Department of Chemistry, New York University, New York, NY 10003, USA
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Abstract |
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Keywords: circular dichroism/divalent metals/DNA binding/high mobility group protein box B (rHMGB1b)/protein stability
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Introduction |
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The isolated domains (boxes A and B) of HMGB1 have been investigated extensively. The structure of each domain has been determined by NMR spectroscopy (Read et al., 1993; Weir et al., 1993
; Hardman et al., 1995
). Each domain contains a highly conserved HMG global fold of three
-helices arranged in an L-shape (Figure 1A
) (Read et al., 1993
; Weir et al., 1993
). The angle subtended by the shorter and longer arms of the L-shaped structure is smaller in the A box (by
14°) than the B box. As with other chromosomal HMG proteins, individual A and B boxes interact weakly with linear duplex DNA and strongly with distorted DNA structures such as four-way junctions (4WJs) (Bianchi, 1988
; Bianchi et al., 1989
), supercoiled (Hamada and Bustin, 1985
; Sheflin et al., 1993
) and cisplatin-modified DNA (Pil and Lippard, 1992
; Chow et al., 1995
). The A box has a higher binding affinity to these distorted DNA substrates (Teo et al., 1995
; Dunham and Lippard, 1997
; Webb and Thomas, 1999
).
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Metal ions play a large role in protein/enzymatic function such as catalysis and redox reactions. It has been estimated that about one-third of all proteins and enzymes require metal ions as cofactors for biological function (Holm et al., 1996). A metal binding site can be generated by inserting two or more properly positioned metal binding side chains (Ghadiri and Choi, 1990
). These amino acids must be located so as to maximize metal binding without destabilizing the native state or significantly reducing the functional properties of the protein or enzyme. Ideally, metal ions would then bind to the folded form of protein, thereby stabilizing the molecule by shifting the foldingunfolding equilibrium towards the native state (Arnold and Haymore, 1991
). While these criteria seemingly make the rational design of metal binding sites into proteins and polypeptides a demanding task, a number of proteins and protein folds have been designed with cysteine or histidine metal binding centers to regulate their activity and stability (Corey and Schultz, 1989
; Muheim et al., 1993
; Wisz et al., 1998
; Benson et al., 2000
; Wray et al., 2000
).
Our strategy to stabilize rHMGB1b is to engineer His residues, into rHMGB1b, as ligand binding sites and to use divalent metal ions as ligands. Histidine residues are inserted at the helical ends, which potentially avoids sacrificing helix stability due to the low helix propensity of His residues (Chakrabartty et al., 1994). Metal binding to a pair of His residues at saturating concentration has been shown to stabilize a native GCN4 coiled-coil helical protein by 1.5 kcal/mol (Krantz and Sosnick, 2001
). It is thus demonstrated that appropriately positioned metal binding residues can stabilize native proteins. We show here that significant stabilizing effects can also be generated by metal binding of engineered pairs of His side chains into the more complex rHMGB1b sequence. Electrophoretic mobility shift assays (EMSAs) were employed to evaluate the effect(s) of introducing metal binding motifs on the binding affinity of the mutant proteins.
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Materials and methods |
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The HMGB1b/pHB1 clone from rat was obtained from Professor S.J.Lippard (Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA). Replacement residues were introduced via site-directed mutagenesis using a mutagenic primer corresponding to the respective amino acid replacement(s) employing the Kunkel method (Kunkel et al., 1991). All mutant HMG1b constructs were amplified by polymerase chain reaction (PCR), prior to DNA sequence analysis, with the sense primer I, 5'-GAC TCA CTA TAG GGA GA-3', and the anti-sense primer II, 5'-TTA ATC TGT ATC AGG CT-3'. The integrity of the mutant constructs was confirmed via DNA sequence analysis performed on an ABI PRISM 377 sequencer (PE Applied Biosystems) using an ABI PrismBigDye Terminator Cycle Reaction Kit (PE Applied Biosystems). All mutagenic and PCR primers were obtained in HPLC-purified form from CyberSyn (Lenni, PA). All PCR products were purified using a QIAquick PCR Purification Kit (Qiagen, Valencia, CA).
