The mechanism of sequence non-specific DNA binding of HMG1/2-box B in HMG1 with DNA

Kouhei Saito, Takeshi Kikuchi1 and Michiteru Yoshida2

Department of Biological Science and Technology and 1 Frontier Research Center for Computer Science, Science University of Tokyo, 2641 Yamazaki, Noda, Chiba 278-8510, Japan


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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
The DNA binding mechanism of box B in HMG1, a member of the sequence non-specific DNA binding HMG1/2-box family of proteins, has been examined by both mutation analyses and molecular modeling techniques. Substitution of the residue 102F, which is characteristically exposed to solvent, with a small hydrophobic amino acid affected its DNA binding activity. However, no additional effect was observed by the further mutation of flanking 101F. Molecular dynamics simulation and modeling studies revealed that 102F intercalates into DNA base-pairs, being supported by the flanking 101F. The mutants with a small hydrophobic residue at position 102 tolerated the substitution for 101F because the side chain at position 102 is too short to intercalate. Thus the intercalation of 102F and the positive effect of the flanking 101F residue are important for the sequence non-specific DNA binding of the HMG1/2-box. The conserved basic residues of 95K, 96R and 109R were also examined for their roles in DNA binding. These residues interacted with DNA mainly by electrostatic interaction and maintained the location of the box on the DNA, which prescribed the intercalation of 102F. The DNA intercalation by HMG1 consists of an ingenious mechanism which brings DNA conformational changes necessary for biological functions.

Keywords: DNA intercalation/HMG1 protein/HMG1/2-box/molecular dynamics/modeling


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
High mobility group protein 1 (HMG1), one of the most abundant nonhistone chromosomal proteins in eukaryotic organisms, has multiple roles in transcription, replication and cellular differentiation. The primary sequences of HMG1 from the various higher organisms, from birds to mammals, have more than 90% homology (reviewed in Bustin et al., 1990Go; Landsman and Bustin, 1993Go; Bustin and Reeves, 1996Go). Porcine HMG1 comprises 214 amino acids and its molecular weight is 24 795 Da (Tsuda et al., 1988Go). The DNA-binding region of the protein consists of two folded domains called the HMG1/2-box. The DNA-binding activity of a single HMG1/2-box in HMG1 is strengthened by its flanking basic region, while whole binding activity of HMG1 is presented by tandem array of the two boxes (Saito et al., manuscript submitted). HMG1 and HMG2 have preferential affinity for unusual structured DNA such as single-stranded DNA (Bidney and Reeck, 1978Go; Isackson et al., 1979Go; Yoshida and Shimura, 1984Go; Hamada and Bustin, 1985Go), cruciform DNA (Hamada and Bustin, 1985Go; Bianchi et al., 1989Go; Waga et al.,1990), B–Z junctions in supercoiled DNA (Waga et al., 1988Go) and anti-cancer drug cisplatin-modified DNA (Pil and Lippard, 1992Go). On binding to DNA they induce unwinding (Makiguchi et al., 1984Go; Yoshida and Shimura, 1984Go; Sheflin and Spaulding, 1989Go) and bending (Paull et al., 1993Go; Pil et al., 1993Go) to double-stranded DNA (reviewed in Bianchi, 1995Go).

The HMG1/2-box is a general DNA-binding motif which consists of about 75 amino acid residues forming three {alpha}-helices and is found in RNA polymerase I transcription factor UBF (Jantzen et al., 1990Go), human male-determining protein SRY (Sinclair et al., 1990Go), lymphoid enhancer binding factor LEF-1 (Travis et al., 1991Go), mitochondrial transcription factor mtTF1 (Parisi and Clayton, 1991Go) and many nuclear and mitochondrial transcription factors (reviewed in Ner, 1992Go; Landsman and Bustin, 1993Go; Grosschedl et al., 1994Go; Bianchi, 1995Go). Binding analyses of the boxes with DNA were mainly conducted for the sequence specific HMG1/2-box proteins (reviewed in Grosschedl et al., 1994Go and references cited therein), because of their anticipated participation in the control of specific gene transcription. The DNA-binding mechanism for sequence-specific proteins containing a single HMG1/2-box has been proposed based on the solution structures of the protein–DNA complexes from nuclear magnetic resonance analyses (Love et al., 1995Go; Werner et al., 1995Go). A significant feature of the binding is its recognition of the DNA minor groove by the intercalation of several hydrophobic residues on a concave surface of the box. In addition, the DNA conformation is altered by the association with the box. In spite of these progressive researches for the sequence-specific HMG1/2-box proteins, it is still not easy to understand the DNA recognition mechanism for the sequence non-specific protein with plural boxes, because of their multiple and complicated DNA-binding process with a low affinity.

