Sulfoquinovosylmonoacylglycerol inhibitory mode analysis of rat DNA polymerase b Nobuyuki Kasai 1 , Yoshiyuki Mizushina 2 , Hiroshi Murata 1 , Takayuki Yamazaki 1 , Tadayasu Ohkubo 3 , Kengo Sakaguchi 1 and Fumio Sugawara 1 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. FEBS Journal 272 (2005) 4349–4361 ª 2005 FEBS 4349 family, 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 C 14 and C 18 , respectively. As shown in Fig. 1, SQMG(C 14:1 ) bears a myristoleic acid (MA) (Fig. 1F) on the glycerol moiety, and SQMG(C 18:1 ) bears an oleic acid on the glycerol moiety. Figure 2A shows the inhibitory dose–response curves of SQMG(C 18:1 ), SQMG(C 14:1 ) and NA against pol b.We measured the DNA polymerization activity under the same condition in order to make precise comparisons between these inhibitors. IC 50 values of SQMG(C 18:1 ), SQMG(C 14:1 ) and NA were determined to be 0.8, 1.8 and 5 lm, respectively. SQMG(C 18:1 ) inhibited pol b activity more strongly than SQMG(C 14:1 ). 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(C 14:1 ) and SQMG(C 18:1 ) 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(C 18:1 ), SQMG(C 14:1 ) and NA are about 32.0, 27.0 and 28.4 A ˚ , respectively, as derived from computer models. The molecular size of SQMG(C 14:1 ) was very similar to that of NA. The difference in the inhibitory potency of O H HO H HO H O OH H H SO 3 H HO O O O H HO H HO H O OH H H SO 3 H HO O O O H HO H HO H O OH H H SO 3 H HO OH C HO O A B C D E F O H HO H HO H OH OH H H OH C HO O Fig. 1. Chemical structures of the compounds (A) sulfoquino- vosylmonoacylglycerol [SQMG(C 14:1 )], (B) sulfoquinovosylmono- acylglycerol [SQMG(C 18:1 )], (C) sulfoquinovosylglycerol (SQG), (D) D-glucose, (E) nervonic acid (NA) and (F) myristoleic acid (MA). Interaction mode between SQMG and DNA Pol b N. Kasai et al. 4350 FEBS Journal 272 (2005) 4349–4361 ª 2005 FEBS SQMG 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(C 18:1 ) influenced the activities more strongly than did SQMG(C 14:1 ); 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 (V max ) was unchanged at 111 pmolÆ 0 20 40 60 80 100 0246810 Compound (µM) C B A DNA polymerase β activity (%) 0.15 nmol SQMG(C 14:1 ) 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 me r 20 me r 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(C 14:1 ) and SQMG(C 18:1 ) 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)orNA(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 lM; 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(C 14:1 ). 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 lM [ 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. N. Kasai et al. Interaction mode between SQMG and DNA Pol b FEBS Journal 272 (2005) 4349–4361 ª 2005 FEBS 4351 h )1 , whereas 242% and 493% increases in the Michael- is constant (K m ) were observed in the presence of 1 and 2 lm SQMG(C 14:1 ), respectively (Table 1). The V max for the dNTP substrate was 62.5 pmolÆh )1 , and the K m for the substrate increased from 3.05 to 20.0 lm in the presence of 2 lm SQMG(C 14:1 ) (Table 1). The inhibitor constant (K i ) values, obtained from Dixon plots, were found to be 0.89 lm and 2.8 lm in the presence of 2mm for the DNA template-primer and the dNTP sub- strate, respectively (Table 1). Similarly, the K i values of SQMG(C 18:1 ) were found to be 0.42 lm and 1.44 l m for the DNA template-primer and dNTP substrates, respectively, and the K i values 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(C 14:1 ) 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(C 14:1 ) (I) and the proteins (P) are repre- sented by I ⁄ P in Fig. 2B. The interference by SQMG(C 14:1 ) 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(C 14:1 ) 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(C 14:1 ) 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(C 18:1 ) instead of SQMG(C 14:1 ) were similar (data not shown). Table 1. Kinetic analysis of the inhibitory effects of sulfoquinovosylmonoacylglycerols (SQMG(C 14:1 ), SQMG(C 18:1 )) 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 (lM) K m a (pmoÆh )1 ) V max a K i b (lM) Inhibitory mode a SQMG(C 14:1 ) DNA template-primer c 0 6.74 111 0.89 Competitive 116.3 233.2 