Protein expression and purification
All mutant proteins were expressed from pHB1Escherichia coli Bl21(DE3)pLysS following procedures described by Chow et al. (Chow et al., 1995). All proteins were purified by FPLC employing an Econo-Pac CM cartridge (Bio-Rad). Crude proteins were loaded on to the CM cartridge in the presence of 50 mM NaCl50 mM TrisHCl (pH 7.0) and eluted with high salt buffer of 500 mM NaCl/50 mM TrisHCl (pH 7.0) using a linear gradient. The purity of the proteins was checked by resolution of protein samples on 15% SDSpolyacrylamide (29:1 acrylamide:bisacrylamide) gels in Tris-Tricine buffer at 196 V for 1 h, followed by Coomassie Brilliant Blue G-250 staining (Schagger and von Jagow, 1987
). Protein concentrations were determined according to Pace et al. (Pace et al., 1995
).
Circular dichroism
Circular dichroism (CD) spectra were recorded on an AVIV 202 spectrometer (Aviv Associates, Lakewood, NJ). CD spectra of 3 µM protein solution in 10 mM HEPES [N-(2-hydroxethyl)piperazine-N'-(2-ethanesulfonic acid)] (Sigma), pH 7.0, in the absence and presence of 1 mM NiSO4.6H2O were recorded in a cell with a 1 cm pathlength from 260 to 200 nm in steps of 0.5 nm at 4°C. HEPES buffer was selected because it does not bind metal ions tightly. The thermal denaturation profile of each protein was monitored under the same protein concentration and buffer condition(s) at a wavelength of 222 nm from 0 to 100°C in 2°C increments. All solutions were filtered with a 0.45 µm filter and degassed prior to analysis.
Four-way junction substrate
The four-way junction (4WJ) J-1 was the DNA substrate employed for the DNA binding assays. J1 was composed of four oligonucleotides described previously (Figure 2B) (Kallenbach et al., 1983
). The sequences of the four strands are 101, 5'-CGCAATCCTGAGCACG-3'; 102, 5'-CGTGCTCACCGAATCGC-3'; 103, 5'-GCATTCGGACTATGGC-3'; and 104, 5'-GCCATAGTGGATTGCG-3'. The oligonucleotide 101 was radiolabeled at its 5'-terminus using [
-32P]ATP and T4 polynucleotide kinase (Promega). After inactivation of T4 kinase, the radiolabeled strand (101) was desalted with a Bio-Spin 6 column (Bio-Rad). The four-way junction, J1, was formed by mixing the radiolabeled strand with a 5-fold excess of the unlabelled strands in 50 mM TrisHCl, pH 7.5, and 10 mM MgCl2. The mixture was incubated for 2 min at 90°C, followed by cooling ato 4°C overnight.
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EMSAs of the rHMGB1b mutants with the 4WJ, J1, were performed as a function of DNAprotein molar ratio. DNA (0.05 µM in the final reaction) and aliquots of rHMGB1b mutants were incubated on ice for 30 min in 20 mM TrisHCl, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 10% (w/v) glycerol and 1 mM NiSO4.6H2O. The reaction mixtures were loaded on to 16.5% acrylamide (29:1 acrylamide:bisacrylamide) in 0.5x TBE (45 mM Trisma, 45 mM boric acid and 1.0 mM EDTA), pH 8.0, at 4°C for 3.5 h. Gels were dried on Whatman 3MM paper and scanned with a PhosphorImager.
