A gel mobility shift assay and a polymerase activity assay showed that SQMG competed with DNA for a binding site on the N-terminal 8-kDa domain of pol b, subsequently inhibiting its cata
Trang 1of rat DNA polymerase b
Nobuyuki Kasai1, Yoshiyuki Mizushina2, Hiroshi Murata1, Takayuki Yamazaki1, Tadayasu Ohkubo3, Kengo Sakaguchi1and Fumio Sugawara1
1 Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba, Japan
2 Department of Nutritional Science, Kobe-Gakuin University, Kobe, Hyogo, Japan
3 Department of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan
We screened many DNA polymerase inhibitors
obtained from natural sources, such as long chain
unsaturated fatty acids, bile acids, terpenoids, and
sulfolipids [1–6] Sulfoquinovosylmonoacylglycerol
(SQMG) (Fig 1A,B), which was isolated from sea
algae, has been shown to be a potent inhibitor of
euk-aryotic DNA polymerases (pol) a, pol b, pol d, pol e,
pol g, pol j, pol k, terminal deoxynucleotidyl
trans-ferase (TdT) and HIV-1 reverse transcriptase, but not
of prokaryotic polymerases such as E coli DNA
polymerase I [7,8] SQMG showed potent antitumor activities in vivo in nude mice transplanted with human adenocarcinoma cells [9,10] and suppressed tumor cell proliferation in vitro [11] We have already reported a pathway of total chemical synthesis of SQMG for bio-chemical and medicinal experiments [12]
Pol b is a key enzyme that protects the cell against DNA damage by base excision repair Eukaryotic DNA polymerases are classified into four group: A, B, X and
Y [13] Pol b is a member of the polymerase X (pol X)
Keywords
binding site; DNA polymerase b; inhibitor;
NMR chemical shift mapping;
sulfoquinovosylmonoacylglycerol
Correspondence
F Sugawara, Department of Applied
Biological Science, Tokyo University of
Science, Noda, Chiba 278–8510, Japan
Fax: +81 4 7123 9767
Tel: +81 4 7124 1501 (ext 3400)
E-mail: sugawara@rs.noda.tus.ac.jp
(Received 11 May 2005, revised 29 June
2005, accepted 6 July 2005)
doi:10.1111/j.1742-4658.2005.04848.x
We have previously reported that sulfoquinovosylmonoacylglycerol (SQMG) is a potent inhibitor of mammalian DNA polymerases DNA polymerase b (pol b) is one of the most important enzymes protecting the cell against DNA damage by base excision repair In this study, we charac-terized the inhibitory action of SQMG against rat pol b SQMG competed with both the substrate and the template-primer for binding to pol b A gel mobility shift assay and a polymerase activity assay showed that SQMG competed with DNA for a binding site on the N-terminal 8-kDa domain
of pol b, subsequently inhibiting its catalytic activity Fragments of SQMG such as sulfoquinovosylglycerol (SQG) and fatty acid (myristoleic acid, MA) weakly inhibited pol b activity and the inhibitory effect of a mixture
of SQG and MA was stronger than that of SQG or MA To characterize this inhibition more precisely, we attempted to identify the interaction interface between SQMG and the 8-kDa domain by NMR chemical shift mapping Firstly, we determined the binding site on a fragment of SQMG, the SQG moiety We observed chemical shift changes primarily at two sites, the residues comprising the C-terminus of helix-1 and the N-terminus
of helix-2, and residues in helix-4 Finally, based on our present results and our previously reported study of the interaction interface of fatty acids, we constructed two three-dimensional models of a complex between the 8-kDa domain and SQMG and evaluated them by the mutational analysis The models show a SQMG interaction interface that is consistent with the data
Abbreviations
HSQC, heteronuclear single quantum coherence; LA, lithocholic acid; MA, myristoleic acid; NA, nervonic acid; oligo(dT), oligo
deoxyribothymidylic acid; Pol, DNA polymerase; SQG, sulfoquinovosylglycerol; SQMG, sulfoquinovosylmonoacylglycerol; TdT, terminal deoxynucleotidyl transferase.
