Department of Chemistry, New York University, New York, NY 10003, USA
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Abstract |
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Keywords: circular dichroism/DNA binding/high mobility group protein I/protein stability/subdomain shuffling
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Introduction |
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For protein engineering, we are interested in whether or how individual domains interact. The role of the number of individual HMG box domains has been most intensively studied in UBF, an RNA polymerase I-specific transcription factor that plays a major role in promoter recognition and assembly of pre-initiation complexes (Bell et al., 1988; Schnapp et al., 1990
; Smith et al., 1990
; Tanaka et al., 1990
; McStay et al., 1991
). The dimerization domain together with HMG box domains 13 of UBF have been shown to be sufficient for its activity as a transcriptional enhancer (Sullivan and McStay, 1998
). DNA supercoiling by xUBFthe Xenopus factorrequires only the first three of its five HMG boxes (Stefanovsky et al., 1996
); only the first HMG box is required for assembly of a cross-over DNA junction (Hu et al., 1998
).
The single HMG domains A and B in HMG1 have been studied in detail. Their structures have been determined by NMR spectroscopy (Read et al., 1993; Weir et al., 1993
; Hardman et al., 1995
). Although their global folds are similar, the angle between the shorter and the longer arms of the L-shaped structure is smaller in the A domain (by ~14°) than in the B domain. The two boxes differ in their binding affinities. While both domains recognize distorted DNA structures including four-way junctions (4WJs) (Bianchi, 1988
; Bianchi et al., 1989
), supercoiled DNA (Hamada and Bustin, 1985
; Sheflin et al., 1993
), and cisplatin-modified DNA (Pil and Lipard, 1992; Chow et al., 1995
), they differ in their ability to discriminate 4WJ DNA from supercoiled DNA. Complexes of the A box with 4WJ DNA are resistant to competition from supercoiled DNA whereas B domain 4WJ complexes are not (Teo et al., 1995
).
Together, the tandem A and B boxes of HMG1 have a higher affinity for supercoiled DNA, double-stranded DNA and 4WJ, and are more effective in DNA cyclization assays than either alone (Grasser et al., 1998). Structure-specific binding of tandem AB HMG boxes to 4WJ DNA appears to be dominated by the higher affinity A domain; foot-printing experiments indicate selective interaction of the A domain with the branch point. Thus, the B domain must lie on one or more of the arms (Webb and Thomas, 1999
). Little is known about the spatial requirements in intact HMG1. Stros reported that a seven-residue N-terminal sequence in combination with an 18-residue C-terminal flanking region near the B box of HMG1 are needed to facilitate strong bending of DNA (Stros, 1998
). Grasser et al. demonstrated that the basic region following the B domain also mediates tighter binding of the AB di-domain to DNA (Grasser et al., 1998
). The role of the basic amino acid sequence linking the two boxes in HMG1 has been analyzed by deletion mutation (Saito et al., 1999
). The linker region can tolerate deletion of a few amino acids and retain appropriate binding of the di-domain to DNA; excessive deletion decreases the DNA-binding affinity. Thus, the affinity of tandem box proteins is a function of the identity of each box and the linker sequence connecting them.
In order to explore the role of the number and arrangement of the boxes in HMG1, we created two new fusion proteins by rearranging individual HMG boxes: in one we invert the order of the boxes in the di-domain, and in the other we fuse an additional A domain to the AB di-domain. In each case we retain the natural linker sequences present in HMG1. The BA di-domain folds and functions very much like the AB combination, implying little interaction between the boxes. However, the ABA triple domain is incompletely folded, and binds less effectively than either AB or BA di-domain. Thus, poly-domains formed from individual HMG boxes do not necessarily retain the activity of dimers or monomers, and a strong interaction between the C-terminal domain with the first box in the di-domain is suggested by the results. This shows that position and orientation of individual boxes in more complex arrangements can be important.
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Material and methods |
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The rHMG1 clone (pRHMG1) was obtained from M.E.Bianchi (Department of Genetics and Microbiology, University of Milan, Italy). The region coding for rHMG1 was subcloned into the plasmid pBluescript-KS whereby the region encoding the His-Taq was removed (HMG1/pBS-KS). Position 84 in the linker sequence between box A and B of HMG1 was mutated to remove a BsmAI restriction site (HMG1/E84E/pBS-KS). The mutation was introduced by site-directed mutagenesis using the mutagenic primer 5'-AAA GGG GAA ACC AAA AAG-3' (from CyberSyn, Lenni, PA) by the method of Kunkel et al., 1991 (mutated base is in italics). An HMG-B/pHB1 clone was obtained from Professor S.J.Lippard (Department of Chemistry, MIT, Cambridge, MA).
PCR primers were obtained in HPLC-purified form from CyberSyn, Lenni, PA. All PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA).
