Metal-dependent stabilization of an active HMG protein

Anthony J. Bell, Jr, Hong Xin, Susann Taudte, Zhengshuang Shi and Neville R. Kallenbach1

Department of Chemistry, New York University, New York, NY 10003, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Using a cloned single domain of the high mobility group protein 1 (HMGB1), we evaluated the effect of introducing metal binding site(s) on protein stability and function. An HMG domain is a conserved sequence of ~80 amino acids rich in basic, aromatic and proline residues that is active in binding DNA in a sequence- or structure-specific manner. The design strategy focuses on anchoring selected regions of the protein, specifically loops and turns in the molecule, using His–metal ligands. Changes in secondary structure, thermostability and DNA binding properties of a series of such mutants were evaluated. The two most stable mutant constructs contain three surface histidine replacements (two metal binding sites) in the regions encompassing both turns of the molecule. On ligation with the divalent nickel cation, the stability of these two triple histidine mutants (I38H/N51H/D55H and G39H/N51H/D55H) increases by 1.3 and 1.6 kcal/mol, respectively, relative to the wild-type protein, although the creation of binding sites per se destabilizes the protein. The DNA-binding properties of the modified proteins are not impaired by the introduction of the metal binding motifs. These results indicate that it is feasible to stabilize protein tertiary structure using appropriate placement of surface His–metal bonds without loss of function.

Keywords: circular dichroism/divalent metals/DNA binding/high mobility group protein box B (rHMGB1b)/protein stability


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
High mobility group protein 1 (HMGB1) is a highly abundant DNA-binding protein in the nucleus, with a variety of putative functions including transcription, replication, repair and cellular differentiation (Zwilling et al., 1995Go; Bustin and Reeves, 1996Go; Zappavigna et al., 1996Go). No unequivocal function for HMGB1 has been assigned because it interacts with a variety of substrates and cofactors (Zappavigna et al., 1996Go; Jayaraman et al., 1998Go; Decoville et al., 2000Go). It has a tripartite structure, consisting of two tandem high mobility group (HMG) box motifs (A and B) and an acidic C-terminus. Each HMG box is a conserved sequence of ~80 amino acids rich in basic, aromatic and proline side chains that has been identified in a large number of DNA-binding proteins (Baxevanis and Landsman, 1995Go; Baxevanis et al., 1995Go). HMG domains are located in single or multiple copies in non-histone chromosomal proteins [HMGB1, HMGB2, UBF (upstream binding factor)] that bind DNA substrates in a structure-specific manner (little or no sequence identity) and transcription factors (LEF-1, SRY) that bind in a sequence-specific manner.

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., 1993Go; Weir et al., 1993Go; Hardman et al., 1995Go). Each domain contains a highly conserved HMG global fold of three {alpha}-helices arranged in an L-shape (Figure 1AGo) (Read et al., 1993Go; Weir et al., 1993Go). 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, 1988Go; Bianchi et al., 1989Go), supercoiled (Hamada and Bustin, 1985Go; Sheflin et al., 1993Go) and cisplatin-modified DNA (Pil and Lippard, 1992Go; Chow et al., 1995Go). The A box has a higher binding affinity to these distorted DNA substrates (Teo et al., 1995Go; Dunham and Lippard, 1997Go; Webb and Thomas, 1999Go).



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Fig. 1. Structure of wild-type rHMGB1b and mutant construct G39H/N51H/D55H. (A) Cylinder representation of wild-type rHMGB1b devoid of surface histidine residues. (B) Cylinder representation of loop I/II mutant G39H/N51H/D55H. The structures were generated with the InsightII molecular modeling program.

 
For protein engineering, we are interested in exploring surface motifs that can stabilize a protein to a desired extent without impairing binding activity, using the B domain of HMGB1 (rHMGB1b) as a model system. An approach to stabilizing proteins via redesigning the non-polar interactions within the core has been reported, which has proven fairly successful (Malakauskas and Mayo, 1998Go). This requires theoretical modeling to select changes likely to prove stabilizing. The premise of our strategy is that the surface offers a simpler context for stabilizing proteins, obviating the need for extensive modeling.

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., 1996Go). A metal binding site can be generated by inserting two or more properly positioned metal binding side chains (Ghadiri and Choi, 1990Go). 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 folding–unfolding equilibrium towards the native state (Arnold and Haymore, 1991Go). 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, 1989Go; Muheim et al., 1993Go; Wisz et al., 1998Go; Benson et al., 2000Go; Wray et al., 2000Go).

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., 1994Go). 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, 2001Go). 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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutagenesis

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., 1991Go). 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 pHB1–Escherichia coli Bl21(DE3)pLysS following procedures described by Chow et al. (Chow et al., 1995Go). 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 NaCl–50 mM Tris–HCl (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% SDS–polyacrylamide (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, 1987Go). Protein concentrations were determined according to Pace et al. (Pace et al., 1995Go).