Trang 2family, which is composed of pol b, pol k, pol l and
TdT Pol X family members share regions that are
similar to the full-length pol b (two helix-hairpin-helix
motifs and a pol X domain) [14] Pol b is the smallest
known DNA polymerase in mammalian cells,
contain-ing 335 amino-acid residues with a molecular mass of
39 kDa, and its structure is highly conserved among
mammals [15] Pol b has two domains with apparent flexibility at a protease-sensitive region between residues 82–86 Trypsin treatment produced an N-terminal 8-kDa domain fragment, which retained binding affinity for ssDNA, and a C-terminal 31-kDa domain fragment with reduced DNA polymerase activity The crystal structure of the full-length pol b [16] and the solution structure of the 8-kDa domain of pol b have been repor-ted [17] The crystal structure of the pol b-DNA com-plex has also been determined, and it reveals important structure-function relationships governing the processes
of DNA polymerization and DNA repair [18,19] Pol b
is one of the most intensively investigated polymerases, particularly among those present in eukaryotic cells
We have determined the binding sites for two types of pol b inhibitors, nervonic acid (NA) (Fig 1E) and litho-cholic acid (LA) by NMR experiments [20,21] These inhibitors bound to the 8-kDa domain of pol b and dis-turbed its binding to the template-primer DNA In this study, we examine the structural interactions of SQMG with rat pol b and discuss the inhibitory action of SQMG against pol b, comparing this with mechanisms
of other inhibitors It is hoped that these studies will aid efforts to design more effective inhibitors of pol b
Results and Discussion
Effects of two SQMGs and NA on the activity
of rat DNA polymerase b
In this study, we examine two types of SQMG, whose fatty acid moieties occur at C14 and C18, respectively
As shown in Fig 1, SQMG(C14:1) bears a myristoleic acid (MA) (Fig 1F) on the glycerol moiety, and SQMG(C18:1) bears an oleic acid on the glycerol moiety Figure 2A shows the inhibitory dose–response curves of SQMG(C18:1), SQMG(C14:1) and NA against pol b We measured the DNA polymerization activity under the same condition in order to make precise comparisons between these inhibitors IC50values of SQMG(C18:1), SQMG(C14:1) and NA were determined to be 0.8, 1.8 and 5 lm, respectively SQMG(C18:1) inhibited pol b activity more strongly than SQMG(C14:1) The inhibi-tory effect of SQMG showed a similar tendency to that
of fatty acid [1] The hydrophobic interaction is import-ant for binding, as the difference of SQMG(C14:1) and SQMG(C18:1) is only in the length of the fatty acid moi-ety The inhibitory effect of SQMG was greater than that of NA The molecular lengths of SQMG(C18:1), SQMG(C14:1) and NA are about 32.0, 27.0 and 28.4 A˚, respectively, as derived from computer models The molecular size of SQMG(C14:1) was very similar to that
of NA The difference in the inhibitory potency of
O
H
HO
H
HO
H
O
OH
SO3H
O
O
H
HO
H
HO
H
O
OH
SO3H
O
O
H
HO
H
HO
H
O
OH
SO3H
C
HO
O
A
B
C
D
E
F
O
H
HO
H
HO
H
OH OH
OH
C
HO
O
Fig 1 Chemical structures of the compounds (A)
sulfoquino-vosylmonoacylglycerol [SQMG(C14:1)], (B)
sulfoquinovosylmono-acylglycerol [SQMG(C18:1)], (C) sulfoquinovosylglycerol (SQG), (D)
D -glucose, (E) nervonic acid (NA) and (F) myristoleic acid (MA).
Trang 3SQMG and NA can be attributed to the relative
hydro-phobicity of the sulfoquinovosyl moiety vs the hydroxyl
moiety
Mode of DNA polymerase b inhibition by SQMG
and NA
In order to elucidate the inhibition mechanism, the
extent of inhibition as a function of DNA
template-primer or dNTP substrate concentrations was studied
(Table 1) SQMG(C18:1) influenced the activities more strongly than did SQMG(C14:1); Table 1 shows a kinetic analysis of the inhibitors In this analysis, poly(dA)⁄ oligo(dT)12)18 and dTTP were used as the DNA template-primer and dNTP substrate, respect-ively Double reciprocal plots of the results show that all of the inhibitors tested for pol b activity competed with the DNA template and the substrate (Table 1) In the case of the DNA template-primer, the apparent maximum velocity (Vmax) was unchanged at 111 pmolÆ
0 20 40 60 80 100
Compound (µM)
C
B
A
0.15 nmol SQMG(C14:1)
conc.
start
DNA + pol β complex M13 ssDNA
DNA + 8-kDa domain complex 0.15 nmol
Lane 1 2 3 4 5 6 7 8 9 10 I/P - 0 0.1 1 10 - 0 0.1 1 10
8-kDa domain full-length pol β
0.5 pmol 1.5 pmol
Lane I/P
17 mer
20 mer
1 100 2 10 3 1 4 0 5 100 6 0
SQMG(C 14:1 ) conc.