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Results |
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The design strategy focuses on inserting metal binding motifs into the target protein. The loops/turns between the -helices were targeted initially because mutations in these regions have shown significant effects on stability of the protein (Taudte et al., 2000
). The amino acids selected for replacement are displayed in bold in Figure 2A
. These residues were selected because they represent non-conserved sites located on the surface of the molecule. Introduction of a histidine residue in positions 3739 creates a potential metal binding site with a naturally occurring histidine in position 33, located in loop I. These residues have also been implicated in binding to 4WJ (Taudte et al., 2000
) and cisplatin-modified DNA substrates (Ohndorf et al., 1999
; He et al., 2000
). Mutant proteins in which a histidine residue replaces functionally relevant residues (3739) are referred to as loop I constructs (Figure 2A
). Proteins with histidine replacements at the non-binding surface of the molecule (residues 51, 55 and 56, encompassing loop II of the native structure) are referred to as loop II constructs (Figure 2A
). Proteins with triple histidine replacements (metal binding sites inserted at both turns simultaneously) are referred to as loop I/II constructs (G39H/N51H/D55H, Figure 1B
). Alteration of the unstructured N-terminus of the protein was avoided in all our experiments. Binding assays of HMG1b alanine mutants at this region displayed a significant reduction of binding affinity (50-fold) to 4WJ DNA (Stros and Muselikova, 2000
; Taudte et al., 2000
).
Secondary structure analysis
The secondary structure of the HMGB1b mutants was monitored by recording the UV CD wavelength scan of each protein. The CD spectra of all the proteins in this study contain two minima in ellipticity, one at 222 nm and the other at 212 nm, indicative of
-helical secondary structure. The ellipticity values were recorded in the absence and presence of 1 mM NiSO4.6H2O in 10 mM HEPES, to determine the effect of metal ligation on secondary structure. These values are listed in Table I
.
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The CD spectra of loop I/II mutants (metal binding sites encompassing both turns) possess ellipticity values that fluctuate above and below wild-type rHMGB1b. In the absence of metal ions, the CD spectrum of the triple mutant G39H/N51H/D55H has an ellipticity value nearly identical with that of wild-type at 222 nm (Figure 3C). In the presence of metal ions, the ellipticity value at 222 nm of this mutant increases by
20%, the largest increase in ellipticity (upon ligation) of all the mutant proteins analyzed. This behavior is again suggestive of a flexible native fold for HMGB: it is hard to imagine how the helix content of a stable native state would vary by so much. The first minimum undergoes a red shift to 212 nm that is independent of the buffer conditions. The ellipticity values of the remaining loop I/II mutants display a range of increases [I38H/N51H/D55H (8.6%)] and decreases [S37H/N51H/D55H (15%); I38H/N51H/D56H (10%); and G39H/N51H/D56H (16%)] compared with wild-type (Table I
). Interestingly, all loop I/II mutants display a relative increase in ellipticity compared with those mutants with replacements solely at loop I (S37H, G39H and I38H).
Thermal denaturation analysis
The thermal stability of each mutant protein was monitored by CD thermal denaturation. Unfolding profiles were measured at a wavelength of 222 nm, from 0 to 100°C. The molar ellipticity values of the respective unfolding assays were normalized to facilitate comparison (Figure 4AC). The respective free energy change (
G,
G), melting temperature (Tm) and enthalpy change (
Hm) values of each protein were estimated according to the procedure described by Pace (Pace, 1988
). The thermodynamic properties of each protein are listed in Table II
. The Tm of wild-type rHMGB1b (40.6°C) is 5.4°C lower than the value previously reported by our laboratory (Taudte et al., 2000
) owing to the difference in buffer conditions used in the experiments. The Tm of wild-type rHMGB1b was identical in the presence and absence of metal ions. The thermal denaturation profiles of all the mutants analyzed show a single sigmoidal transition, consistent with a two-state unfolding transition. However, the mutant proteins (G39H and G39H/N51H/D55H) have slightly elevated baseline(s) at lower temperatures, which suggests that these mutants are less stable. The effect of the mutational replacements on the relative stability of each mutant was determined by measuring the thermostability of each mutant in the absence of metal ions (depicted as open symbols in Figure 4AC
); these values are also listed in Table II
.