The present study was conducted to elucidate the DNA recognition mechanism of box B in HMG1, which binds sequences non-specifically, by DNA-binding analyses using electrophoretic mobility shift assays and surface plasmon resonance, and by model building of the box B–DNA complex using molecular dynamics simulation.


    Materials and methods
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 Introduction
 Materials and methods
 Results
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Preparation of peptide

The mutants of peptide Bm (85–164 amino acid residues in HMG1) containing HMG1/2-box B flanked by a part of the linker (l) region in HMG1 were constructed using the expression plasmid pGEM-1Bm (Saito et al., manuscript submitted). The procedures for mutation were a modification of Deng and Nickoloff (1992). Denatured PCR fragments between the 95K96R primer (5'-cga agg agg CBT CYY ggg tgc att gg-3'; the mutation sites are written in capital letters), 101F102F primer (5'-aga aca aaa caa GAN ADA ggc cga agg a-3') or 109R primer (5'-cc ttt gat ttt tgg TTB ata ctc aga aca aaa-3') and mutXbaI primer (5'-tttccc TCTAGT actagttgatcctaggag-3'; Figure 2aGo) were annealed with the denatured pGEM-1Bm. The mutant plasmids were synthesized in vitro using Escherichia coli DNA polymerase III (Toyobo) and T4 DNA ligase. Using E.coli strains BMH 71-18 mutS and JM109, the objective plasmids were selected by their sensitivity to XbaI. The peptides were expressed in E.coli BL21(DE3) using a T7 expression system (Studier and Moffatt, 1986Go). At an OD600 of 0.3 to 0.5, the cultured cells were induced with 1 mM of isopropyl-1-thio-ß-D-galactopyranoside (IPTG). The E.coli cells were harvested after 6–10 h of induction and suspended in a sonication buffer (50 mM Tris–HCl, pH 8.0, 1 mM EDTA, 2 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride and 200 mM NaCl) and sonicated. After centrifugation at 10 000 g for 40 min, polyethylenimine P-70 solution was added to the supernatant to a final concentration of 0.5%. After centrifugation at 10 000 g for 10 min, the supernatant was then fractionated with ammonium sulfate. Protein in the supernatant in the presence of 70% saturation of ammonium sulfate was fractionated by alkyl Superose column chromatography with a Pharmacia FPLC system. The peptide fractions thus obtained were homogeneous on tricine-SDS–PAGE (Schagger and von Jagow, 1987Go).



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Fig. 2. (a) Schematic illustration of annealing sites for three mutation inducing primers (95K96R, 101F102F and 109R) and selection primer (mutXbaI). PCR fragments between mutation inducing primer and selection primer were used as described in Materials and methods. (b) Schematic illustration of peptide Bm mutants prepared for this study. Five amino acid residues at three positions were chosen as targets for mutation analyses of DNA binding of box B. Four mutants for 95K96R, four mutants for 101F102F and two mutants for 109R were prepared.

 
Electrophoretic mobility shift assay

The peptides were analyzed for their DNA-binding activity by an electrophoretic mobility shift assay using the pBR322 form III DNA linearized by EcoRV digestion. DNA (0.3 µg) and each peptide in various amounts were incubated in 20 µl of a reaction solution (10 mM Tris–HCl, pH 7.8, 10 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 20 µg bovine serum albumin and 100 mM NaCl) at 25°C for 60 min. The mixture was run on a 1.2% agarose gel in 40 mM Tris–acetate, pH 8.0, containing 1 mM EDTA (1xTAE). After electrophoresis, the gel was stained with ethidium bromide.

Surface plasmon resonance (SPR) measurement

A biotinylated 30 bp DNA (5'-tgtatgaaatctaacaatgcgctcatcgtc-3') in TES (10 mM Tris–HCl, pH 7.5, 1 mM EDTA and 300 mM NaCl) was immobilized on to a streptavidin-modified sensorchip SA (BIAcore AB) surface (Bondeson et al., 1993Go), resulting in the capture of approximately 1000 RU of DNA. Each peptide at various concentrations in 10 mM Tris–HCl, pH 7.8, 10 mM MgCl2, 1 mM EDTA and 100 mM NaCl was injected over the immobilized DNA at a constant flow rate of 2 µl/min, and the SPR response was monitored for 600 s. The dissociation of peptide from the surface-bound complex was monitored for another 400 s at the same flow rate of buffer. After each measurement, the sensorchip surface of fixed DNA was regenerated with an injection of 8 µl of 2 M NaCl. Data were analyzed using a BIAevalution software package, which was supplied with BIAcore (BIAcore AB). Kinetic analyses of protein–DNA interactions by SPR measurement have been previously reported, regarding, for example, lactose operator–repressor DNA interaction (Bondeson et al., 1993Go), ETS1–target sequence DNA (Fisher et al., 1994Go) and the methionine repressor–operator complex (Parsons et al., 1995Go). According to these studies, all data were treated. Here, kass is the apparent association rate constant of DNA–HMG1 peptides, kdiss the apparent dissociation rate constant, and Kd the apparent equilibrium constant to be calculated, Kd = kdiss/kass (Bondeson et al., 1993Go; Parsons et al., 1995Go).