full-length pol β 31-kDa domain
Fig 2 (A) Dose–response curves of SQMG(C14:1) and SQMG(C18:1) and nervonic acid Rat DNA polymerase b (0.05 units) was preincubated with the indicated concentrations (0–10 l M ) of SQMG(C 14:1 ) (j), SQMG(C 18:1 ) (d) or NA (n) DNA polymerase activity in the absence of added compounds was taken to be 100% (B) Gel mobility shift analysis Gel mobility shift analysis of binding between M13 ssDNA and DNA polymerase b M13 plasmid ssDNA (2.2 nmol; nucleotide, single strand and singly primed) was mixed with purified proteins and SQMG(C 14:1 ) Lanes 2–5 contained the full-length DNA polymerase b at a concentration of 7.5 l M ; lanes 7–10 contained the 8-kDa domain
at a concentration of 7.5 l M ; lanes 1 and 6 contained no protein Lanes 2, 3, 4, 5, 7, 8, 9 and 10 were mixed with various concentrations of SQMG(C14:1) The concentrations were as follows: lanes 2 and 7, lanes 3 and 8, lanes 4 and 9, and lanes 5 and 10 were zero, 0.75, 7.5 and
75 l M , respectively (C) Analysis of the poly(dA) ⁄ oligo(dT) 16 template ⁄ primer synthetic products DNA synthetic reactions were carried out with 20 l M poly(dA) ⁄ oligo(dT) 16 (¼ 2 ⁄ 1) and 20 l M [ 32 P]dTTP[aP] (60 CiÆmmol)1), and the products were examined by gel electrophoresis and imaging analysis as described in the Experimental procedures section The protein concentrations were as follows: lanes 1–4, 25 n M of the full-length DNA polymerase b; lanes 5 and 6, 75 n M of the 31-kDa domain SQMG(C 14:1 ) concentrations were as follows: lanes 1–6 were
2500, 250, 25, 0, 7500 and 0 n M , respectively Markers indicate the positions of the extended primer.
Trang 4h)1, whereas 242% and 493% increases in the
Michael-is constant (Km) were observed in the presence of 1 and
2 lm SQMG(C14:1), respectively (Table 1) The Vmax
for the dNTP substrate was 62.5 pmolÆh)1, and the Km
for the substrate increased from 3.05 to 20.0 lm in the
presence of 2 lm SQMG(C14:1) (Table 1) The inhibitor
constant (Ki) values, obtained from Dixon plots, were
found to be 0.89 lm and 2.8 lm in the presence of
2 mm for the DNA template-primer and the dNTP
sub-strate, respectively (Table 1) Similarly, the Kivalues of
SQMG(C18:1) were found to be 0.42 lm and 1.44 lm
for the DNA template-primer and dNTP substrates,
respectively, and the Kivalues of NA were found to be
4.0 lm and 3.5 lm for the DNA template-primer and
dNTP substrates, respectively All of the pol b
inhibi-tors examined competed with both the DNA
template-primer and the dNTP substrate
Binding analysis comparing SQMG and the
N-terminal 8-kDa domain of pol b by a gel
mobility shift assay
We investigated the interaction between the 8-kDa
domain of pol b and SQMG in detail The DNA binding
activity of the 8-kDa domain was analyzed using a gel
mobility shift assay Figure 2B shows results of a gel mobility shift assay demonstrating M13 single stranded DNA (ssDNA) binding to the full-length pol b (lane 2),
as well as to the 8-kDa domain (lane 7) The full-length pol b and the 8-kDa domain formed complexes with the M13 ssDNA, leading to changes in the DNA mobility that appeared as shifts in its position However, the 31-kDa domain, the polymerization domain without a DNA-binding site, was not shifted [23] SQMG(C14:1) interfered with complex formation between M13 ssDNA and pol b (left panel) and between M13 ssDNA and the 8-kDa (right panel) to the same extent The molecular ratios of SQMG(C14:1) (I) and the proteins (P) are repre-sented by I⁄ P in Fig 2B The interference by SQMG(C14:1) is shown with the I⁄ P ratios in lanes 2, 3,
4 and 5, and in lanes 7, 8, 9 and 10 of 0, 0.1, 1 and 10, respectively The interference by SQMG(C14:1) was nearly complete at an I⁄ P ratio of 1, and it disappeared
at the ratio of 0.1, suggesting that one molecule of SQMG(C14:1) competed with one molecule of M13 ssDNA and subsequently interfered with the binding of DNA to the full-length pol b or to the 8-kDa domain The results of the gel mobility shift assay using SQMG(C18:1) instead of SQMG(C14:1) were similar (data not shown)
Table 1 Kinetic analysis of the inhibitory effects of sulfoquinovosylmonoacylglycerols (SQMG(C14:1), SQMG(C18:1)) and NA on the activities
of DNA polymerase b, as a function of the DNA template-primer and the nucleotide substrate concentrations Rat DNA polymerase b was 0.05 units.
Compound Substrate conc (l M ) Compound (l M ) K ma(pmoÆh)1) V maxa K ib(l M ) Inhibitory mode a SQMG(C14:1)
SQMG(C18:1)
NA
a From Lineweaver–Burke plot b From Dixon plot c Poly (dA) ⁄ oligo(dT)12)18 d dTTP.