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The thermal denaturation profile of the loop II mutant N51H/D55H displays an increase in Tm of 6.2°C upon ligation (closed triangles, Figure 4B). The histidine replacements at the helical termini produce a 0.5°C residual increase in the Tm of this protein (open triangles, Figure 4B
). A similar replacement mutant N51/D56H also displays a Tm enhancement of 6.2°C vs wild-type in the presence of metal ions (Table II
). Unlike N51H/D55H, N51/D56H is destabilized by 2.4°C in the absence of metal ions.
The thermal denaturation profile of the loop I/II mutant G39H/N51H/D55H shows an increase in Tm of 4.1°C in the presence of metal ions (closed squares, Figure 4C). A similar mutant, I38H/N51H/D55H, undergoes a Tm increase of 5.5°C in the presence of metal ions (Table II
). These and the remaining loop I/II mutants (S37H/N51H/D55H, I38H/N51H/D56H and G39H/N51H/D56H) are less stable than wild-type in the absence of metal ions, even in the presence of metal ions for the latter constructs (Table II
).
Electrophoretic mobility shift assays
To test the functional capability of the rHMGB1b mutants, we monitored their DNA binding using electrophoretic mobility shift assays (EMSAs). Intact HMGB1, isolated boxes (A and B) and tandem boxes bind to distorted DNA structures including 4WJs (Bianchi et al., 1989, 1992
; Falciola et al., 1994
; Taudte et al., 2000
) and supercoiled DNA (Stros and Reich, 1998
) with no obvious sequence specificity. However, the isolated A box has a strong preference for the bases adjacent to cisplatin-modified DNA (Dunham and Lippard, 1997
; Thomas and Travers, 2001
). The 4WJ J1 was employed as the substrate for the gel shift assays (Figure 5AC
). Wild-type rHMGB1b binds J1 with 4:1 stoichiometry at higher proteinDNA ratios (Xin et al., 2000
). A 4:1 binding pattern is represented by the higher order complexes formed at proteinDNA ratios of 100:1 and higher.
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Discussion |
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The effect of metal-mediated stabilization on the function of the proteins was assessed by gel shift assays. Isolated HMG boxes (A and B) bind to 4WJ DNA with 1:1 stoiochiometry at lower proteinDNA ratios (Bianchi et al., 1989) and 4:1 stoichiometry at higher proteinDNA ratios (Xin et al., 2000
). The metal binding site(s) did not significantly alter the binding properties of the mutant proteins. The loop I mutant, G39H, showed the most significant loss in binding affinity of the mutants analyzed. The reduction in binding affinity may be induced by the larger histidine side chain at the binding surface; this mutant retains 4:1 binding capacity at elevated proteinDNA ratios (Figure 5A
). As with G39H, the remaining loop I mutants (S37H and I38H) also display 4:1 binding at higher proteinDNA ratios. Moreover, S37H and I38H do not display a loss in binding affinity at lower proteinDNA ratios (binding data not shown). The binding affinity of the most stable mutant that we produced, N51H/D55H (loop II mutant), is very similar to wild-type, with 4:1 binding at higher proteinDNA ratios (Figure 5B
). The remaining loop II mutant (N51H/D56H) displays a similar binding pattern (data not shown). The loop I/II mutant G39H/N51H/D55H also displays 4:1 binding at higher proteinDNA ratios (lanes 78, Figure 5C
).