Model building of a box B–DNA complex

In order to build a model of the complex structure of HMG1 box B (and its mutants) with DNA, solution structures of box B of HMG1 (1hme; Weir et al., 1993Go) and the LEF-1–DNA complex (1lef; Love et al., 1995Go) were obtained from the Brookhaven Protein Data Bank (PDB; Bernstein et al., 1977Go). All the calculations were performed with Insight II/Discover 3 (Molecular Simulations Inc.), using amber forcefield. A 9.5 Å cut-off for van der Waals interaction and cell multipole mode for coulomb interaction with a distance-dependent dielectric function of {varepsilon} = 1xr were used, where r is the distance in angstroms between interacting atoms. The PDB structure of the isolated box B (1hme) solvated in a 6 Å layer of water was subjected to 500 steps of energy minimization followed by 130 ps molecular dynamics simulation at 298 K for equilibration. The obtained structure was used for the modeling of mutants of HMG1 box B. The modeling of mutant structures was conducted using the same values of corresponding dihedral angles of wild type side chains as far as possible. The final structures of mutant peptides free from DNA were obtained by another 500 steps of energy minimization followed by 50 ps molecular dynamics simulation. The obtained tertiary structure of isolated box B model or its mutants was superimposed on that of LEF-1 bound with DNA (1lef), suiting the positions of backbone atoms of five amino acid residues 9K, 10R, 15F, 16F and 23R in 1hme derivatives to 4K, 5K, 10F, 11M and 18R in 1lef, respectively, which were exposed to the DNA-binding surface. These residues were corresponding ones to each other in a sequence alignment between box B in HMG1 and LEF-1. Thereafter this structure, solvated in a 6 Å layer of water, was subjected to 500 steps of energy minimization followed by 70 ps molecular dynamics simulation at 298 K for equilibration. Final structures thus obtained were adopted as the complex models of HMG1 box B (and its mutants) with DNA.


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 Results
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 References
 
Design of the mutants for box B in HMG1

The HMG1/2-box proteins have been classified into three groups by their DNA recognition specificity (Bianchi, 1995Go); the first group contains chromosomal proteins bound to DNA with no sequence specificity (class I), the second ribosomal and mitochondrial transcription factors which show sequence specificity in the presence of another associating factor when bound with DNA (class II), and the third gene-specific transcription factors which show sequence specific DNA binding (class III). HMG1 is a member of the class I HMG1/2-box family. Although the solution structure of each HMG1/2-box in HMG1 has been reported (Read et al., 1993Go; Weir et al., 1993Go; Hardman et al., 1995Go), the complex structure with DNA has not been determined and therefore the mechanism of DNA recognition is still unclear. On the other side, the complex structures of members of the class III box in SRY and LEF-1 with DNA have been reported (Love et al., 1995Go; Werner et al., 1995Go), indicating that the concave surface of the HMG1/2-box structure is a DNA binding interface.

In order to determine the relevant residues for DNA binding of HMG1, several primary sequences of HMG1/2-boxes in the family proteins supplied from the SWISS-PROT amino acid sequence database (Bairoch and Boeckmann, 1991Go) were aligned. As shown in Figure 1Go, several basic and hydrophobic amino acid residues are highly conserved in all classes of HMG1/2-box family proteins. Comparing the multiple alignment with the structural data for class III protein, 95K, 96R, 101F, 102F and 109R in HMG1 were chosen as target residues for the mutation analyses of DNA recognition by box B in HMG1. These five amino acid residues are highly conserved and are located on the concave surface which is predicted as DNA interface.



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Fig. 1. Amino acid sequence alignment of HMG1/2-boxes in HMG1/2-box family proteins. The sequential numbers of amino acid residue for box B in HMG1 are listed on the top of the alignment. The HMG1/2-box family proteins were classified into three classes by their DNA-binding properties.

 
Thus, several mutants of these amino acid residues in peptide Bm (amino acid residues 85–164), which contains the minimal HMG1/2-box B with three Lys residues at the N-terminus, were prepared. Considering the structural characteristics and the side-chain charges of the amino acids, four mutant peptides for 95K96R (mutants 95G96T, 95K96T, 95K96K and 95R96R), four mutant peptides for 101F102F (mutants 101S102F, 101Y102F, 101Y102V and 101F102V), and two mutant peptides for 109R (mutants 109E and 109Q) were expressed using a T7 expression system (Studier and Moffatt, 1986Go) in E.coli (Figure 2bGo). The structures substituted for these amino acid residues were confirmed not to be distorted using the intrinsic fluorescence spectrum derived from a Trp residue at position 132 (data not shown), which is present in the hydrophobic core formed of three helices (Weir et al., 1993Go).