Trang 5Product analysis after DNA synthesis on
poly(dA)⁄ oligo(dT)
We examined whether the catalytic activity on the
31-kDa domain was inhibited by SQMG The 31-kDa
domain can bind to the DNA template-primer
(although weakly), and it retains the DNA
polymeriza-tion activity We used poly(dA)oligo(dT)16 as the
tem-plate-primer, and analyzed newly synthesized DNA
fragments produced by the 31-kDa domain (Fig 2C)
The reaction products in vitro were investigated by
using denaturing polyacrylamide gel electrophoresis
Figure 2C shows the products formed by the
full-length pol b (lanes 1–4) or the 31-kDa domain (lanes
5–6) It is known that DNA polymerase b is a
distribu-tive enzyme [24], which adds a single nucleotide and
then dissociates from the template-product complex
The 31-kDa domain can replicate DNA in a similar
manner to the full-length pol b
Within a 10-minute incubation period, most of the
primers were elongated (lane 4) With 1.5 pmol of the
31-kDa domain, DNA replication was observed (lane
6) The 8-kDa domain fragment was incapable of
repli-cating DNA [23] At an I⁄ P ratio of more than 10,
SQMG(C14:1) (lanes 1–2) completely suppressed DNA
polymerization by the full-length pol b At an I⁄ P ratio
of 1 for the protein (lane3), DNA synthesis hardly
occurred However, the 31-kDa domain synthesized
DNA without interference from SQMG(C14:1) (lane 5)
At the range of the SQMG(C14:1) concentrations that
influence the template-primer-binding site on the 8-kDa
domain, SQMG(C14:1) is thus thought to indirectly
inhi-bit DNA polymerization at the 31-kDa catalytic site
because the site lacks a template-primer, and it is also
thought to compete with the substrate The results of
the products analysis using SQMG(C18:1) instead of
SQMG(C14:1) were similar (data not shown)
Biochemical characterization of fragments
of SQMG
To determine the inhibitory mechanism of pol b
by SQMG(C14:1), two separated fragments of
SQMG(C14:1), the sulfoquinovosylglycerol (SQG)
(Fig 1C) moiety and the myristoleic acid (MA) moiety,
were prepared SQG weakly inhibited the DNA
poly-merization activity of pol b with the IC50 value of
7.95 mm (Fig 3A) The inhibition dose–response curves
of SQG and MA against pol b were shown in Fig 3B
In the range of 0–1 mm, SQG did not influence pol b
activity, although MA inhibited it with the IC50value
of 375 lm The inhibitory effect of a mixture of SQG
and MA was stronger than that of SQG or MA, and
the IC50value was 120 lm When SQG was present in the polymerase reaction mixture, the MA inhibitory effect on pol b was approximately 2.6-fold stronger The pol b inhibitory effect of SQMG(C14:1) was stron-ger than that of a mixture of SQG and MA (Fig 3B)
An excessive amount of SQG or MA (i.e I⁄ P ¼ 10) did not inhibit the ssDNA binding activity of the 8-kDa domain of pol b (Lanes 3 and 4 of Fig 3C) On the other hand, a mixture of SQG and MA inhibited the activity (Lane 5 of Fig 3C) As the mode of the pol b inhibition by SQG and MA was competitive against both DNA template-primer and dNTP sub-strate (data not shown), it was suggested that a mix-ture of SQG and MA could also competitively inhibit the binding activity of DNA template-primer These results suggested that the SQG moiety could enhance the inhibitory activities of the DNA polymerization and ssDNA binding by MA
NMR experiment to determine the interaction interface between SQMG(C14:1) and the 8-kDa domain
A titration experiment using the 8-kDa domain and a
1 mm stock solution of SQMG(C14:1) was performed as follows Two-dimensional1H-15N HSQC spectra of the 8-kDa domain-SQMG(C14:1) complex at SQMG(C14:1) concentrations of 0.05, 0.1, 0.15 and 0.2 mm were recorded As the concentration of SQMG(C14:1) increased, the cross-peaks of the 8-kDa domain broad-ened At an SQMG(C14:1) concentration of 0.1 mm, most of the cross-peaks disappeared and some broad cross-peaks appeared at 7.8–8.5 p.p.m SQMG(C14:1) may aggregate at the millimolar concentration required for NMR experiments, and the 8-kDa domain may interact with micelle-like forms of SQMG(C14:1) [25]