The A box was amplified by PCR using a sense primer, I: 5'-CGTGGATCCCCGGGCAAAGGAGATCCTAAGAAGCCGAGAGG-3' (the BamHI site is in italic) and the antisense primer, II: 5'-CCGGGAATTCGGGGTTACTCCCCTTTGGGGGGGATGTA-3' (the EcoRI site is in italics and the termination codon is underlined) on an HMG1/pBS-KS template. After double-digestion with the restriction enzymes BamHI and EcoRI the box was cloned into the BamHIEcoRI restriction fragment of pGEX-4T-3 (Pharmacia Biotech) yielding the GST-A box DNA template.
The AB di-domain DNA template was obtained by cloning of a double-digested PCR fragment (amplified using the sense primer I and an antisense primer, 5'-CCGGGAATTCGGGGTTATTTAGCTCTGTAGGCAGCAATATCC-3' (again the EcoRI site is in italics and the termination codon is underlined) on the HMG1/pBS-KS DNA template, cut by BamHI and EcoRI) into the BamHIEcoRI restriction fragment of pGEX-4T-3.
The full-length GST-HMG1 construct was built with a BamHIEcoRI trimmed PCR fragment resulting from DNA amplification using the sense primer I and the antisense primer, 5'-CCGGGAATTCGGGGTTATTCATCATCATCATCTTCTTCTTC-3' (the EcoRI site is in italics and the termination codon is underlined) on a HMG1/pBS-KS template. Cloning of this fragment into the BamHIEcoRI restriction fragment of pGEX-4T-3 produced a GST-HMG1 vector.
For construction of the BA combination the B box followed by the linker sequence was amplified using a sense primer, 5'-CGTGGATCCCCGAAAAAAAAGTTCAAGGACCCCAATGC-3' (the BamHI site is in italics) with the antisense primer, III: 5'-CTTTTTGGTCTCCCC TTTTTTA-GCTCTGTAGGCAGCAATA-3' (the BsmAI site is in italics and the termination codon is underlined) on a HMG1/pBS-KS template, with subsequent digestion by EcoRI and BsmAI. The A box with the linker sequence in front was obtained by amplification using 5'-CTAAAAAAGGGGAGACCAAAAAGGGCAAAGGAGATCCTAAGAAGC-3' (the BsmAI site is in italics) and II as primers with HMG1/pBS-KS as the template, followed by double-digestion with BsmAI and EcoRI. The two pieces were cloned together into the BamHIEcoRI restriction fragment of pGEX-4T-3, producing the GST-BA DNA template.
For construction of the ABA combination the di-domain AB followed by the linker sequence was amplified using primers I and III with a HMG1/E84E/pBS-KS DNA template. The PCR product was trimmed with BamHI and BsmAI. The A box with the linker in front was obtained as described for the BA construct. Both restriction fragments (AB and the A box) were cloned together into the BamHIEcoRI restriction fragment of pGEX-4T-3 yielding a GST-ABA template.
DNA sequence analysis was performed on an ABI PRISM 377 Sequencer (PE Applied Biosystems) using an ABI PrismBigDye Terminator Cycle Reaction Kit (PE Applied Biosystems) to confirm the correct assembly of HMG-box combinations.
Protein expression and purification
The B box of rHMG1 was expressed from pHB1/Escherichia coli Bl21(DE3)pLysS (Chow et al., 1995). The pGEX-4T-3 vectors containing the required protein insert were transformed into Epicurean Coli BL21-Codon PlusTM (DE3)-RIL (Stratagene). All GST fusion proteins were then expressed and purified according to standard procedures described in the GST manual from Pharmacia Biotech. The fusion proteins were cleaved with thrombin (Pharmacia Biotech). Application onto Glutathione Sepharose 4B columns (Pharmacia Biotech) yielded HMG box proteins containing three additional N-terminal amino acids (glycineserineproline). All proteins, except HMG1, were repurified by FPLC using an Econo-Pac CM cartridge (Bio-Rad). The purity of the HMG box proteins was checked by resolution of protein samples on 15% SDSpolyacrylamide gels in Tris-Tricine buffer at 196 V for 1 h, followed by Coomassie 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 obtained using AVIV 60DS or AVIV 202 SF CD spectrometers (Aviv Associates, Lakewood, NJ). CD spectra of 0.5, 1 and 2 mM protein solutions in a 10 mM potassium phosphate buffer, pH 7.0, containing 0.5 mM dithiothreitol (DTT) and 0.5 mM EDTA were recorded in a cell with a 1 cm pathlength from 260 to 200 nm at 4°C. Thermal denaturation was followed at a wavelength of 222 nm under the above conditions over the range of 494°C.