Nucleotide substrate d 0 3.05 62.5 2.8 Competitive 1 4.95 220.0 SQMG(C 18:1 ) DNA template-primer 0 6.74 111 0.42 Competitive 0.5 18.4 134.8 Nucleotide substrate 0 3.05 62.5 1.44 Competitive 0.5 6.34 123.5 NA DNA template-primer 0 6.74 111 4.0 Competitive 417.2 631.0 Nucleotide substrate 0 3.05 62.5 3.5 Competitive 4 4.80 618.7 a From Lineweaver–Burke plot. b From Dixon plot. c Poly (dA) ⁄ oligo(dT) 12)18 . d dTTP. Interaction mode between SQMG and DNA Pol b N. Kasai et al. 4352 FEBS Journal 272 (2005) 4349–4361 ª 2005 FEBS Product 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(C 14:1 ) (lanes 1–2) completely suppressed DNA polymerization by the full-length pol b.AtanI⁄ P ratio of 1 for the protein (lane3), DNA synthesis hardly occurred. However, the 31-kDa domain synthesized DNA without interference from SQMG(C 14:1 ) (lane 5). At the range of the SQMG(C 14:1 ) concentrations that influence the template-primer-binding site on the 8-kDa domain, SQMG(C 14:1 ) 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(C 18:1 ) instead of SQMG(C 14:1 ) were similar (data not shown). Biochemical characterization of fragments of SQMG To determine the inhibitory mechanism of pol b by SQMG(C 14:1 ), two separated fragments of SQMG(C 14:1 ), 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 IC 50 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 IC 50 value of 375 lm. The inhibitory effect of a mixture of SQG and MA was stronger than that of SQG or MA, and the IC 50 value 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(C 14:1 ) 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(C 14:1 ) and the 8-kDa domain A titration experiment using the 8-kDa domain and a 1mm stock solution of SQMG(C 14:1 ) was performed as follows. Two-dimensional 1 H- 15 N HSQC spectra of the 8-kDa domain-SQMG(C 14:1 ) complex at SQMG(C 14:1 ) concentrations of 0.05, 0.1, 0.15 and 0.2 mm were recorded. As the concentration of SQMG(C 14:1 ) increased, the cross-peaks of the 8-kDa domain broad- ened. At an SQMG(C 14:1 ) 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(C 14:1 ) may aggregate at the millimolar concentration required for NMR experiments, and the 8-kDa domain may interact with micelle-like forms of SQMG(C 14:1 ) [25]. If the experiment could be carried out at micromolar concentrations, SQMG(C 14:1 ) would not aggregate, as SQMG(C 14:1 ) 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(C 14:1 ). 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(C 14:1 ) 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 N. Kasai et al. Interaction mode between SQMG and DNA Pol b FEBS Journal 272 (2005) 4349–4361 ª 2005 FEBS 4353 several 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 1 H- 15 N 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 15 N signals of the 8-kDa domain upon complex formation. This was achieved by recording a series of 1 H- 15 N 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 15 N 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 15 N chemical shift chan- ges of 0.15–0.25 p.p.m. (A6, T10, L11, G13, V20, L22, 0 20 40 60 80 DNA polymerase β activity (%) 100 SQG (mM) 0 20 40 60 80 100 0 20 40 60 80 DNA polymerase β a ctivity (%) 100 Compound (mM) 0 0.2 0.4 0.6 0.8 1 AB C 8-kDa domain 7.5 µM 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(C 14:1 ). 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. Interaction mode between SQMG and DNA Pol b N. Kasai et al. 4354 FEBS Journal 272 (2005) 4349–4361 ª 2005 FEBS F25, 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 15 N 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 15 N 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 15 N-{ 1 H} 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 (d av ) of the chemical shifts for the free and the bound forms (d b ) of the K35 resonance. Similarly, the K D 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 K D values determined by K35 and G66 were 59 and 79 mm, respectively (Fig. 6). The average K D value was 69 mm. We have reported that the K D 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 (K i ) values of SQMG(C 14:1 ) and SQMG(C 18:1 ) 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 and 15 N 