Effective DNA binding of the side-chain of 102F depending on the flanking 101F

In box B of the HMG1 protein, the side chain of 102F is considered to be a typical hydrophobic residue exposed to solvent (Read et al., 1993Go; Weir et al., 1993Go) and likewise for other HMG1/2-box family proteins (Jones et al., 1994Go; Love et al., 1995Go; van Houte et al., 1995Go; Werner et al., 1995Go). The DNA intercalation by this side chain is an essential characteristic at least for the interaction between sequence specific HMG1/2-box and DNA (Love et al., 1995Go; Werner et al., 1995Go). In order to examine whether the side chain 102F and the flanking 101F participate also in the case of interaction between the sequence non-specific HMG1/2-box and DNA, mutational analyses for 102F and 101F were conducted. The effect of substitution for these amino acid residues in peptide Bm on the affinity to DNA was examined by an electrophoretic mobility shift assay using form III (linearized) plasmid pBR322 DNA (Figure 3aGo). In the assay, mutant 101S102F practically lost its DNA-binding activity. The retardation by mutant 101Y102F was smaller than that of wild type. On the other hand, in mutants 101F102V and 101Y102V the retardation was much closer to that of wild type, suggesting that the mutant substitution of 102F with Val does not have an effect on DNA-binding activity, whether the amino acid residue at 101 position is Phe or Tyr.



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Fig. 3. Electrophoretic mobility shift assay for the mutant peptides. Form III pBR322 DNA (0.3 µg DNA linearized by EcoRV digestion) was mixed with the respective peptide of (a) 101F102F mutants, (b) 95K96R mutants and (c) 109R mutants at peptide/DNA molar ratios of 0, 50, 100, 200, 300, 400 and 500 (from left to right lanes, respectively), and then separated by 1.2% agarose gel electrophoresis (left). Relative retardation of the complexes was plotted against the respective peptide/DNA molar ratio (right).

 
The SPR measurement (Table IGo) showed that the Kd value for all of the 101F102F mutants were consistent with the result obtained by electrophoretic mobility shift assay (Figure 3aGo). These experimental results demonstrated that 101F as well as 102F is important for the DNA binding of HMG1.


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Table I. Kd values determined by SPR measurement and modeling data
 
Model building of a box B–DNA complex by molecular dynamics simulation and the analysis of the role of 102F

The solution structures of HMG1/2-boxes in SRY (Werner et al., 1995Go) and LEF-1 (Love et al., 1995Go) in the complex with DNA, of box A (Hardman et al., 1995Go) and box B (Read et al., 1993Go; Weir et al., 1993Go) in HMG1, and of those in HMG-D (Jones et al., 1994Go) and Sox-4 (van Houte et al., 1995Go) have been determined by NMR. However, the structure of the HMG1/2-box in HMG1 complexed with DNA has not been solved. Therefore, a model of the tertiary structure of the box B–DNA complex was built using PDB data for 1lef (LEF-1–DNA; Love et al., 1995Go) and 1hme (box B of HMG1; Weir et al., 1993Go).

The superimposition of the modeled structures for all mutants at 101F102F free from DNA to the wild type backbone is presented in Figure 4aGo. The backbone model structures were similar between wild type and the mutants. R.m.s. deviations of the atoms in the backbone structure of respective mutants from the wild type were around 1.8 Å or less (Table IGo). Examination of the wild type and mutants structures revealed no obvious differences in the direction of the probable base-stacking side chain Phe at 102 position against the backbones of helix I (Figure 4bGo). The flanking hydrophobic side chain of Phe at 101 position faces a hydrophobic core in HMG1 box B. This residue seems to be involved in keeping the direction of the 102F side chain.



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Fig. 4. DNA-free models of box B and the mutants for 101F102F residues simulated by molecular dynamics. (a) Backbone of each peptide is overlapped on that of wild type box B. For clarity, only backbone structures are presented in the ribbon model. Indigo ribbon shows wild type; green, mutant SF; sky blue, mutant YF; magenta, mutant YV; and red, mutant FV. (b) Magnified view of side chains in respective mutant for 101F102F. Only two side chains positioned at 101 and 102 are presented in the stick model.