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 2BGo) (Kallenbach et al., 1983Go). 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 [{gamma}-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 Tris–HCl, 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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Fig. 2. Schematic of the HMG global fold and the binding substrate, 4WJ, J1. (A) Sequence alignment of the A and B domain of rat HMG1 and schematic representation of the structure of the proteins indicating the position of the three {alpha}-helices, including representative non-conserved and conserved regions. (B) Schematic of the four-way junction, 4WJ, binding substrate J1.

 
Electrophoretic mobility shift assays (EMSAs)

EMSAs of the rHMGB1b mutants with the 4WJ, J1, were performed as a function of DNA–protein 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 Tris–HCl, 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Design strategy

The design strategy focuses on inserting metal binding motifs into the target protein. The loops/turns between the {alpha}-helices were targeted initially because mutations in these regions have shown significant effects on stability of the protein (Taudte et al., 2000Go). The amino acids selected for replacement are displayed in bold in Figure 2AGo. These residues were selected because they represent non-conserved sites located on the surface of the molecule. Introduction of a histidine residue in positions 37–39 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., 2000Go) and cisplatin-modified DNA substrates (Ohndorf et al., 1999Go; He et al., 2000Go). Mutant proteins in which a histidine residue replaces functionally relevant residues (37–39) are referred to as loop I constructs (Figure 2AGo). 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 2AGo). 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 1BGo). 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, 2000Go; Taudte et al., 2000Go).

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 {alpha}-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 IGo.


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Table I. Mean residue ellipticities and the change in ellipticity values of the mutant proteins vs wild-type rHMGB1b in the absence and presence of Ni2+
 
The CD spectra of the mutant proteins containing a single histidine replacement at loop I display a significant reduction in secondary structure vs wild-type. The CD scan of G39H reflects a severe loss of intensity in the bands at 222 and 212 nm of ~40% (open circles, Figure 3AGo). Interestingly, the CD spectrum of this protein indicates a further loss in helix content in the presence of metal ions (closed circles, Figure 3AGo). G39H also possesses a red shift of the first band from 208 to 212 nm vs wild-type (a similar red shift is present in the CD spectra of all the mutant proteins analyzed). The ellipticity values of the remaining loop I mutants (S37H and I38H) display similar losses in secondary structure, with S37H having the largest loss (~50%) in ellipticity. The ellipticity values of these mutants do not undergo a significant change in the presence of metal ions (Table IGo). This behavior is consistent with the fact that HMGB1b is marginally stable and stabilized on interacting with its DNA target. Such behavior suits this protein well for evaluating the effects of metal ligands.



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Fig. 3. CD spectra of wild-type rHMGB1b and mutant constructs. Spectra of protein solutions in a 10 mM HEPES buffer, pH 7 in the absence and presence of NiSO4.6H2O were recorded in a cell with a 1 cm pathlength from 260 to 200 nm at 4°C. Mean residue ellipticity is plotted against wavelength. (A) CD spectra of 3 mM protein solutions of wild-type rHMGB1b and the loop I construct G39H; (B) CD spectra of 3 mM protein solutions of wild-type and the loop II mutant N51H/D55H; (C) CD spectra of a 3 mM wild-type rHMGB1b and the loop I/II construct G39H/N51H/D55H.

 
The CD spectra of the mutant proteins with amino acid replacements encompassing loop II are comparable to wild-type. The CD scan of the double mutant N51H/D55H possesses an ellipticity value at 222 nm close to that of the wild-type (open triangles, Figure 3BGo). Upon metal ligation, the ellipticity of the red-shifted band at 212 nm increases slightly in intensity (closed triangle, Figure 3BGo). The ellipticity values of a similar mutant, N51H/D56H, show a slight increase in the intensity of the bands at 222 and 212 nm, indicative of a higher helical content in the absence and presence of metal ions (Table IGo).

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 3CGo). 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 IGo). 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 4A–CGo). The respective free energy change ({Delta}G, {Delta}{Delta}G), melting temperature (Tm) and enthalpy change ({Delta}Hm) values of each protein were estimated according to the procedure described by Pace (Pace, 1988Go). The thermodynamic properties of each protein are listed in Table IIGo. 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., 2000Go) 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 4A–CGo); these values are also listed in Table IIGo.



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Fig. 4. Temperature unfolding of wild-type rHMGB1b and mutant constructs monitored by CD. (A) Unfolding of wild-type rHMGB1b and the loop I mutant construct G39H in the absence and presence of NiSO4.6H2O; (B) unfolding of wild-type rHMGB1b and the loop II mutant construct N51H/D55H in the absence and presence of NiSO4.6H2O; (C) unfolding of wild-type rHMGB1b and the loop I/II mutant construct G39H/N51H/D55H in the absence and presence of NiSO4.6H2O.

