Partial B-to-A DNA transition upon minor groove binding of protein Sac7d monitored by Raman spectroscopy

Members of the Sso7d/Sac7d protein family and other related proteins are believed to play an important role in DNA packaging and maintenance in archeons. Sso7d/Sac7d are small, abundant, basic, and nonspecific DNA-binding proteins of the hyperthermophilic archeon Sulfolobus. Structures of several complexes of Sso7d/Sac7d with DNA octamers are known. These structures are characterized by sequence unspecific minor groove binding of the proteins and sharp kinking of the double helix. Corresponding Raman vibrational signatures have been identified in this study. A Raman spectroscopic analysis of Sac7d binding to the oligonucleotide decamer d(GAGGCGCCTC)(2) reveals large conformational perturbations in the DNA structure upon complex formation. Perturbed Raman bands are associated with the vibrational modes of the sugar phosphate backbone and frequency shifts of bands assigned to nucleoside vibrations. Large changes in the DNA backbone and partial B- to A-form DNA transitions are indicated that are closely associated with C2'-endo/anti to C3'-endo/anti conversion of the deoxyadenosyl moiety upon Sac7d binding. The major spectral feature of Sac7d binding is kinking of the DNA. Raman markers of minor groove binding do not largely contribute to spectral differences; however, clear indications for minor groove binding come from G-N2 and G-N3 signals that are supported by Trp24 features. Trp24 is the only tryptophan present in Sac7d and binds to guanine N3, as has been demonstrated clearly in X-ray structures of Sac7d-DNA complexes. No changes of the Sac7d secondary structure have been detected upon DNA binding.     

Structural characterization of the functional regions in the archaeal protein Sso7d

Sso7d from the extreme thermophilic crenarchaeon Sulfolobus solfataricus is a multifunctional protein in in vitro assays, whose in vivo role is still puzzling. Crystals of Sso7d in complex with DNA elucidated the protein surface involved in the binding to the nucleic acid, whereas the locations of the Sso7d regions responsible for a chaperone activity in renaturing protein aggregates (i.e., the protein-binding surface and the site of ATPase activity) are still unknown. We identified the regions of Sso7d involved in protein-binding by limited proteolysis experiments associated to advanced mass spectrometric procedures performed on isolated Sso7d and Sso7d in complex with the peptide melittin. By affinity labeling of Sso7d with the ATP analogue 5'-p-fluorosulfonylbenzoyl adenosine and characterization of the labeled tryptic peptides by tandem mass spectrometry, we found that Y7 and K39 are residues involved in ATP binding/hydrolysis. Insights into the positions of the ligands melittin and ATP were achieved by a molecular modeling study; the models obtained were in agreement with most experimental data. A comparison among the complexes of Sso7d with DNA, with melittin, and with ATP showed that the DNA-binding surface and the protein-binding surface overlap, whereas the ATPase site is mostly independent of the binding sites for the nucleic acid and melittin.     

The Sso7d protein of Sulfolobus solfataricus: in vitro relationship among different activities

The physiological role of the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus is unknown. In vitro studies have shown that Sso7d promotes annealing of complementary DNA strands (Guagliardi et al. 1997), induces negative supercoiling (Lopez-Garcia et al. 1998), and chaperones the disassembly and renaturation of protein aggregates in an ATP hydrolysis-dependent manner (Guagliardi et al. 2000). In this study, we examined the relationships among the binding of Sso7d to double-stranded DNA, its interaction with protein aggregates, and its ATPase activity. Experiments with 1-anilinonaphthalene-8-sulfonic acid as probe demonstrated that exposed hydrophobic surfaces in Sso7d are responsible for interactions with protein aggregates and double-stranded DNA, whereas the site of ATPase activity has a non-hydrophobic character. The interactions of Sso7d with double-stranded DNA and with protein aggregates are mutually exclusive events, suggesting that the disassembly activity and the DNA-related activities of Sso7d may be competitive in vivo. In contrast, the hydrolysis of ATP by Sso7d is independent of the binding of Sso7d to double-stranded DNA or protein aggregates.     