 
The models of the box B mutants in the complex with DNA are represented in Figure 5aGo, superimposing the structure of DNA. When focused on the side chain at position 102, obvious differences were observed between the wild type and the mutants (Figure 5bGo). The side chain of 102F in the wild-type model intercalated into base-pairs, while those of the mutants did not. These differences are reflected upon the {chi}1 dihedral angle at position 102 (Table IGo). For example, {chi}1 of wild type is about –90° while that of the most inactive mutant 101S102F is about –180°. This suggests that {chi}1 of the 102F side chain in box B complexed with DNA depends on the hydrophobic environment formed by the flanking 101 residue which also exists at the boundary of a hydrophobic core (Figure 5cGo). It is noted that helices II and III are close to DNA in the wild-type box model compared with the case in mutants. The intercalation of 102F seems to cause a shift in the whole location of box B on DNA. It was also considered that the probable base-stacking side chains of Val in mutants 101F102V and 101Y102V were too short to intercalate even though the direction of one of two C{gamma} atoms was suitable for an interaction. The complex models with DNA for mutants of box A-type (101Y102A), SRY-type (101F102I) and LEF-1-type (101F102M) were also built (Figure 5dGo). It was observed that the side chain of 102A in the box A-type mutant was too short to intercalate. The side chains of 102I in SRY-type and 102M in LEF-1-type partially intercalated into DNA base-pairs independent of the flanking residue 101 of Phe or Tyr.



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Fig. 5. DNA–complex models of box B and the mutants for 101F102F residues simulated by molecular dynamics. (a) Each DNA–complex model is superimposed for DNA atoms with that of wild type box B. For clarity, only the DNA in wild type box B complex model is presented in the stick model and backbone structures of respective peptides are presented in the ribbon model. Indigo ribbon shows wild type; green, mutant SF; sky blue, mutant YF; magenta, mutant YV; and red, mutant FV. (b) Magnified view of mutated side chains for 101F102F in respective mutants. Two side chains positioned at 101 and 102 are presented in the stick model. DNA in wild type box B complex model is also presented. (c) Mechanism of DNA base-stacking by side chain of 102F in box B. 104F, 132W and 143Y forms a hydrophobic core between three helices (Weir et al., 1993Go). 102F is exposed to solvent and surrounded by two hydrophobic residues, 101F and 103L. (d) Magnified view of the side chains for 101Y102A (sky blue), 101F102A (magenta), 101F102I (red), 101Y102I (orange), 101F102M (dark green) and 102Y102M (light green) mutants. Two side chains of residues positioned at 101 and 102 are presented in the stick model. DNA in wild-type box B complex model (indigo) is also presented.

 
DNA-binding analysis for box B mutants substituted for three conserved basic amino acid residues of 95K, 96R and 109R

Since the multiple alignment revealed that the basic amino acid residues 95K, 96R and 109R were especially well conserved (Figure 1Go), the role of those residues was also examined by an electrophoretic mobility shift assay using form III (linearized) plasmid pBR322 DNA (Figure 3bGo). All the mutants for 95K96R showed a smaller gel shift (less retardation) of the respective complexes with DNA compared with the wild type. Even the mutants 95K96K and 95R96R, which were replaced with a basic amino acid residue, showed less retardation, as well as the mutant 95K96T in which 96R was substituted with a neutral residue. This result suggests that these two amino acid residues and their sequential array are essential for tight DNA binding of box B. This possibility was supported by the observation that substitution with 95G96T had the greatest effect on its DNA binding. The DNA affinity of these mutants was also analyzed by SPR measurement using 30 bp DNA (Table IGo). As expected from the gel retardation assays, mutants 95G96T and 95K96T showed larger dissociation constants (Kd) than wild type. In addition, mutants 95K96K and 95R96R also gave larger Kd values than wild type, even though they showed a slightly higher affinity compared with mutants replaced with a neutral amino acid residue.

The substitution of 109R with 109Q or 109E has a dramatic effect on their retardation (Figure 3cGo). The mutants also gave larger Kd values compared with wild type when measured by the SPR (Table IGo). These experimental results demonstrated that the three conserved basic amino acid residues of 95K, 96R and 109R are also important for the DNA binding of HMG1.