If the experiment could be carried out at micromolar concentrations, SQMG(C14:1) would not aggregate, as SQMG(C14:1) inhibited the DNA polymerization activ-ity of the full-length pol b but not the 31-kDa domain This finding indicated that pol b was not denatured by surface-active effects of SQMG(C14:1) Consequently, the NMR relaxation time shortening was due to the increase of the apparent molecular weight, leading to the appearance of cross-peaks of unstructured residues For this reason, we could not directly identify the SQMG(C14:1) interaction interface of the 8-kDa domain To avoid the aggregation of SQMG, we used chemically synthesized SQG, which did not bind the fatty acid moiety
The fragment linking method is commonly used in the NMR-based drug design process [26] A strongly binding compound can be synthesized by combining
Trang 6several low affinity compounds with different binding
sites for a target protein By applying this fragment
linking method inversely, we attempted to identify the
interaction interface of SQMG with the 8-kDa domain
Firstly, we determined the interaction interfaces of the
SQG and fatty acid separately We then combined
these two interaction interfaces and identified the
SQMG interaction interface of the 8-kDa domain
Analysis of the SQG interface with the 8-kDa
domain by NMR chemical shift changes
The solution structure of the 8-kDa domain has been
determined by Mullen et al [17] According to their
results, the 8-kDa domain (residues 1–87) formed four
a-helices packed as two antiparallel pairs The pairs of
a-helices crossed each other at 60, producing a V-like
shape The 8-kDa domain contains a helix-hairpin-helix
motif that is classified as a DNA binding domain There
is a hydrophobic region between helix-1 and helix-2
The 8-kDa domain was titrated with a 1 m stock
solution of SQG Two-dimensional 1H-15N HSQC
spectra were recorded for the 8-kDa domain-SQG complex at SQG concentrations of 10, 30, 60 and
100 mm The pol b-SQG complex was in the fast exchange region on the NMR time scale, permitting us
to follow the chemical shift changes of the backbone
NH and 15N signals of the 8-kDa domain upon complex formation This was achieved by recording a series of 1H-15N HSQC spectra of the uniformly 15 N-labeled 8-kDa domain in the presence of increasing amounts of SQG Of the 80 amides in residues 6–87
of the 8-kDa domain, 76 amides were assigned in the SQG complex using the CBCA(CO)NH and HNCACB spectra to confirm the reported assignments [17] NH and 15N chemical shift differences along the amino-acid sequence of the 8-kDa domain in the pres-ence of 100 mm SQG are indicated in Fig 4
The residues displaying chemical shift changes upon binding to SQG in the structure of the 8-kDa domain with or without SQG are shown in Fig 5A The surfa-ces of residues with NH chemical shift changes in the range of 0.02–0.03 p.p.m and 15N chemical shift chan-ges of 0.15–0.25 p.p.m (A6, T10, L11, G13, V20, L22,
0
20
40
60
80
100
SQG (mM)
0 20 40 60 80 100
0 20 40 60 80
100
Compound (mM)
0 0.2 0.4 0.6 0.8 1
Lane 1 2 3 4 5 I/P - 0 10 10 10
SQG MA SQG+MA
M13 ssDNA
DNA+8-kDa domain
complex start
Fig 3 (A) Dose–response curve of SQG Rat DNA polymerase b (0.05 units) was pre-incubated with the indicated concentrations (0–100 m M ) of SQG DNA polymerase activ-ity in the absence of added compounds was taken to be 100% (B) Dose–response curves of SQG, MA, and a mixture of SQG and MA.Rat DNA polymerase b (0.05 units) was preincubated with the indicated con-centrations (0–1 m M) of SQG (d), MA (n) or
a mixture of SQG and MA (s) The DNA polymerase activity in the absence of added compounds was taken to be 100% (C) Gel mobility shift analysis Gel mobility shift ana-lysis of binding between M13 ssDNA and the 8-kDa domain of pol b M13 plasmid ssDNA (2.2 nmol; nucleotide, single strand and singly primed) was mixed with purified proteins and SQMG(C14:1) Lanes 2–5 con-tained the 8-kDa domain at a concentration
of 7.5 l M ; lanes 1 contained no protein The compounds (75 l M each) were as follows: lanes 3, 4, and 5 were SQG, MA, and a mix-ture of SQG and MA, respectively.
