Þ
of 2.1 kcal/mol, half of that lost in the N119A mutant
(4.3 kcal/mol, Table 2). These free energies suggest that the
side chain of asparagine simultaneously participates in
two hydrogen bonds, acting as both a donor and acceptor
(Fig. 2), while that of aspartate participates in only one
hydrogen bond, as an acceptor.
Table 2
Factors by which KIslope of nucleotides increase, and increases in the free energies of binding of nucleotides in mutants of MutT
Enzyme
8-oxo-dGMP
dGMP
dAMP
Specificity for 8-oxo-dGMPa
KIslope
DDG8binding
(kcal/mol)
KIslope
DDG8binding
(kcal/mol)
KIslope
DDG8binding
(kcal/mol)
lDDDG8bindingl
(kcal/mol)
(fold increase)
(fold increase)
(fold increase)
Wild type
N119A
1.0
0
1.0
0
1.0
0
6.1 ^ 0.9b
3.9 ^ 0.4
1.3 ^ 0.3
0.5 ^ 0.3
1.0 ^ 0.3
5.4 ^ 0.3
1650 ^ 670
36.7 ^ 11.7
6.6 ^ 2.1
4.3 ^ 0.3
2.1 ^ 0.2
1.1 ^ 0.2
1.7 ^ 0.2
6.5 ^ 0.2
1.84 ^ 0.63
3.87 ^ 1.24
3.01 ^ 0.90
3.14 ^ 1.06
7.13 ^ 2.20
0.36 ^ 0.21
0.79 ^ 0.20
0.64 ^ 0.18
0.67 ^ 0.20
1.15 ^ 0.18
1.69 ^ 0.42
2.52 ^ 0.34
1.38 ^ 0.11
2.96 ^ 0.17
4.80 ^ 1.66
0.31 ^ 0.15
0.54 ^ 0.08
0.19 ^ 0.05
0.63 ^ 0.04
0.92 ^ 0.21
N119D
R78A
R78K
16.8 ^ 5.6
63300 ^ 21900
R78K þ N119A
Derived from the data in Table 1.
a
Specificity ¼ DDG8binding (8-oxo-dGMP) 2 DDG8 binding (dGMP).
From the ratio of the KIslope values of dGMP and 8-oxo-dGMP.
b

M.A. Massiah et al. / Journal of Molecular Structure 700 (2004) 247–254
253
As with N119A, the weakening effect of N119D was
selective for 8-oxo-dGMP, with order of magnitude smaller
weakening effects on the binding of dGMP (3.9-fold) and
dAMP (2.5-fold) (Table 2). The residual binding selectivity
of 1.3 kcal/mol of the N119D mutant for 8-oxo-dGMP
combined in the R78K þ N119A double mutant. This
double mutant showed kcat and Km values for dGTP
indistinguishable from those of the wild type enzyme,
indicating an intact active site. The 1H–15N HSQC
spectrum showed chemical shift changes for 21 out of 113
detectable resonances, predominantly surrounding the sites
of the two mutations, indicating an otherwise intact protein
(DDG8binding ¼ 2:1 kcal/mol) versus dGMP (DDG8binding
¼
0:8 kcal/mol) (Table 2) could well result from Asp-119
accepting a single hydrogen bond from N7H of 8-oxo-
dGMP. The selective effects on 8-oxo-dGMP binding by
both the N119A and N119D mutations support bifunctional
interaction of Asn-119 with 8-oxo-dGMP, as shown in
Fig. 2A and B, and argue against the structures shown in
Fig. 2C and D.
structure. However, as measured by the increases in KIslope
;
the R78K þ N119A double mutant weakened 8-oxo-dGMP
binding 63,000-fold, while the single mutants R78K and
N119A weakened 8-oxo-dGMP binding by factors of 17 and
1650, respectively (Table 2). Within experimental error, the
product of the effects of the two single mutants on the KIslope
of 8-oxo-dGMP (28,000 ^ 15,000) overlaps with the effect
of the double mutant on this parameter (63,000 ^ 22,000).