Roles of 95K, 96R and 109R in DNA binding analyzed by molecular dynamics simulation

The molecular dynamics simulation was performed also for the mutants of 95K, 96R and 109R in order to clarify their roles in DNA binding. The side chain of 95K in wild-type box B was close to a DNA phosphate (3.6 Å between N{xi} of 95K and the nearest P of DNA) suggesting the presence of a salt-bridge between them (Figure 6aGo). The side chain of 96R was deeply inserted into a DNA minor groove. The directions of these side chains in the mutants of these positions free from DNA were similar to those in the wild type. The basic side chain at position 95 in mutants 95K96K and 95R96R in the complex with DNA was also close to a DNA phosphate, which was the next DNA phosphate different from the case of the wild type (Figure 6aGo). For example, the N{xi} of 95K was 5.9 Å distant from the P of DNA which corresponded to the case for wild type, but 4.9 Å distant from the nearest P for mutant 95K96K. This migration was caused by the alteration of the relative location of box B on DNA and by a loss of intercalation of the side chain of 102F. Only a shallow intercalation was observed in the mutant 95R96R–DNA complex (Figure 6bGo). The side chain of 96K in mutant 95K96K was not inserted into a DNA minor groove but close to a DNA phosphate by 3.6 Å and the next phosphate by 3.9 Å (Figure 6aGo). Similar phenomena were observed in mutant 95K96T and these migrations of the box on DNA were reflected in the results of DNA-binding analysis of these mutants (Table IGo).



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Fig. 6. DNA–complex models of box B and the mutants for 95K96R and 109R residues. Magnified views of (a) side chains at 95 and 96; (b) 102F of respective 95K96R mutant; (c) a side chain at 109; and (d) 102F of 109R mutant models are superimposed for DNA atoms with that of wild-type box B. For clarity, only the DNA in wild-type box B complex model is presented in the stick model. Indigo shows wild type; sky blue, mutant 95G96T; orange, mutant 95K96K; red, mutant 95K96T; magenta, mutant 95R96R; yellow, mutant 109E; and green, mutant 109Q.

 
The side chain of 109R in wild-type box B in the complex with DNA was also considered to make a salt-bridge with a DNA phosphate (3.6 Å distant from an N{eta} of 109R to a P of DNA; Figure 6cGo). The directions of the side chain at 109 in mutants 109Q and 109E free from DNA and complexes with DNA did not vary compared with that of wild type (Figure 6cGo). Therefore, the loss of binding affinity of 109Q and 109E with DNA can be attributed to electrostatic repulsion by the substitution of the residues with opposite charge. In addition, as observed for 95K96R mutants, the amino acid substitution at the 109 position with Glu also influenced the intercalation of the side chain of 102F, while the substitution with Gln did not (Figure 6dGo).


    Discussion
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 Abstract
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 Materials and methods
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 References
 
We demonstrated that the intercalation of 102F is essential for the non-specific binding of HMG1 box B with DNA in this study. Based on the simulation results, it is interesting to calculate the binding energy as follows; the potential energy of the peptide free from DNA as E[free protein], that of DNA independently calculated from standard B–DNA as E[free DNA] and that of the box–DNA complex as E[complex]. Then, the binding energy of each peptide with DNA was calculated by the following equation; {Delta}E = E[complex] – (E[free protein] + E[free DNA]). The {Delta}{Delta}E values for each mutant against the wild type were positively correlated with the Kd value for the respective mutants (correlation coefficient r2 = 0.67). Hence, it is suggested that the intercalation of 102F in the box B–DNA complex is stabilized mainly by energy.

Our model for the box B–DNA complex indicated that the intercalation of the side-chain of 102F with DNA base-pairs depends on the presence of the flanking side-chain at position 101 (Figure 5Go). Even the substitution with Tyr for Phe at position 101 influenced the intercalation. A similar intercalation was observed for LEF-1, i.e. the side chain of 10M forms a so-called wedge composed of three hydrophobic side chains of 6L, 10M and 13M (Love et al., 1995Go). In the case of box B in HMG1, a similar hydrophobic wedge may be formed by 102F (as the tip), 101F and 103L (Figure 5cGo). In other words, the hydrophobic environment around 102F steers its intercalation into DNA. When the tip of the hydrophobic wedge was substituted with a smaller side-chain such as Val or Ala, the effect of additional substitution of the flanking 101 residue with Tyr was small (Figure 3Go, Table IGo and data not shown). This seems to be the result of the tip of the wedge being too short for it to interact with the DNA. The tip residue of the hydrophobic wedge was substituted with Met or Ile, the partial intercalation by the wedge was observed even though the flanking 101 side chain was replaced with Tyr. In other words, the effect of the flanking residue of position 101 on binding to DNA seems to be small when the intercalating side chain at position 102 forms a more flexible conformation than Phe which contains an aromatic ring. A similar phenomenon can be observed in the primary sequence alignment of HMG1/2-box family proteins (Figure 1Go). That is, in this case the amino acid residue of the wedge head is Phe, the amino acid residue at –1 position is always Phe (see box B of HMG1, 2 and T). Most proteins containing this type of HMG1/2-box bind with DNA sequences non-specifically. In the case of the wedge head residues, other hydrophobic residues such as Ile, Met and Ala, Tyr can adapt as a residue at position 101 as well as Phe. Noticeably, most boxes of this type have another hydrophobic amino acid at the –4 position from the wedge head (Figure 1Go), and most of the sequence-specific DNA-binding proteins have this type of box (see LEF-1 and SRY). These primary structural characteristics suggest that the mechanism of intercalation is different between the tip of the hydrophobic wedge of Phe and that of other hydrophobic amino acids, because Phe contains an aromatic ring. This is true for the wedge of box B, which is composed of Phe at the head, Phe at the –1 position but Pro at the –4 position. It means that the hydrophobic residues relevant to the mechanism of intercalation by the sequence non-specific HMG1/2-box B in HMG1 are different from those in the sequence specific HMG1/2-boxes in SRY and LEF-1.