Thermodynamics of core hydrophobicity and packing in the hyperthermophile proteins Sac7d and Sso7d

The influence of core hydrophobicity and packing on the structure and stability of the hyperthermophile proteins Sac7d and Sso7d have been studied by calorimetry, circular dichroism, and NMR. Valine 30 is positioned in Sac7d to allow a cavity-filling Val --> Ile substitution which occurs naturally in the homologous more thermostable Sso7d. The cavity-filling mutation in Sac7d has been characterized and compared to the reciprocal Ile --> Val mutation in Sso7d. A detailed analysis of the stability of the proteins was obtained by globally fitting the variation of DSC parameters and circular dichroism intensities as a function of temperature (0-100 degrees C), salt (0-0.3 M), and pH (0-8). A global analysis over such a range of conditions permitted an unusually precise measure of the thermodynamic parameters, as well as the separation of the thermodynamics of the intrinsic unfolding reaction from the linked effects of protonation and chloride binding associated with acid-induced folding. The results indicate differences in the energetics of unfolding Sac7d and Sso7d that would not be apparent from an analysis of DSC data alone using conventional methods. The sign and magnitude of the changes in DeltaG, DeltaH, TDeltaS, and DeltaC(P) of unfolding resulting from core Ile/Val substitutions in the two proteins were consistent with differences in hydrophobicity of Val and Ile and negligible changes in packing (van der Waals) interactions. The benefit of increased hydrophobicity of the core increased with temperature, with maximal effect around 116 degrees C. Increased hydrophobicity of the core achieved not only an increase in the free energy of unfolding, but also a lateral shift of the temperature of maximal stability to higher temperature.     

The solution structure of the Sac7d/DNA complex: a small-angle X-ray scattering study.

Small-angle X-ray scattering has been used to study the structure of the multimeric complexes that form between double-stranded DNA and the archaeal chromatin protein Sac7d from Sulfolobus acidocaldarius. Scattering data from complexes of Sac7d with a defined 32-mer oligonucleotide, with poly[d(GC)], and with E. coli DNA indicate that the protein binds along the surface of an extended DNA structure. Molecular models of fully saturated Sac7d/DNA complexes were constructed using constraints from crystal structure and solution binding data. Conformational space was searched systematically by varying the parameters of the models within the constrained set to find the best fits between the X-ray scattering data and simulated scattering curves. The best fits were obtained for models composed of repeating segments of B-DNA with sharp kinks at contiguous protein binding sites. The results are consistent with extrapolation of the X-ray crystal structure of a 1:1 Sac7d/octanucleotide complex [Robinson, H., et al. (1998) Nature 392, 202-205] to polymeric DNA. The DNA conformation in our multimeric Sac7d/DNA model has the base pairs tilted by about 35 degrees and displaced 3 A from the helix axis. There is a large roll between two base pairs at the protein-induced kink site, resulting in an overall bending angle of about 70 degrees for Sac7d binding. Regularly repeating bends in the fully saturated complex result in a zigzag structure with negligible compaction of DNA. The Sac7d molecules in the model form a unique structure with two left-handed helical ribbons winding around the outside of the right-handed duplex DNA.     

DNA bending,compaction and negative supercoiling by the architectural protein Sso7d of Sulfolobus solfataricus

Members of the Sso7d/Sac7d family are small, abundant, non-specific DNA-binding proteins of the hyperthermophilic Archaea Sulfolobus. Crystal structures of these proteins in complex with oligonucleotides showed that they induce changes in the helical twist and marked DNA bending. On this basis they have been suggested to play a role in organising chromatin structures in these prokaryotes, which lack histones. We report functional in vitro assays to investigate the effects of the observed Sso7d-induced structural modifications on DNA geometry and topology. We show that binding of multiple Sso7d molecules to short DNA fragments induces significant curvature and reduces the stiffness of the complex. Sso7d induces negative supercoiling of DNA molecules of any topology (relaxed, positively or negatively supercoiled) and in physiological conditions of temperature and template topology. Binding of Sso7d induces compaction of positively supercoiled and relaxed DNA molecules, but not of negatively supercoiled ones. Finally, Sso7d inhibits the positive supercoiling activity of the thermophile-specific enzyme reverse gyrase. The proposed biological relevance of these observations is that these proteins might model the behaviour of DNA in constrained chromatin environments.     

The Sso7d DNA-binding protein from Sulfolobus solfataricus has ribonuclease activity

Sso7d is a small, basic, abundant protein from the thermoacidophilic archaeon Sulfolobus solfataricus. Previous research has shown that Sso7d can bind double-stranded DNA without sequence specificity by placing its triple-stranded beta-sheet across the minor groove. We previously found RNase activity both in preparations of Sso7d purified from its natural source and in recombinant, purified protein expressed in Escherichia coli. This paper provides conclusive evidence that supports the assignment of RNase activity to Sso7d, shown by the total absence of activity in the single-point mutants E35L and K12L, despite the preservation of their overall structure under the assay conditions. In keeping with our observation that the residues putatively involved in RNase activity and those playing a role in DNA binding are located on different surfaces of the molecule, the activity was not impaired in the presence of DNA. If a small synthetic RNA was used as a substrate, Sso7d attacked both predicted double- and single-stranded RNA stretches, with no evident preference for specific sequences or individual bases. Apparently, the more readily attacked bonds were those intrinsically more unstable.     