Fluorescence
Guanidine hydrochloride (GdnHCl)-induced unfolding was monitored using an Automated Temperature/Titration Filter Fluorometer, ATFF-212 (Aviv Associates), using an excitation wavelength of 280 nm and an emission wavelength of 340 nm. Samples containing 5 mM protein solutions were titrated with a 4.8 M GdnHCl stock solution at 5°C. Both solutions were buffered in 10 mM potassium phosphate, pH 7.0, containing 0.5 mM DTT and 0.5 mM EDTA. The GdnHCl concentration was increased from 0 to 4 M in 0.1 M increments, while the sample volume and protein concentration were held constant.
Binding to four-way DNA junctions
The oligonucleotides used in this study were synthesized and HPLC purified by CyberSyn Inc. (Lenni, PA). The 4WJ (J1), displayed in Figure 1B, was composed of four oligonucleotides as described previously (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). The strands were annealed by combining 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 to 4°C overnight.
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EMSA of the complexes of the 4WJ (J1) with HMG box proteins were performed as a function of DNAprotein molar ratio. DNA (0.0154 µM in the final reaction) and aliquots of HMG box protein were incubated in 20 ml of a reaction solution (0.5 mM DTT, 100 mM NaCl, 10% w/v glycerol, 25 mM TrisHCl, pH 8.0, 2 mM MgCl2) on ice for 30 min. The complexes were subjected to electrophoresis on 6% polyacrylamide gels in 0.5x TBE (45 mM Trisma, 45 mM boric acid and 1.0 mM EDTA), pH 8.1, at 4°C. Gels were dried on Whatman 3MM paper and scanned with a PhosphorImager.
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Results |
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The thermal stability of the HMG box combinations was measured via CD thermal denaturation. The denaturation profile was monitored at 222 nm from 4 to 94°C. The respective melting temperatures of each protein (Tm), displayed in Table I, were estimated according to the procedure described by Pace (Pace, 1988
). The Tm values of the A domain (unpublished data) and B domain (Taudte et al., 2000
) are analogous to previously reported values. The separate domains, full-length HMG1 and all box combinations show only a single transition during temperature-induced unfolding (Figure 4AD
), which suggests each protein unfolds via a two-state mechanism. While all unfolding curves are similar in shape, the mean residue ellipticity differs slightly between single domains, di-domains and full-length HMG1, and is significantly lower for the ABA box combination.
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Discussion |
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The mean residue ellipticity value of the full-length protein HMG1 is lower than that of the di-domains (Table I), as anticipated from the contributions of the basic region following the B domain together with the disordered C-terminal tail. However, the additional sequences influence the secondary structure of the protein: a red shift of the shorter wavelength band in the CD spectrum results (Figure 3C
). This suggests that some structural components in HMG1 differ from the A and B boxes, individually or together. The ABA box combination has the lowest ellipticity value of all the protein constructs monitored. ABA seems to be partially unfolded, as indicated by the reduction in intensity of both minima in the CD spectrum (Figure 3D
).
Individual HMG boxes unfold in a single transition, with the A domain slightly more stable than the B domain, according to thermal denaturation or GdnHCl-induced unfolding. The tandem AB and BA di-domains unfold in a single overlapping process. Monitoring the fluorescence emission during GdnHCl-induced unfolding reveals that the tryptophan emission maxima shifts to the red upon unfolding which has also been observed for the unfolding of the isolated B domain. The transition midpoint of GdnHCl-induced unfolding of HMG1 [m]1/2 is calculated to be 2.1 M at 4°C. This is below the [m]1/2 values computed for either of the di-domains (AB, 2.3 M and BA, 2.5 M at 5°C).
A second difference is in the apparent cooperativity of the transition. While the di-domains unfold over a range of approximately 1.2 molarity units (BA) or 1.5 molarity units of GdnHCl (AB), the transition window for the full-length HMG1 extends over three molarity units in GdnHCl. Differences in unfolding between the di-domain and holo-HMG1 protein were reported by Ramstein et al. (Ramstein et al., 1999), and led them to assert that the two domains in the di-domain protein AB are equivalent and unfold independently. However, in HMG1 one domain seems to be destabilized by interaction with the acidic tail, while the second is unaffected (Read et al., 1994
). Grasser et al. found no evidence for any domaindomain interactions between tandem boxes (Grasser et al., 1998
). Comparison of 2D 1H-15N HMQC spectra of the A domain, B domain and AB di-domain show that the cross-peaks in the 2D spectrum correspond to the superposition of the spectra of single A and B boxes. There are no chemical shift differences between an isolated B domain and the B domain in AB. Relaxation data confirm this picture: the linker region between the two boxes appears to be highly flexible, consistent with the idea that interaction between the two boxes is minimal. Additional evidence comes from thermal stability studies using differential scanning calorimetry and CD (Ramstein et al., 1999
). The melting temperatures of A and B are identical to those of the AB di-domain (in their study an 11 amino acid basic stretch was added to the B domain). Conservation of denaturation enthalpies indicates that the two boxes in the di-domain unfold independently. Therefore, the A and B domains appear to be energetically uncoupled and the structure of the isolated boxes is maintained in the AB di-domain. It should be noted that tandem HMG boxes, single HMG domains, ABA, as well as HMG1, all unfold with single sigmoidal profiles.