The amino acid residue corresponding to position 95K in HMG1 is highly conserved in the HMG1/2-box family proteins, except for a few proteins containing Arg (Figure 1Go). The residue interacts with a DNA phosphate in the complex with the box in class III protein (Love et al., 1995Go). In the models presented in Figure 6aGo, the side chain of 95K faced in the direction of a DNA phosphate. The model shows that the length of the Lys side chain at this position is suitable for interaction with a DNA phosphate (the distance between N{xi} of Lys and the nearest P of DNA was 3.6 Å), while that of Arg was too long to interact with the DNA phosphate precisely. The simulation analysis for the mutant 95R96R indicated that the side chain of 95R was also close to the next DNA phosphate (the distance between N{eta} of 95R and the P of DNA was 5.2 Å) compared with the case of wild type (7.5 Å). In addition, 96R was also close to a DNA phosphate (the distance between N{eta} of 96R and the nearest P of DNA was 4.1 Å). This alteration was caused by a shift of the relative location of the mutated box B against DNA. Our simulation also showed that the side chain of 96R is highly important to the DNA binding in a different participation from that of 95K (Figure 5aGo). The side chain was deeply inserted into a minor groove of DNA, but did not interact with a DNA phosphate. The amino acid residue in many boxes corresponding to this residue is Arg except for a few substitutions with Lys (Figure 1Go). The box B binding with DNA was influenced by the substitution for Lys (Figure 3aGo and Table IGo). In addition, the side chain of 96K in mutant 95K96K as well as 96T in mutant 95K96T did not enter into the DNA minor groove (Figure 6aGo). Thus it was concluded that the wild-type sequence of 95K96R is the most appropriate for the effective binding of the box with DNA.

Another conserved side chain of 109R on the interface between HMG1 box B and DNA (Figure 5aGo) is positioned in helix I mainly with electrostatic interaction in class III box family proteins (Love et al., 1995Go; Werner et al., 1995Go). These boxes have another basic side chain, which also interacts with DNA, at the –3 position from this amino acid residue. On the other hand, the class I box proteins lack such a basic amino acid residue and likewise box B in HMG1 (Figure 1Go). The class I box differs from the class III box in having many essential side chains to recognize DNA phosphate and bases to create sequence specificity. Thus the roles of 109R as well as 95K and 96R are mainly to have direct electrostatic interaction with DNA in order to bring about the correct array of the box on DNA into which the side chain of 102F intercalates.


    Notes
 
2 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bairoch,A. and Boeckmann,B. (1991) Nucleic Acids Res., 19, 2247–2249.[ISI][Medline]

Bernstein,F.C., Koetzle,T.F., Williams,G.J., Meyer,E.F., Brice,M.D., Rodgers,J.R., Kennard,O., Shimanouchi,T. and Tasumi,M. (1977) Eur. J. Biochem., 80, 319–324.[Abstract]

Bianchi,M.E. (1995) In Lilley,D.M.J. (ed.), DNA–Protein: Structural Interactions. IRL Press, London, pp. 177–200.Bianchi,M.E. (1995) In Lilley,D.M.J. (ed.), DNA–Protein: Structural Interactions. IRL Press, London, pp. 177–200.

Bianchi,M.E., Beltrame,M. and Paonessa,G. (1989) Science, 243, 1056–1059.[ISI][Medline]

Bidney,D.L. and Reeck,G.R. (1978) Biochem. Biophys. Res. Commun., 85, 1211–1218.[ISI][Medline]

Bondeson,K., Frostell-Karlsson,Å, Fägerstam,L. and Magnusson,G. (1993) Anal. Biochem., 214, 245–251.[ISI][Medline]

Bustin,M. and Reeves,R. (1996) Prog. Nucleic Acid Res. Mol. Biol., 54, 35–100.[ISI][Medline]

Bustin,M., Lehn,D.A. and Landsman,D. (1990) Bichim. Biophys. Acta, 1049, 231–243.[ISI][Medline]

Deng,W.P. and Nickoloff,J.A. (1992) Anal. Biochem., 200, 81–88.[ISI][Medline]