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. N. Kasai et al. Interaction mode between SQMG and DNA Pol b FEBS Journal 272 (2005) 4349–4361 ª 2005 FEBS 4355 domain 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 15 N 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 F25 V29 V29 K35 K35 D74 D74 L77 L77 H51 H51 E26 E26 A70 A70 G66 G66 G64 G64 K60 K60 I33 I33 L62 L62 Q31 Q31 K27 K27 Site I Site I Site II Site II K35 K35 E26 E26 Y39 Y39 S30 S30 Q31 Q31 N24 N24 F25 F25 V29 V29 N28 N28 L22 L22 K60 K60 D74 D74 Site I Site I V29 V29 K35 K35 D74 D74 L77 L77 H51 H51 E26 E26 I33 I33 K52 K52 T79 T79 K35 K35 E26 E26 Y39 Y39 S30 S30 V29 V29 L22 L22 D74 D74 H51 H51 F76 F76 G80 G80 K52 K52 T79 T79 L77 L77 Site III Site III Site I Site I F25 F25 V29 V29 K35 K35 D74 D74 L77 L77 H51 H51 E26 E26 A70 A70 G66 G66 G64 G64 K60 K60 I33 I33 L62 L62 Q31 Q31 K27 K27 K52 K52 T79 T79 K35 K35 E26 E26 Y39 Y39 S30 S30 Q31 Q31 N24 N24 F25 F25 V29 V29 N28 N28 L22 L22 K60 K60 D74 D74 H51 H51 F76 F76 G80 G80 K52 K52 T79 T79 L77 L77 K35 K35 F25 F25 K60 K60 E71 E71 K68 K68 K72 K72 I73 I73 I69 I69 G66 G66 Site III Site III Site I Site I 90 o F25 F25 K35 K35 G64 G64 K60 K60 L62 L62 E71 E71 K72 K72 K68 K68 G66 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-terminal (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 and 15 N 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 the 1 H- 15 N 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 020406080100 SQG (mM) NH chemical shift difference (ppm) 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 1 H- 15 N HSQC spectra at 750 MHz (25 °C). The average K D value of SQG was 69 mM. Interaction mode between SQMG and DNA Pol b N. Kasai et al. 4356 FEBS Journal 272 (2005) 4349–4361 ª 2005 FEBS We 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 or 15 N 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(C 14:1 ) inhibitory effects of the DNA polymerization activity against these four mutants (Table 2). The IC 50 value of SQMG(C 14:1 ) against the wild type pol b protein was 1.8 lm. On the other hand, the IC 50 values 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(C 14:1 ) on all the mutants decreased significantly, these four residues may be involved in the interaction with SQMG(C 14:1 ). The SQMG(C 14:1 ) inhibitory effect on the G66P mutant was approximately 50-fold weaker compared to that of the wild type pol b protein. Moreover, the IC 50 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(C 14:1 ).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(C 14:1 ) 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(C 14:1 ) 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). N. Kasai et al. Interaction mode between SQMG and DNA Pol b FEBS Journal 272 (2005) 4349–4361 ª 2005 FEBS 4357 A may be more reasonable to represent the interaction interface between SQMG(C 14:1 ) 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 15 N 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(C 18:1 ) showed a larger inhibitory effect on pol b than did SQMG(C 14:1 ). 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 15 N-NH 4 Cl 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)18 and dTTP were used as DNA template-primer Table 2. IC 50 values of SQMG(C 14:1 ) against the DNA polymeriza- tion activity of mutants of DNA polymerase b. SQMG(C 14:1 )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%. Pol b IC 50 values of SQMG(C 14:1 )(lM) Wild type 1.8 Mutants E26A 10.6 K35A > 200 G66P 89.2 A70P 11.8 Interaction mode between SQMG and DNA Pol b N. Kasai et al. 4358 FEBS Journal 272 (2005) 4349–4361 ª 2005 FEBS [...]