Trang 7F25, K27, N28, Q31, Y36, N37, V45, K60, L62, G64,
D74, L77, L82 and K84) are colored yellow Those
with NH chemical shift changes of 0.03–0.04 p.p.m
and 15N chemical shift changes of 0.25–0.35 p.p.m
(N24, V29, S30, I33, Y39 and I69) are colored orange
NH chemical shift changes exceeding 0.04 p.p.m and
15N chemical shift changes exceeding 0.35 p.p.m (E9, A23, E26, K35, H51, G66, A70, R83 and L85) are colored red
In the presence of SQG, the cross-peaks were shifted
as follows: A6, E9 and L11 were in the unstructured segment G13, V20, L22, A23, N24, F25, E26, K27 and N28 were in helix-1; V29, S30 and Q31 were in the loop between helix-1 and helix-2; I33, K35, Y36 and N37 were in helix-2; H51 was in the loop between helix-2 and helix-3; K60 and L62 were in helix-3; G64 and G66 were in the loop between helix-3 and helix-4; I69, A70, D74 and L77 were in helix-4; L82, R83, K84 and L85 were in the unstructured linker segment that connected to the 31-kDa catalytic domain The N- (residues 1–10) and C-termini (residues 83–87) were disordered, as judged from the heteronuclear 15N-{1H} NOE data (values < 0.4) [17] As the chemical shifts
of the residues in the disordered regions are changed easily by minor changes in the environment (buffer, etc.), we excluded the residues in the disordered regions from our analysis These chemical shift chan-ges could be explained in terms of SQG contact and perturbations of the electrostatic charge distribution at the surface Surface residues displaying chemical shift changes were predominantly, although not entirely, clustered at two sites of the 8-kDa domain (Fig 5A), e.g Site I: L22, F25, E26, N28, I33, K35, N37 and Y39, and Site II: K60, L62, G64, G66 and A70 The cross-peak for K35 at Site I was sufficiently resolved during the titration to determine the mole fraction of protein bound to SQG The backbone amide proton of K35 displayed a long chemical shift change upon complex formation The change in the chemical shift of the K35 resonance was interpreted as resulting from an average (dav) of the chemical shifts for the free and the bound forms (db) of the K35 resonance Similarly, the KD value was determined by the chemical shift change of G66 at Site II Assuming that SQG binds to the 8-kDa domain as a 2 : 1 com-plex with each site having the same affinity, the KD values determined by K35 and G66 were 59 and
79 mm, respectively (Fig 6) The average KD value was 69 mm We have reported that the KD value of
NA was 1.02 mm [20] Linking of two moieties that each has a millimolar affinity has been reported to cre-ate a compound with a micromolar affinity [26] Thus,
it was reasonable that the inhibitor constant (Ki) values of SQMG(C14:1) and SQMG(C18:1) were found
to be 0.89 lm and 0.42 lm, respectively (Table 1) In order to determine whether or not the chemical shift change induced by SQG was specific, the 8-kDa
A
B
C
Fig 4 Chemical shift changes of HN and15N for the pol b 8-kDa
domain upon complex formation with SQG (A) Overlay of the
1 H- 15 N HSQC spectra of the 15 N-labeled N-terminal 8-kDa domain
of pol b (0.1 m M ) in the absence (blue) and presence (red) of
100 m M SQG (B and C) Chemical shift changes, |D 1H | (panel B) and
|D 15N | (panel C), are plotted vs residue number, where D 1H and
D 15N are the differences in p.p.m between the free and bound
chemical shifts.
Trang 8domain was titrated with d-glucose (Fig 1D) at con-centrations of 10, 50 and 100 mm d-glucose is a pyra-nose, as is SQG, but it possesses neither a sulfonyl nor
a glycerol moiety No NH or 15N chemical shift chan-ges were observed upon addition of d-glucose There-fore, the interaction of SQG with the 8-kDa domain was specific
Analysis of the SQMG binding site on the pol b 8-kDa domain
We determined the interaction interface between fatty acids and the 8-kDa domain of pol b in the previous report [20] We analyzed the binding site of SQMG based on the results of the NMR chemical shift map-pings of SQG (Fig 5A) and the fatty acid (Fig 5B) [20] We propose two possible models of the SQMG-pol b complex We constructed both models of the complex between the 8-kDa domain and SQMG based
on the following analysis (Fig 7)
In the first model (hereafter referred to as Model A), the sulfoquinovosylglycerol moiety of SQMG interacts with the 8-kDa domain at Site I and the alkyl moiety
of SQMG interacts with the C-terminus of helix-4 (Site III) In the model of the fatty acid complex with the 8-kDa domain, the carboxyl moiety of the fatty acid interacts with Site I and the alkyl moiety interacts at Site III (Fig 7A) There is a hydrophobic region between helix-1 and helix-2 (Fig 5C) There is an over-lap at Site I in the interaction interfaces of SQG and the fatty acid In Model A, the sulfoquinovosylglycerol moiety of SQMG is bound to Site I instead of the carboxyl moiety of the fatty acid, and the alkyl moiety binds to Site III
F25 V29 K35
D74
L77
H51
E26
A70
G66
G64
K60
I33
L62
Q31
K27
Site I Site II
K35 E26 Y39
S30 Q31
N24 F25 V29 N28 L22 K60
D74
Site I
V29 K35
D74
L77
H51
E26
I33
K52
T79
K35 E26 Y39
S30 V29 L22 D74 H51
F76 G80
K52 T79 L77
Site III
Site I
F25 V29 K35
D74
L77
H51
E26
A70
G66
G64
K60
I33
L62
Q31 K27
K52
T79
K35 E26 Y39
S30 Q31
N24 F25 V29 N28 L22 K60
D74 H51
F76 G80
K52 T79 L77
K35 F25
K60 E71
K68 K72 I73 I69 G66
Site III
Site I
F25 K35
G64
K60
L62 E71
K72 K68 G66
A
B
C
D
Fig 5 Interaction interfaces between DNA polymerase b and SQG,
fatty acid and ssDNA, and hydrophobicity representation; the
N-ter-minal (1–10) and C-terN-ter-minal (81–87) unstructured regions were
removed for clarity (A) Interaction interface between SQG and the
8-kDa domain The amino-acid residues of the major shifted
cross-peaks from the 1 H- 15 N HSQC spectra are indicated NH chemical
shift changes of 0.02–0.03 p.p.m and15N chemical shift changes of
0.15–0.25 p.p.m are depicted in yellow NH chemical shift changes
of 0.03–0.04 p.p.m and 15 N chemical shift changes of 0.25–
0.35 p.p.m are indicated in orange NH chemical shift changes of
more than 0.04 p.p.m and 15 N chemical shift changes of more than
0.35 p.p.m are indicated in red (B) Interaction interface between
fatty acids and the 8-kDa domain The amino-acid residues of the
major shifted cross-peaks from the1H-15N HSQC spectra are
indica-ted in red (C) These images show the hydrophobicity of the
molecular surfaces (i.e blue is hydrophilic and red is hydrophobic).