Hence the loss of binding free energy for 8-oxo-dGMP
3.8. Properties of mutants of Arg-78
The otherwise intact R78K mutation weakened 8-oxo-
dGMP binding 17-fold, corresponding to a DDG8binding of
1.7 kcal/mol, with smaller (3-fold) effects on dGMP and
dAMP binding (Tables 1 and 2), indicating a lesser role of
Arg-78 than of Asn-119 in the selective binding of 8-oxo-
dGMP [22]. The 1.0 kcal/mol difference in contributions of
Arg-78 to the binding of 8-oxo-dGMP (1.7 kcal/mol) and
dGMP (0.7 kcal/mol) is a measure of the contribution of
Arg-78 to selectivity. Hence, Asn-119 plays a much larger
role than Arg-78 in both the binding of 8-oxo-dGMP
(4.3 kcal/mol versus 1.7 kcal/mol) and in the selectivity of
MutT for 8-oxo-dGMP versus dGMP (3.9 kcal/mol versus
1.0 kcal/mol) (Table 2). The 2.6 kcal/mol greater contri-
bution by Asn-119 to the binding of 8-oxo-dGMP is
consistent with bifunctional hydrogen bonding between
Asn-119 and the purine ring of 8-oxo-dGMP, and mono-
functional hydrogen bonding by Arg-78 (Fig. 2). The
2.9 kcal/mol greater contribution of Asn-119 to the
selectivity for 8-oxo-dGMP over dGMP is consistent with
the structure shown in Fig. 2A and B in which Asn-119 both
donates a hydrogen bond to C8vO and accepts a hydrogen
bond from N7H of 8-oxo-dGMP, while Arg-78 donates a
hydrogen bond to C6vO. The opposite orientation of bound
8-oxo-dGMP shown in Fig. 2C and D makes, at most, a two
order of magnitude smaller contribution to the structure,
consistent with the substantially greater contribution of
Asn-119 than of Arg-78 to the selectivity for 8-oxo-dGMP,
since Fig. 2C and D predict that Arg-78 alone contributes to
the selectivity by donating a single hydrogen bond to
C8vO, and that Asn-119 does not contribute to selectivity
[22]. Surprisingly, the R78A mutation was less damaging to
introduced
by
the
N119A
single
mutant
(DDG8binding ¼ 4:3 ^ 0:3 kcal/mol) and the R78K single
mutant (DDG8binding ¼ 1:7 ^ 0:2 kcal/mol) were, within
error, additive in the double mutant (DDG8binding
¼
6:5 ^ 0:2 kcal/mol). Similarly, the much smaller decreases
in affinity of MutT for dGMP and dAMP in the single
mutants also show additivity in the double mutant (Table 2).
Such additivities of the effects of the two single mutations
in the double mutant are most simply explained by
the independent functioning of Asn-119 and Arg-78 in
the binding of 8-oxo-dGMP, dGMP and dAMP [26]. The
independent functioning of Asn-119 and Arg-78 in the
binding of nucleotides is consistent with their somewhat
remote locations from each other, on opposite sides of the
nucleotide binding cleft, with side chain N to N distances
˚
between Asn-119-NdH2 and Arg-78-NhH2 of 8.4 ^ 0.5 A
in the 8-oxo-dGMP complex (Fig. 2) [14].
The R78K þ N119A double mutant weakens the binding
of 8-oxo-dGMP by 6.5 kcal/mol, but weakens the binding of
dGMP by only 1.1 kcal/mol (Table 2) indicating a selective
effect of the two mutations of 5.4 kcal/mol on 8-oxo-dGMP
binding. The magnitude of this selective effect approaches
the 6.1 kcal/mol greater affinity of the wild type enzyme for
8-oxo-dGMP than for dGMP (Table 2) [4]. These data
suggest that hydrogen bonding by Asn-119 and Arg-78, as
shown in Fig. 2A and B provides the major driving force for
the tight and selective binding of 8-oxo-dGMP by the wild
type enzyme (DG8binding ¼ 29:8 kcal/mol), and for the
resulting conformational changes [4,14]. These confor-
mation changes result in the protection of 45 residues
against NH exchange suggesting the tightening of numerous
hydrogen bonds, consistent with the highly favorable
DHbinding (232 kcal/mol). The resulting increased ordering
of the system would explain the unfavorable 2TDS8binding
(þ22 kcal/mol) [4].
the binding of 8-oxo-dGMP (6.6-fold increase in KIslope
)
than the more conservative R78K mutation (17-fold
increase in KIslope), reinforcing the modest role of Arg-78
in the selective binding of 8-oxo-dGMP [22].
Finally, it is noted that the important roles of Arg-78 and
Asn-119, found here to provide the structural basis for the
selective binding of 8-oxo-dGMP by E. coli MutT, are not
universal [27–29], even among MutT homologs [27]. Thus
in the human, mouse, and rat homologs of MutT, Arg-78 is
3.9. Properties of the R78K þ N119A double mutant
The single mutations of Arg-78 and Asn-119 which were
most damaging to the binding of 8-oxo-dGMP were

254
M.A. Massiah et al. / Journal of Molecular Structure 700 (2004) 247–254
[9] K.C. Cheng, D.S. Cahill, H. Kasai, S. Nishimura, L.A. Loeb, J. Biol.
Chem. 267 (1992) 166.
replaced by Ser, and Asn-119 is replaced by Asp [27].
Further, Trp-117 is conserved among mammalian MutT
homologs, and the W117A mutation of the human homolog
of MutT decreased both the 8-oxo-dGTPase and the 2-oxo-
dATPase activities by at least an order of magnitude,
suggesting the importance of aromatic ring stacking in these
homologs [27]. However, in E. coli MutT, Trp-117 is
replaced by Pro. Although the purine rings of bound
nucleotides approach Tyr-73 and Phe-75 in E. coli MutT,
they do not stack onto them [3,14].