... the chemical shift of < /b> the protein when fully bound by inhibitor, [P]t is the total concentration of < /b> the protein, [I]t is the total concentration of < /b> inhibitor, and n is the number of < /b> inhibitor binding sites on the protein [36,37] Structural models of < /b> the complex Molecular docking of < /b> the 8-kDa domain of < /b> pol b and SQMG was performed using the affinity program within the insight ii software (Accelrys,... The inhibitory < /b> action of < /b> fatty acids on DNA < /b> polymerase < /b> beta Biochim Biophys Acta 1336, 509–521 24 Abbotts J, SenGupta DN, Zmudzka B, Widen SG, Notario V & Wilson SH (1988) Expression of < /b> human DNA < /b> polymerase < /b> beta in Escherichia coli and characterization of < /b> the recombinant enzyme Biochemistry 27, 901–909 25 Matsumoto K, Takenouchi M, Ohta K, Ohta Y, Imura T, Oshige M, Yamamoto Y, Sahara H, Sakai H, Abe... Crystal structures of < /b> human DNA < /b> polymerase < /b> beta complexed with DNA:< /b> implications for catalytic mechanism, processivity, and fidelity Biochemistry 35, 12742–12761 20 Mizushina Y, Ohkubo T, Date T, Yamaguchi T, Saneyoshi M, Sugawara F & Sakaguchi K (1999) Mode < /b> analysis < /b> of < /b> a fatty acid molecule binding to the N-terminal 8-kDa domain of < /b> DNA < /b> polymerase < /b> beta A 1: 1 complex and binding surface J Biol Chem 274,... empirical approach for structure-based prediction of < /b> carbohydrate-binding sites on proteins Protein Eng 16, 467–478 28 Prasad R, Beard WA, Chyan JY, Maciejewski MW, Mullen GP & Wilson SH (1998) Functional analysis < /b> of < /b> the amino-terminal 8-kDa domain of < /b> DNA < /b> polymerase < /b> beta as revealed by site-directed mutagenesis DNA < /b> binding and 5¢-deoxyribose phosphate lyase activities J Biol Chem 273, 11121–11126 29 Hu... novel DNA < /b> polymerase < /b> inhibitor group, synthetic sulfoquinovosylacylglycerols: inhibitory < /b> action on cell proliferation Mutat Res 467, 139–152 Hanashima S, Mizushina Y, Yamazaki T, Ohta K, Takahashi S, Sahara H, Sakaguchi K & Sugawara F (2001) Synthesis of < /b> sulfoquinovosylacylglycerols, inhibitors of < /b> eukaryotic DNA < /b> polymerase < /b> alpha and beta Bioorg Med Chem 9, 367–376 Burgers PM, Koonin EV, Bruford E, Blanco... titration curves were analyzed by nonlinear leastsquare fitting to the following equations dob À df ¼ ðdsat =½PŠt ÞfðKD þ ½PŠt þ n½IŠt Þ À ½ðKD þ ½PŠt þ n½IŠt Þ2 À 4½PŠt n½IŠt Š1=2 g=2 dsat ¼ db À df ð1Þ ð2Þ 4359 Interaction mode < /b> between SQMG and DNA < /b> Pol b where dob is the chemical shift of < /b> the protein at each titration point and df is the chemical shift of < /b> the protein in the absence of < /b> inhibitor, db... standard reaction mixtures, and incubation was carried out at 37 °C for 60 min The activity without the inhibitor was considered to be 100%, and the remaining activities at each concentration of < /b> inhibitor were determined as percentages of < /b> this value One unit of < /b> each DNA < /b> polymerase < /b> activity was defined as the amount of < /b> enzyme that catalyzes the incorporation of < /b> 1 nmol of < /b> deoxyribonucleotide triphosphates (i.e... concentrations of < /b> inhibitors were added to the binding mixture, followed by incubation at 25 °C for 10 min Samples were run on a 1.0% agarose gel in 0.1 m Trisacetate buffer, pH 8.3, containing 5 mm EDTA at 50 V for 2 h Interaction mode < /b> between SQMG and DNA < /b> Pol b Expression and purification of < /b> the mutant proteins The expression vectors of < /b> the mutant proteins were constructed by QuikChange II (Stratagene,... Backbone dynamics and refined solution structure of < /b> the N-terminal domain of < /b> DNA < /b> polymerase < /b> beta Correlation with DNA < /b> binding and dRP lyase activity J Mol Biol 296, 229–253 18 Sawaya MR, Prasad R, Wilson SH, Kraut J & Pelletier H (1997) Crystal structures of < /b> human DNA < /b> polymerase < /b> beta complexed with gapped and nicked DNA:< /b> evidence for an induced fit mechanism Biochemistry 36, 11205– 11215 19 Pelletier H,... Sato N & Sakaguchi K (2004) Design of < /b> vesicles of < /b> 1,2-di-O-acyl-3-O-(beta-D-sulfoquinovosyl) -glyceride bearing two stearic acids (beta-SQDG-C18), a novel immunosuppressive drug Biochem Pharmacol 68, 2379–2386 FEBS Journal 272 (2005) 4349–4361 ª 2005 FEBS Interaction mode < /b> between SQMG and DNA < /b> Pol b 26 Lepre CA, Moore JM & Peng JW (2004) Theory and applications of < /b> NMR-based screening in pharmaceutical . SQMG(C 18:1 )(d)orNA(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. . 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. Sulfoquinovosylmonoacylglycerol inhibitory mode analysis of rat DNA polymerase b Nobuyuki Kasai 1 , Yoshiyuki Mizushina 2 , Hiroshi Murata 1 , Takayuki Yamazaki 1 , Tadayasu Ohkubo 3 , Kengo