 

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Table II. Thermal denaturation properties of histidine mutants vs wild-type rHMGB1b
 
The thermal denaturation profile of the loop I mutant, G39H, indicates an increase in Tm of 4.3°C upon ligation vs wild-type (closed circles, Figure 4AGo). Replacement of Gly with His at this position does not alter the relative stability of the molecule vs wild-type (open circles, Figure 4AGo). The single mutant, I38H, is stabilized slightly upon ligation, with an increase in Tm of 1.3°C vs wild-type (Table IIGo). However, the relative stability of I38H and the remaining loop I mutant S37H are both reduced, the former being destabilized by 4.4°C and the latter by 10.2°C in the absence of metal ions (Table IIGo). Upon ligation, the Tm of S37H is still 6.2°C less stable than wild-type (Table IIGo).

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 4BGo). The histidine replacements at the helical termini produce a 0.5°C residual increase in the Tm of this protein (open triangles, Figure 4BGo). 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 IIGo). 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 4CGo). A similar mutant, I38H/N51H/D55H, undergoes a Tm increase of 5.5°C in the presence of metal ions (Table IIGo). 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 IIGo).

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., 1989Go, 1992Go; Falciola et al., 1994Go; Taudte et al., 2000Go) and supercoiled DNA (Stros and Reich, 1998Go) 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, 1997Go; Thomas and Travers, 2001Go). The 4WJ J1 was employed as the substrate for the gel shift assays (Figure 5A–CGo). Wild-type rHMGB1b binds J1 with 4:1 stoichiometry at higher protein–DNA ratios (Xin et al., 2000Go). A 4:1 binding pattern is represented by the higher order complexes formed at protein–DNA ratios of 100:1 and higher.




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Fig. 5. Binding of wild-type rHMGB1b and mutant constructs. Aliquots of the proteins were combined with DNA in a 20 ml reaction volume also containing 1 mM NiSO4.6H2O, 100 mM NaCl, 10% (w/v) glycerol, 25 mM Tris–HCl, pH 8.0, and 2 mM MgCl2. Reaction mixtures were incubated on ice for 40 min prior to loading on to a 15% polyacrylamide gel (29:1 acrylamide:bis ratio) in 0.5x TBE. Samples were electrophoresed for 3–4 h, dried, scanned on a Molecular Imager System GS-525 (Bio-Rad) and visualized using Molecular Analyst Software Version 1.4.1 (Bio-Rad). (A) Binding of the loop I mutant G39H and wild-type rHMGB1b to J1. Lane 1, single strand 101 (0.05 µM); lane 2, free J1 (05 µM); lane 3–8, G39H incubated with J1 at DNA/protein ratios of 1:1, 1:10, 1:20, 1:40, 1:100, 1:200; lanes 9–14, rHMGB1b incubated with J1 at DNA/protein ratios of 1:1, 1:10, 1:20, 1:40, 1:100, 1:200. (B) Binding of the loop II mutant N51H/D55H and wild-type rHMGB1b to J1. Lane 1, single strand 101 (0.05 µM); lane 2, free J1 (05 µM); lanes 3–8, N51H/D55H incubated with J1 at DNA/protein ratios of 1:1, 1:10, 1:20, 1:40, 1:100, 1:200; lanes 9–14, rHMGB1b incubated with J1 at DNA/protein ratios of 1:1, 1:10, 1:20, 1:40, 1:100, 1:200. (C) Binding of the loop I/II mutant G39H/N51H/D55H and wild-type rHMBG1b to 4WJ J1. Lane 1, single strand 101 (0.05 µM); lane 2, free J1 (05 µM); lanes 3–8, G39H/N51H/D55H incubated with J1 at DNA/protein ratios of 1:1, 1:10, 1:20, 1:40, 1:100, 1:200; lanes 9–14, rHMGB1b incubated with J1 at DNA/protein ratios of 1:1, 1:10, 1:20, 1:40, 1:100, 1:200.