The gel shift assays and stability measurements are consistent with a relationship between stability and function in HMGB proteins. It has been hypothesized that enzymatic activity and proteinligand interactions depend on flexibility: enzymes must have internal motions. An enzyme with a flexible molecular structure has a variety of conformations that increase potential enzymesubstrate interactions. Such increased substrate interactions could serve to enhance the efficiency of the enzyme (Aghajari et al., 1996; Feller et al., 1997
; Gerday et al., 2000
). On the other hand, structural rigidity has been proposed to be one component in enhanced protein stability of thermophilic proteins (Davail et al., 1994
; Zavodszky et al., 1998
). The behavior of HMGB1b mutants indeed is consistent with a relatively loosely folded native state in the sense that single amino acid substitutions can significantly influence its helix content, as we see in both our present constructs and previous mutational experiments (Taudte et al., 2000
). This is not seen, for example, in helical proteins such as myoglobin with a highly stable native fold (Lin et al., 1993
). One reason for the difference is probably the fact that HMGB is a protein of marginal stability so that any stabilizing or destabilizing interaction will have a significant effect on helicity. The binding properties of the stabilized mutants are not compromised by stabilizing mutations, hence the lack of defined native structure is not itself required for functional activity. We surmise that HMGB acquires a more stable fold after substrate binding. Investigations of similar behavior in strongly thermophilic enzymes (Van den Berg et al., 1998
; Miyazaki et al., 2000
) suggest a similar conclusion.
Although stabilization of this molecule with retention of functionality has been achieved, there are several issues associated with introducing metal binding sites into a protein as a general strategy. Certain constructs, such as loop I mutants (S37H, I38H and G39H), undergo significant losses in both secondary structure and stability. Thus how to select sites of substitution a priori is not obvious. Destabilization might result from disruption of the N-cap motif at the amino terminus of helix II, for example. N-caps have been shown to stabilize proteins (Serrano and Fersht, 1989; Lu et al., 1999
) and peptides (Gong et al., 1995
; Aurora and Rose, 1998
). The N-cap motif is deleted in the S37H and S37H/N51H/D55H constructs. These mutants display the greatest reduction in secondary structure (
50%: S37H) and stability (12.5°C: S37H/N51H/D55H) of those which we have examined. The loop II mutants, N51H/D55H and N51H/D56H, on the other hand, have structural and stability properties similar to wild-type. Inserting His side chains in this region does not significantly perturb the protein. These mutants have a proline residue in close proximity (position 59) to the N-terminal histidine. Proline residues have been implicated in reducing the conformational freedom of cytochrome c by orienting the histidine residues in a proper ligand binding position (Mathews, 1985
). Proline residues have also been implicated as rigidifying elements in loop regions of other proteins (Suzuki, 1989
; Watanabe and Suzuki, 1998
; Zhu et al., 1999
; Muslin et al., 2002
), effectively reducing the conformational entropy of the local region. However, the proline residue at position 59 of HMGB may or may not have a role in our loop II mutants as we observe that they have structural and stability properties similar to wild-type. The secondary structure of the loop I/II mutants (i.e. G39H/N51H/D55H, etc.) reflects the relatively loose native state(s) associated with rHMG1b. Three of the five loop I/II mutant proteins have lower helical content than wild-type, while two others show increased helicity (Table I
). Most mutants that we analyzed (nine of 10) were destabilized relative to wild-type in the absence of metal (Table II
). The majority of mutants (six of 10) are more stable than wild-type in the presence of metal ions (Table II
). Hence metal-mediated stabilization via histidine metal binding sites can potentially serve as a strategy to enhance protein stability. The extent will depend on local effects that we are only beginning to understand at this point: deleting helix N caps may be one such factor. It is tempting to consider the effects of metal ligation simply in terms of some sort of rigidification of a protein such as HMGB. However, this is likely to be misleading, since it is not clear mechanistically how stabilization occurs. If we assume that metal binding does rigidify the molecule, it necessarily reduces the entropy of the folded state and must be compensated for by favorable enthalpic interactions accompanying metal binding. Since this process does not entail loss of function, we believe our strategy may be generally applicable in natively unfolded or weakly folded proteins (Cavagnero et al., 1999
).
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Notes |
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Acknowledgments |
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Received May 8, 2002; revised July 15, 2002; accepted July 18, 2002.