Binding the separate boxes A and B to 4WJs yields 1:1 complexes at lowest protein to DNA ratios, although higher order complexes are formed at increased protein to DNA ratios (Bianchi et al., 1989; Read et al., 1994
; Teo et al., 1995
; Xin et al., 2000
). It has been suggested that di-domain binding to 4WJs is directed by the A rather than the B domain (Webb and Thomas, 1999
). As in the case of the individual domains, a 1:1 complex forms at low di-domain to DNA ratios, and higher order complexes form at increased protein to DNA ratios as indicated by a progressive shift in mobility of the di-domainJ1 complex (Figure 6BC
). Binding of the BA di-domain is indistinguishable from that of the AB tandem HMG boxes. Thus, the order of the HMG boxes seems not only to have little influence on secondary structure and unfolding of the two components, but it is also insignificant functionally in terms of recognizing 4WJs.
Adding the basic region and acidic tail to the AB di-domain to generate the full-length HMG1 alters the binding specificity of the protein. Binding to the 4WJ is not only significantly tighter, but the mobility shift of the HMG14WJ complex bands at higher protein to DNA molar ratios is considerably reduced (Figure 6D). A similar effect has also been reported in binding HMG-D to 4WJs (Ramstein et al., 1999
). While the HMG domain of HMG-D alone does not bind to the 4WJ, the HMG domain with the basic region as well as the full-length HMG-D protein does. The direction of this effect is not what would be predicted from simple electrostatics: adding a strongly negative charged tail to a box should weaken binding if anything. The fact that it strengthens binding points to a change in conformation or dynamics that is transmitted to the binding face of the box. With full-length HMG-D, as with HMG1, single complexes can be detected. However, at least four complexes are observed in the case of the HMG domain plus the basic region (Payet and Travers, 1997
). Thus, the stoichiometry of binding is also influenced by the presence of the additional linker and tail segments.
The ABA box combination fails to maintain the individual folding characteristics of the di-domains, and is unfolded relative to single boxes, AB or BA. While ABA still binds selectively to 4WJs, its binding affinity to 4WJs is lower than for HMG1 and either di-domain. More importantly, the binding pattern differs significantly from that of the other HMG proteins of this study. Single HMG boxes A and B, the two di-domains AB and BA and full-length HMG1 all reveal one dominant complex at a given protein to DNA ratio, yet three different complexes can be detected simultaneously in binding of ABA to J1 over the same concentration range. This binding pattern is similar to that observed for the HMG-D domain with its basic region bound to the 4WJ (Payet and Travers, 1997), as well as for binding of Rox1 to 4WJs (Xin et al., 2000
) and NHP6A to a linear DNA fragment (Allain et al., 1999). Moreover, the presence of multiple-binding species (Figure 6BE
) and the residual presence of single strand 101 precludes the determination of binding constants for the HMG constructs; multiple-binding species were also detected in HMG-B/4WJ binding assays (Xin et al., 2000
).
Taken together these results show, that while the order of pairs of boxes is unimportant, extension of any subdomain to the C-terminus of a pair involves a strong interaction of that domain with one or both of the N-terminal boxes. In the case of the acidic tail terminal domain, the interaction favors binding of the 4WJ despite the added negative charge. In the case of a basic HMG box domain, the interaction destabilizes one of the two boxesmost likely the firstand impedes folding and function. Such an effect might reflect spatial rigidity in positioning the additional domain or direct interaction of one di-domain by the third. However, since no rigid coupling is detected in linking A with B, or B with A, it seems unlikely that an extra linker will be rigid. This makes it unlikely that the third domain is mechanically forced to fold back so as to make contact with one or the other of the independent pair. Alternatively, the presence of the third box destabilizes or unfolds the first or second. Evidence for a functionally favorable interaction between the C-terminal tail in HMG1 with the A and B boxes in the protein is seen in the enhanced binding by the holoprotein. In our hands ABA drastically impairs binding by the di-domain. How then does UBF avoid a similar problem? One possibility is that the linker sequences in UBF are different. The linker sequences in UBFs are of different lengths (711 amino acids) with no obvious sequence similarity, giving no clues to significant functional differences. The other is that the interactive surfaces of the boxes in UBF are different from those in HMG1. This question can be addressed by fusing boxes from UBF with these from HMG1.
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Notes |
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Acknowledgments |
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References |
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Received April 4, 2001; revised July 23, 2001; accepted July 24, 2001.