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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Keywords: DNA intercalation/HMG1 protein/HMG1/2-box/molecular dynamics/modeling
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Introduction |
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The HMG1/2-box is a general DNA-binding motif which consists of about 75 amino acid residues forming three -helices and is found in RNA polymerase I transcription factor UBF (Jantzen et al., 1990
), human male-determining protein SRY (Sinclair et al., 1990
), lymphoid enhancer binding factor LEF-1 (Travis et al., 1991
), mitochondrial transcription factor mtTF1 (Parisi and Clayton, 1991
) and many nuclear and mitochondrial transcription factors (reviewed in Ner, 1992
; Landsman and Bustin, 1993
; Grosschedl et al., 1994
; Bianchi, 1995
). Binding analyses of the boxes with DNA were mainly conducted for the sequence specific HMG1/2-box proteins (reviewed in Grosschedl et al., 1994
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 proteinDNA complexes from nuclear magnetic resonance analyses (Love et al., 1995
; Werner et al., 1995
). 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 BDNA complex using molecular dynamics simulation.
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Materials and methods |
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The mutants of peptide Bm (85164 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 2a) 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, 1986
). 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 610 h of induction and suspended in a sonication buffer (50 mM TrisHCl, 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-SDSPAGE (Schagger and von Jagow, 1987
).
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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 TrisHCl, 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 Trisacetate, 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 TrisHCl, 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., 1993), resulting in the capture of approximately 1000 RU of DNA. Each peptide at various concentrations in 10 mM TrisHCl, 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 proteinDNA interactions by SPR measurement have been previously reported, regarding, for example, lactose operatorrepressor DNA interaction (Bondeson et al., 1993
), ETS1target sequence DNA (Fisher et al., 1994
) and the methionine repressoroperator complex (Parsons et al., 1995
). According to these studies, all data were treated. Here, kass is the apparent association rate constant of DNAHMG1 peptides, kdiss the apparent dissociation rate constant, and Kd the apparent equilibrium constant to be calculated, Kd = kdiss/kass (Bondeson et al., 1993
; Parsons et al., 1995
).
Model building of a box BDNA 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., 1993) and the LEF-1DNA complex (1lef; Love et al., 1995
) were obtained from the Brookhaven Protein Data Bank (PDB; Bernstein et al., 1977
). 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
= 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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The HMG1/2-box proteins have been classified into three groups by their DNA recognition specificity (Bianchi, 1995); 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., 1993
; Weir et al., 1993
; Hardman et al., 1995
), 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., 1995
; Werner et al., 1995
), 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, 1991) were aligned. As shown in Figure 1
, 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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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., 1993; Weir et al., 1993
) and likewise for other HMG1/2-box family proteins (Jones et al., 1994
; Love et al., 1995
; van Houte et al., 1995
; Werner et al., 1995
). 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., 1995
; Werner et al., 1995
). 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 3a
). 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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The solution structures of HMG1/2-boxes in SRY (Werner et al., 1995) and LEF-1 (Love et al., 1995
) in the complex with DNA, of box A (Hardman et al., 1995
) and box B (Read et al., 1993
; Weir et al., 1993
) in HMG1, and of those in HMG-D (Jones et al., 1994
) and Sox-4 (van Houte et al., 1995
) 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 BDNA complex was built using PDB data for 1lef (LEF-1DNA; Love et al., 1995
) and 1hme (box B of HMG1; Weir et al., 1993
).
The superimposition of the modeled structures for all mutants at 101F102F free from DNA to the wild type backbone is presented in Figure 4a. 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 I
). 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 4b
). 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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Since the multiple alignment revealed that the basic amino acid residues 95K, 96R and 109R were especially well conserved (Figure 1), the role of those residues was also examined by an electrophoretic mobility shift assay using form III (linearized) plasmid pBR322 DNA (Figure 3b
). 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 I
). 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 3c). The mutants also gave larger Kd values compared with wild type when measured by the SPR (Table I
). 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 of 95K and the nearest P of DNA) suggesting the presence of a salt-bridge between them (Figure 6a
). 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 6a
). For example, the N
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 95R96RDNA complex (Figure 6b
). 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 6a
). 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 I
).
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Discussion |
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Our model for the box BDNA 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 5). 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., 1995
). In the case of box B in HMG1, a similar hydrophobic wedge may be formed by 102F (as the tip), 101F and 103L (Figure 5c
). 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 3
, Table I
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 1
). 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 1
), 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 1). The residue interacts with a DNA phosphate in the complex with the box in class III protein (Love et al., 1995
). In the models presented in Figure 6a
, 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
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
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
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 5a
). 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 1
). The box B binding with DNA was influenced by the substitution for Lys (Figure 3a
and Table I
). 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 6a
). 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 5a) is positioned in helix I mainly with electrostatic interaction in class III box family proteins (Love et al., 1995
; Werner et al., 1995
). 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 1
). 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.
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Notes |
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References |
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Received August 31, 1998; revised October 28, 1998; accepted November 30, 1998.