These images were prepared using the computer program INSIGHT II
(D) Interaction interface between ssDNA and the 8-kDa domain The
amino acid residues related to DNA binding are depicted in cyan.
0 0.01 0.02 0.03 0.04 0.05 0.06
SQG (mM)
Fig 6 Determination of K D for SQG binding to the 8-kDa domain
of DNA polymerase b Titration of SQG was performed to measure the chemical shift change at the nondegenerate K35 (diamonds) and G66 (triangles) NH in 1H-15N HSQC spectra at 750 MHz (25 C) The average K D value of SQG was 69 m M
Trang 9We examined the general binding mode of the
sulfo-nyl moiety by analysis of crystal structures of
com-plexes between proteins and sugars containing the
sulfate moiety We analyzed 35 crystal structures deposited in the PDB, which were collected based on the criteria listed in a previous report [27] The sulfonyl moieties interacted with the sidechain of arginine and lysine in 12 and 10 crystal structures, respectively This implies that the sulfoquinovosylglycerol moiety of SQMG would interact with residues in Site I K35 is the only basic amino-acid residue in Site I and the NH chemical shift of K35 was greatly changed by addition
of SQG Thus, the sulfonyl moiety of SQMG may form a salt bridge to the amino moiety of the side-chain of K35 The hydroxyl moieties of the sugar of SQMG might interact with the sidechain carboxyl moi-ety of E26 The NH chemical shift of E26 was also changed greatly by addition of SQG
In the second model (hereafter referred to as Model B), the sulfoquinovosylglycerol moiety of SQMG inter-acted with the 8-kDa domain at Site II and the alkyl moiety of SQMG interacted at Site III (Fig 7B) At Site II, the residues in which NH or15N chemical shift were greatly changed were G66, I69 and A70, which does not possess any amino moiety The survey of crystal structures showed that the sulfonyl moieties interacted with the backbone amide in 10 out of 35 crystal structures Thus, the sulfonyl moiety of SQMG might bind to the backbones of these residues, as shown in this model
To examine which is a more reasonable model, we performed a mutational analysis of pol b We altered four residues whose chemical shifts were greatly chan-ged by addition of SQG In Site I, we mutated E26 and K35 to alanine to remove the charged moieties of the sidechains In Site II, we altered G66 and A70 to proline to remove the backbone amide protons All the mutants of pol b retained the DNA polymerization activity We measured the SQMG(C14:1) inhibitory effects of the DNA polymerization activity against these four mutants (Table 2) The IC50 value of SQMG(C14:1) against the wild type pol b protein was 1.8 lm On the other hand, the IC50values against the E26A, G66P and A70P mutants were determined to be 10.6, 89.2 and 11.8 lm, respectively, whereas that against the K35A mutant was more than 200 lm As the inhibitory effects of SQMG(C14:1) on all the mutants decreased significantly, these four residues may be involved in the interaction with SQMG(C14:1) The SQMG(C14:1) inhibitory effect on the G66P mutant was approximately 50-fold weaker compared
to that of the wild type pol b protein Moreover, the
IC50 value against the K35A mutant was more than two times that against the G66P mutant The K35A mutant was influenced most weakly among the four mutants Therefore, these results suggested that Model
K35
A
B
F25
G66
A70
Fig 7 Possible structures of the complex formed between the
8-kDa domain and SQMG(C14:1).The sulfurs, carbons, oxygens, and
hydrogens in the inhibitor structures are indicated in orange, green,
red, and white, respectively (A) Model A SQMG(C 14:1 ) binds to
the 8-kDa domain of pol b at Site I and Site III The molecular
orien-tation of pol b-SQMG(C14:1) is almost the same as that in Fig 5 in
the left column image (B) Model B SQMG(C 14:1 ) binds to the
8-kDa domain at Site II and Site III The molecular orientation of pol
b-SQMG(C14:1) is almost the same as that in Fig 5 in the right
col-umn image These images were prepared using PYMOL (DeLano
Sci-entific, CA, USA).