[10] H.P. Treffers, V. Spinelli, N.O. Belser, Proc. Natl Acad. Sci. USA 40
(1954) 1064.
[11] C. Yanofsky, E.C. Cox, V. Horn, Proc. Natl Acad. Sci. USA 55 (1966)
274.
[12] T. Tsuzuki, A. Egashira, H. Igarashi, T. Iwakuma, Y. Nakatsuru, Y.
Tominaga, H. Kawate, K. Nakao, K. Nakamura, F. Ide, S. Kura, Y.
Nakabeppu, M. Katsuki, T. Ishikawa, M. Sekiguchi, Proc. Natl Acad.
Sci. USA 98 (2001) 11456.
[13] M.L. Tassotto, C.K. Mathews, J. Biol. Chem. 277 (2002) 15807.
[14] M.A. Massiah, V. Saraswat, H.F. Azurmendi, A.S. Mildvan,
Biochemistry 42 (2003) 10140.
[15] M. Ottiger, F. Delaglio, A. Bax, J. Magn. Reson. 131 (1998) 373.
[16] M. Ru¨ckert, G. Otting, J. Am. Chem. Soc. 122 (2000) 7793.
[17] G. Cornilescu, F. Delaglio, A. Bax, J. Biomol. NMR 13 (1999) 289.
[18] J.R. Tolman, J.M. Flanagan, M.K. Kennedy, J.H. Prestegard, Proc.
Natl Acad. Sci. USA 92 (1995) 9279.
Acknowledgements
These studies were supported by National Institutes of
Health Grant DK28616. We are grateful to Gregory Lopez,
and L. Mario Amzel for valuable collaboration in the
calorimetric measurements.
[19] N. Tjandra, A. Bax, Science 278 (1997) 1111.
[20] A.T. Brunger, P.D. Adams, G.M. Clore, W.L. Delano, P. Gros, R.W.
Grosse-Kunstleve, J.-S. Jiang, J. Kuszewski, M. Nilges, N.S. Pannu,
R.J. Read, L.M. Rice, T. Simonson, G.L. Warren, Acta Crystallogr.
D54 (1998) 905.
[21] Y. Bai, J.S. Milne, L. Mayne, S.W. Englander, Proteins, Struct.,
Funct., Genet. 17 (1993) 75.
References
[22] V. Saraswat, H.F. Azurmendi, A.S. Mildvan, Biochemistry 43 (2004)
3404.
[1] A.S. Mildvan, D.J. Weber, C. Abeygunawardana, Adv. Enzymol.
Relat. Areas Mol. Biol. 73 (1999) 183.
[23] O. Zhang, L.E. Kay, P.J. Olivier, J.D. Foreman-Kay, J. Biomol. NMR
4 (1994) 845.
[2] C. Abeygunawardana, D.J. Weber, A.G. Gittis, D.N. Frick, J. Lin,
A.-F. Miller, M.J. Bessman, A.S. Mildvan, Biochemistry 34 (1995)
14997.
[24] C. Abeygunawardana, D.J. Weber, D.N. Frick, M.J. Bessman, A.S.
Mildvan, Biochemistry 32 (1993) 13071.
[3] J. Lin, C. Abeygunawardana, D.N. Frick, M.J. Bessman, A.S.
Mildvan, Biochemistry 36 (1997) 1199.
[25] D.N. Frick, D.J. Weber, C. Abeygunawardana, A.G. Gittis, M.J.
Bessman, A.S. Mildvan, Biochemistry 34 (1995) 5577.
[26] A.S. Mildvan, D.J. Weber, A. Kuliopulos, Arch. Biochem. Biophys.
294 (1992) 327.
[4] V. Saraswat, M.A. Massiah, G. Lopez, L.M. Amzel, A.S. Mildvan,
Biochemistry 41 (2002) 15566.
[5] T.K. Harris, G. Wu, M.A. Massiah, A.S. Mildvan, Biochemistry 39
(2000) 1655.
[27] Y. Sakai, M. Furuichi, M. Takahashi, M. Mishima, S. Iwai, M.
Shirakawa, Y. Nakabeppu, J. Biol. Chem. 277 (2000) 8579.
[28] S.D. Bruner, D.P.G. Norman, G.L. Verdine, Nature 403 (2000) 859.
[29] J.C. Fromme, G.L. Verdine, Nat. Struct. Biol. 9 (2002) 544.
[30] P.J. Kraulis, J. Appl. Crystallogr. 24 (1991) 946.
[6] M.J. Bessman, D.N. Frick, S.F. O’Handley, J. Biol. Chem. 271 (1996)
25059.
[7] H. Maki, M. Sekiguchi, Nature 355 (1992) 273.
[8] S. Shibutani, M. Takeshita, A.P. Grollman, Nature 349 (1991) 431.