Role of a surface tryptophan in defining the structure, stability, and DNA binding of the hyperthermophile protein Sac7d

Sac7d is a small, chromatin protein from Sulfolobus acidocaldarius which induces a sharp kink in DNA with intercalation of valine and methionine side chains. The crystal structure of the protein-DNA complex indicates that a surface tryptophan (W24) plays a key role in DNA binding by hydrogen bonding to the DNA at the kink site. We show here that substitution of the solvent-exposed tryptophan with alanine (W24A) led to a significant loss in not only DNA binding affinity but also protein stability. The W24A substitution proved to be one of the most destabilizing surface substitutions in Sac7d. A global linkage analysis of the pH and salt dependence of stability indicated that the protein stability surface (DeltaG vs temperature, pH, and salt concentration) was lowered overall by 2 kcal/mol (from 0 to 100 degrees C, pH 0 to 7, and 0 to 0.3 M KCl). The lower free energy of unfolding could not be attributed to significant structural perturbations of surface electrostatic interactions. Residual dipolar coupling of partially aligned protein and the NMR solution structure of W24A confirmed that the surface substitution resulted in no significant change in structure. Stabilization of this hyperthermophile protein and its DNA complex by a surface cluster of hydrophobic residues involving W24 and the two intercalating side chains is discussed.     

Probing the DNA kink structure induced by the hyperthermophilic chromosomal protein Sac7d

Sac7d, a small, abundant, sequence-general DNA-binding protein from the hyperthermophilic archaeon Sulfolobus acidocaldarius, causes a single-step sharp kink in DNA (~60°) via the intercalation of both Val26 and Met29. These two amino acids were systematically changed in size to probe their effects on DNA kinking. Eight crystal structures of five Sac7d mutant–DNA complexes have been analyzed. The DNA-binding pattern of the V26A and M29A single mutants is similar to that of the wild-type, whereas the V26A/M29A protein binds DNA without side chain intercalation, resulting in a smaller overall bending (~50°). The M29F mutant inserts the Phe29 side chain orthogonally to the C2pG3 step without stacking with base pairs, inducing a sharp kink (~80°). In the V26F/M29F-GCGATCGC complex, Phe26 intercalates deeply into DNA bases by stacking with the G3 base, whereas Phe29 is stacked on the G15 deoxyribose, in a way similar to those used by the TATA box-binding proteins. All mutants have reduced DNA-stabilizing ability, as indicated by their lower T_m values. The DNA kink patterns caused by different combinations of hydrophobic side chains may be relevant in understanding the manner by which other minor groove-binding proteins interact with DNA.     

Carboxyl pK(a) values, ion pairs, hydrogen bonding, and the pH-dependence of folding the hyperthermophile proteins Sac7d and Sso7d

Sac7d and Sso7d are homologous, hyperthermophile proteins with a high density of charged surface residues and potential ion pairs. To determine the relative importance of specific amino acid side-chains in defining the stability and function of these Archaeal chromatin proteins, pK(a) values were measured for the acidic residues in both proteins using (13)C NMR chemical shifts. The stability of Sso7d enabled titrations to pH 1 under low-salt conditions. Two aspartate residues in Sso7d (D16 and D35) and a single glutamate residue (G54) showed significantly perturbed pK(a) values in low salt, indicating that the observed pH-dependence of stability was primarily due to these three residues. The pH-dependence of backbone amide NMR resonances demonstrated that perturbation of all three pK(a) values was primarily the result of side-chain to backbone amide hydrogen bonds. Few of the significantly perturbed acidic pK(a) values in Sac7d and Sso7d could be attributed to primarily ion pair or electrostatic interactions. A smaller perturbation of E48 (E47 in Sac7d) was ascribed to an ion pair interaction that may be important in defining the DNA binding surface. The small number (three) of significantly altered pK(a) values was in good agreement with a linkage analysis of the temperature, pH, and salt-dependence of folding. The linkage of the ionization of two or more side-chains to protein folding led to apparent cooperativity in the pH-dependence of folding, although each group titrated independently with a Hill coefficient near unity. These results demonstrate that the acid pH-dependence of protein stability in these hyperthermophile proteins is due to independent titration of acidic residues with pK(a) values perturbed primarily by hydrogen bonding of the side-chain to the backbone. This work demonstrates the need for caution in using structural data alone to argue the importance of ion pairs in stabilizing hyperthermophile proteins.     