Trang 10A may be more reasonable to represent the interaction
interface between SQMG(C14:1) and the 8-kDa
domain
Proposed inhibitory mode of SQMG against pol b
Figure 5D shows the interaction interface of the
8-kDa domain with ssDNA This model is based on
site-directed mutagenesis assays [28] and NMR
experi-ments [17] According to the report of Prasad et al
[28], point mutants at F25, K35, K60, and K68
showed impaired ssDNA binding activity The NMR
experiment indicated which residues (K60, L62, G64,
G66, I69, E71, K72, I73 and R83) had NH chemical
shift changes over 0.2 p.p.m and 15N chemical shift
changes over 1.0 p.p.m upon addition of 5
mer-ssDNA, p(dT)8 or 9 mer-ssDNA [17] In Model A,
SQMG competes with template DNA for binding to
Site I, and subsequently inhibits the template DNA
binding to the 8-kDa domain Binding of SQMG to
K35 would disrupt its interaction with ssDNA In
Model B, SQMG competes with template DNA for
binding to Site II Subsequently, SQMG blocks
bind-ing of template DNA to pol b In both models,
SQMG would prevent template DNA binding to the
8 kDa domain at Site I or Site II Consequently,
SQMG would inhibit the DNA polymerization
activ-ity of pol b
We have previously reported the interaction
inter-face of lithocholic acid (LA) with the 8-kDa domain
of pol b [21] LA binds to the 8-kDa domain at helix-3
and helix-4, but not at Site I Many other inhibitors,
such as glycyrrhizic acid, bind to Site II [29]
Gly-cyrrhizic acid would compete with template DNA for
binding to Site II of the 8-kDa domain Most
inhibi-tors of pol b, whose interaction interfaces are known
thus far, bind competitively to the DNA binding site
of the 8-kDa domain The hydrophilic part of SQMG
would interact with DNA binding site and compete with DNA in a similar fashion, whereas the hydropho-bic part of SQMG would bind to and then anchor at Site III Both hydrophobic and hydrophilic types of affinity contribute to the formation of the SQMG-pol
b complex SQMG(C18:1) showed a larger inhibitory effect on pol b than did SQMG(C14:1) Their structural difference was just in the length of the fatty acid moi-ety This suggests that the fatty acid moiety contributes
to the binding affinity to some extent In the case of fatty acids, the inhibitory effect increased in propor-tion to the number of carbons comprising the alkyl chain [1] These three-dimensional structural models could facilitate the design of more potent inhibitors for DNA pol b
SQMG inhibited not only pol b, but also pol a, pol
d, pol e, pol g, pol j, pol k and TdT [8] It was sug-gested that similar binding sites were present in these mammalian polymerases For example, they might possess hydrophobic cores adjacent to DNA binding sites where SQMG could interact Their amino-acid sequences differ, but they might have similar three-dimensional structures The binding site might be essential for their DNA polymerase activity, and such
a region might have been conserved evolutionarily among the mammalian polymerases Low molecular weight organic compounds may prove useful as molecular probes to investigate the structural homo-logy and the structure-function relationships of enzymes whose three-dimensional structures are as yet unknown
Experimental procedures
Materials Sulfoquinovosylmonoacylglycerol and sulfoquinovosylglyc-erol were chemically synthesized according to our previ-ously reported method [12] NA was purchased from Sigma (St Louis, MO, USA), and 15N-NH4Cl was pur-chased from Cambridge Isotope Laboratory (Andover,
MA, USA) Nucleotides and chemically synthesized tem-plate-primers such as poly(dA), poly(rA), oligo(dT)12)18, and oligo(dT)16 were purchased from Amersham Bio-science (Uppsala, Sweden) The other reagents of ana-lytical grade were purchased from Junsei Kagaku (Tokyo, Japan)
DNA polymerase assays Activity of pol b was measured by the methods described previously [1,23,30] For DNA polymerases, poly(dA)⁄ oli-go(dT)12)18and dTTP were used as DNA template-primer
Table 2 IC50values of SQMG(C14:1) against the DNA
polymeriza-tion activity of mutants of DNA polymerase b SQMG(C14:1) was
incubated with each enzyme (0.05 units) The enzymatic activity
was measured as described under Experimental procedures.
Enzyme activity in the absence of the compound was taken as
100%.
Mutants