Fisher,R. J., Fivash,M. Casas-Finet,J., Erickson,J.W., Kondoh,A., Bladen,S.V., Fisher,C., Watson,D.K. and Papas,T. (1994) Protein Sci., 3, 257–266.[Abstract/Free Full Text]

Grosschedl,R., Giese,K. and Pagel,J. (1994) Trends Genet., 10, 94–100.[ISI][Medline]

Hamada,H. and Bustin,M. (1985) Biochemistry, 24, 1428–1433.[ISI][Medline]

Hardman,C.H., Broadhurst,R.W., Raine,A.R., Grasser,K.D., Thomas,J.O. and Laue,E.D. (1995) Biochemistry, 34, 16596–16607.[ISI][Medline]

Isackson,P. J., Fishback,J.L., Bidney,D.L. and Reeck,G.R. (1979) J. Biol. Chem., 254, 5569–5572.[Abstract]

Jantzen,H.-M., Admon,A., Bell,S.P. and Tjian,R. (1990) Nature, 344, 830–836.[ISI][Medline]

Jones,D.N., Searles,M.A., Shaw,G.L., Churchill,M.E., Ner,S.S., Keeler,J., Travers,A.A. and Neuhaus,D. (1994) Structure, 2, 609–627.[ISI][Medline]

Landsman,D. and Bustin,M. (1993) BioEssays, 15, 539–546.[ISI][Medline]

Love,J.J., Li,X., Case,D.A., Giese,K., Grosschedl,R. and Wright,P.E. (1995) Nature, 376, 791–795.[ISI][Medline]

Makiguchi,K., Chida,Y., Yoshida,M. and Shimura,K. (1984) J. Biochem., 95, 423–429.[Abstract]

Ner,S.S. (1992) Curr. Biol., 2, 208–210.

Parisi,M.A. and Clayton,D.A. (1991) Science, 252, 965–969.[ISI][Medline]

Parsons,I.D., Persson,B., Mekhalfia,A., Blackburn,G.M. and Stockley,P.G. (1995) Nucleic Acids Res., 23, 211–216.[Abstract]

Paull,T.T., Haykinson,M. J. and Johnson,R.C. (1993) Genes Dev., 7, 1521–1534.[Abstract]

Pil,P.M. and Lippard,S.J. (1992) Science, 256, 234–237.[ISI][Medline]

Pil,P.M., Chow,C.S. and Lippard,S.J. (1993) Proc. Natl Acad. Sci. USA, 90, 9465–9469.[Abstract]

Read,C.M., Cary,P.D., Crane-Robinson,C., Driscoll,P.C. and Norman,D.G. (1993) Nucleic Acids Res., 21, 3427–3436.[Abstract]

Schagger,H. and von Jagow,G. (1987) Anal. Biochem., 166, 368–379.[ISI][Medline]

Sheflin,L.G. and Spaulding,S.W. (1989) Biochemistry, 28, 5658–5664.[ISI][Medline]

Sinclair,A.H., Berta,P., Palmer,M.S., Hawkins,J.R., Griffiths,B.L., Smith,M. J., Foster,J.W., Frischauf,A.-M., Lovell-Badge,R. and Goodfellow,P.N. (1990) Nature, 346, 240–244.[ISI][Medline]

Studier,F.W. and Moffatt,B.A. (1986) J. Mol. Biol., 189, 113–130.[ISI][Medline]

Travis,A., Amsterdam,A., Belanger,C. and Grosschedl,R. (1991) Genes Dev., 5, 880–894.[Abstract]

Tsuda,K., Kikuchi,M., Mori,K., Waga,S. and Yoshida,M. (1988) Biochemistry, 27, 6159–6163.[ISI][Medline]

van Houte,L.P., Chuprina,V.P., van der Wetering,M., Boelens,R., Kaptein,R. and Clevers,H. (1995) J. Biol. Chem., 270, 30516–30524.[Abstract/Free Full Text]

Waga,S., Mizuno,S. and Yoshida,M. (1988) Biochem. Biophys. Res. Commun., 153, 334–339.[ISI][Medline]

Waga,S., Mizuno,S. and Yoshida,M. (1990) J. Biol. Chem., 265, 19424–19428.[Abstract/Free Full Text]

Weir,H.M., Kraulis,P. J., Hill,C.S., Raine,A.R.C., Laue,E.D. and Thomas,J.O. (1993) EMBO J., 12, 1311–1319.[Abstract]

Werner,M.H., Huth,J.R., Gronenborn,A.M. and Clore,G.M. (1995) Cell, 81, 705–714.[ISI][Medline]

Yoshida,M. and Shimura,K. (1984) J. Biochem., 95, 117–124.[Abstract]

Received August 31, 1998; revised October 28, 1998; accepted November 30, 1998.