 
Wild-type rHMGB1b and the mutant proteins display comparable binding behavior (4:1 binding) at higher protein–DNA ratios (100:1 and 200:1). The binding affinity of the loop I mutant G39H is lower than wild-type at lower protein–DNA ratios (lanes 3–6), but at higher protein concentration(s) the binding pattern appears to be analogous to wild-type (lanes 7–8 vs 13–14). The true binding stoichiometry of this mutant is difficult to determine at the lower protein concentrations (lanes 3–7) owing to the diffuse nature of the bands, indicative of intermediate binding species (Figure 5AGo). The binding affinity of the loop II mutant N51H/D55H is very similar to wild-type with 4:1 binding at higher protein–DNA ratios (lanes 6–8). The binding affinity of this mutant at lower DNA–protein ratios is again difficult to characterize owing to the diffuse nature of the bands (lanes 3–5, Figure 5BGo). As in the case of the other proteins studied, the loop I/II mutant G39H/N51H/D55H also displays 4:1 binding at higher protein–DNA ratios (lanes 7–8) but forms a number of intermediate species at lower concentrations (lanes 3–6, Figure 5CGo). In each case, there is a residual amount of single strand present owing to the lack of MgCl2 in the running buffer leading to dissociation of the unbound 4WJ (J1). This behavior has been reported previously (Xin et al., 2000Go) and does not affect assessment of the relative binding characteristics of the mutant proteins analyzed here.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our results indicate that it is possible to stabilize the protein rHMGB1b by introducing metal binding ligands at turn or loop regions on the protein surface. Thermodynamically the effect reflects preferential ligation of the metal to the two histidines in the folded protein relative to the unfolded protein (Krantz and Sosnick, 2001Go). All experiments with metal were run with 1 mM Ni2+; we believe the concentration is reaching the saturating range based on our unpublished Ni2+ titration results on a bi-His mutant of GCN4 coiled-coil helical protein (Z.Shi, T.R.Sosnick and N.R.Kallenbach, unpublished results). From Table IIGo, it is obvious that the majority of the mutant constructs (nine of 10) are less stable than the wild-type in the absence of metal; however, for those mutants with properly positioned histidine residues, the lost stability can be over-recovered by the addition of the divalent nickel cation. The two most stable mutant constructs contain three surface histidine replacements (two metal binding sites) in the regions encompassing both turns of the molecule. On ligation with the divalent nickel cation, the stability of these two triple histidine mutants (I38H/N51H/D55H and G39H/N51H/D55H) increases by 1.3 and 1.6 kcal/mol, respectively, relative to the wild-type protein. From the perspective of melting temperature, several mutant constructs (six of 10) are more stable than wild-type in the presence of metal ions (Table IIGo). This is evident in the moderate increase in stability of 4.3°C of the loop I mutant G39H. The maximum increase in Tm is associated with the loop II mutants, N51H/D55H and N51H/D56H, each of which displays an increase of 6.2°C. Surprisingly, the loop I/II mutants do not produce an additive increase in stability, with I38H/N51H/D55H, having an increase in Tm of 5.5°C. Also, the moderate increase in melting temperature upon metal binding does not correlate with a large change in enthalpy ({Delta}Hm) (Table IIGo). This suggests that the addition of surface clips might not be associated with changes in the hydrophobic core of the protein, which is modest in MHGB proteins and centers around the tryptophan and lysine residues located in positions 49 and 57, respectively (Weir et al., 1993Go).

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 protein–DNA ratios (Bianchi et al., 1989Go) and 4:1 stoichiometry at higher protein–DNA ratios (Xin et al., 2000Go). 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 protein–DNA ratios (Figure 5AGo). As with G39H, the remaining loop I mutants (S37H and I38H) also display 4:1 binding at higher protein–DNA ratios. Moreover, S37H and I38H do not display a loss in binding affinity at lower protein–DNA 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 protein–DNA ratios (Figure 5BGo). 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 protein–DNA ratios (lanes 7–8, Figure 5CGo).

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 protein–ligand interactions depend on flexibility: enzymes must have internal motions. An enzyme with a flexible molecular structure has a variety of conformations that increase potential enzyme–substrate interactions. Such increased substrate interactions could serve to enhance the efficiency of the enzyme (Aghajari et al., 1996Go; Feller et al., 1997Go; Gerday et al., 2000Go). On the other hand, structural rigidity has been proposed to be one component in enhanced protein stability of thermophilic proteins (Davail et al., 1994Go; Zavodszky et al., 1998Go). 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., 2000Go). This is not seen, for example, in helical proteins such as myoglobin with a highly stable native fold (Lin et al., 1993Go). 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., 1998Go; Miyazaki et al., 2000Go) 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, 1989Go; Lu et al., 1999Go) and peptides (Gong et al., 1995Go; Aurora and Rose, 1998Go). 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, 1985Go). Proline residues have also been implicated as rigidifying elements in loop regions of other proteins (Suzuki, 1989Go; Watanabe and Suzuki, 1998Go; Zhu et al., 1999Go; Muslin et al., 2002Go), 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 IGo). Most mutants that we analyzed (nine of 10) were destabilized relative to wild-type in the absence of metal (Table IIGo). The majority of mutants (six of 10) are more stable than wild-type in the presence of metal ions (Table IIGo). 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., 1999Go).


    Notes
 
1 To whom correspondence should be addressed. E-mail: nrk{at}nyu.edu Back


    Acknowledgments
 
This research was supported by grant CA24101 from the National Cancer Institute, NIH and the Margaret and Herman Sokol Science Research Fund from New York University.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received May 8, 2002; revised July 15, 2002; accepted July 18, 2002.





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