Engineering a thermostable protein with two DNA-binding domains using the hyperthermophile protein Sac7d

The acid- and thermostable Sac7d is a small, non-specific DNA-binding protein of the hyperthermophile archaea Sulfolobus acidocaldarius. In this study, Sac7d was employed as a structural unit in the design of a thermostable protein containing two putative DNA-binding domains. By linking two Sac7d proteins together and comparing the DNA interaction of dimer to that of monomer, this study may provide structural insights into other dimeric DNA-binding proteins. The engineered protein, Sac7dK66C, was over-expressed and purified. Dimeric Sac7d was obtained by cross-linking two mutant Sac7d molecules through the C-terminal disulfide bond. Thermal stability and DNA-binding ability of dimeric Sac7d were assessed and compared to those of wild type Sac7d by gel retardation assay, circular dichroism spectroscopy, and crystallization experiments. Dimeric Sac7d was shown to be equally thermostable as wild type, and its ability to stabilize DNA duplex is the same as wild type. However, the interaction of dimeric Sac7d with DNA diverged from that of wild type, suggesting different DNA-binding modes for dimeric Sac7d. In addition, a large difference in extinction coefficient was observed in all dimer/DNA CD spectra, which was reminiscent of the spectrum of Psi-DNA. Conjugation of various chemical groups to mutant Sac7d is possible through the C-terminal thiol group. This offers a possible approach in the design of a thermostable biomolecule with novel functions.     

Effect of mutation of the Sac7d intercalating residues on the temperature dependence of DNA distortion and binding thermodynamics

Sac7d is a small chromatin protein from the hyperthermophile Sulfolobus acidocaldarius which kinks duplex DNA by approximately 66 degrees at a single base pair step with intercalation of V26 and M29 side chains. Site-directed mutagenesis coupled with calorimetric and spectroscopic data has been used to characterize the influence of the intercalating side chains on the structure and thermodynamics of the DNA complex from 5 to 85 degrees C. Two single-alanine substitutions (V26A and M29A) and five double-glycine, -alanine, -leucine, -phenylalanine, and -tryptophan substitutions of the surface residues have been created. NMR and fluorescence titrations indicated that the substitutions had little effect on the structure of the protein or DNA binding site size. Each of the mutant proteins demonstrated a temperature-dependent binding enthalpy which was correlated with a similar temperature dependence in the structure of the complex reflected by changes in fluorescence and circular dichroism. A positive heat capacity change (DeltaC(p)) for DNA binding was observed for only those mutants which also demonstrated a thermotropic structural transition in the complex, and the temperature range for the positive DeltaC(p) coincided with that observed for the structural transition. The thermodynamic data are interpreted using a model in which binding is linked to an endothermic distortion of the DNA in the complex. The results support the proposal that the unfavorable enthalpy of binding of Sac7d at 25 degrees C is due in part to the distortion of DNA.     

In vitro DNA binding of the archaeal protein Sso7d induces negative supercoiling at temperatures typical for thermophilic growth.

The topological state of DNA in hyperthermophilic archaea appears to correspond to a linking excess in comparison with DNA in mesophilic organisms. Since DNA binding proteins often contribute to the control of DNA topology by affecting DNA geometry in the presence of DNA topoisomerases, we tested whether the histone-like protein Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus alters DNA conformation. In ligase-mediated supercoiling assays carried out at 37, 60, 70, 80 and 90 degrees C we found that DNA binding of increasing amounts of Sso7d led to a progressive decrease in plasmid linking number (Lk), producing negative supercoiling. Identical unwinding effects were observed when recombinant non-methylated Sso7d was used. For a given Sso7d concentration the DNA unwinding induced was augmented with increasing temperature. However, after correction for the overwinding effect of high temperature on DNA, plasmids ligated at 60-90 degrees C exhibited similar sigma values at the highest Sso7d concentrations assayed. These results suggest that Sso7d may play a compensatory role in vivo by counteracting the overwinding effect of high temperature on DNA. Additionally, Sso7d unwinding could be involved in the topological changes observed during thermal stress (heat and cold shock), playing an analogous role in crenarchaeal cells to that proposed for HU in bacteria.     

Engineering a Thermostable Protein with Two DNA-binding Domains Using the Hyperthermophile Protein Sac7d

Abstract

The acid- and thermostable Sac7d is a small, non-specific DNA-binding protein of the hyperthermophile archaea Sulfolobus acidocaldarius. In this study, Sac7d was employed as a structural unit in the design of a thermostable protein containing two putative DNA-binding domains. By linking two Sac7d proteins together and comparing the DNA interaction of dimer to that of monomer, this study may provide structural insights into other dimeric DNA-binding proteins. The engineered protein, Sac7dK66C, was over-expressed and purified. Dimeric Sac7d was obtained by cross-linking two mutant Sac7d molecules through the C-terminal disulfide bond. Thermal stability and DNA-binding ability of dimeric Sac7d were assessed and compared to those of wild type Sac7d by gel retardation assay, circular dichroism spectroscopy, and crystallization experiments. Dimeric Sac7d was shown to be equally thermostable as wild type, and its ability to stabilize DNA duplex is the same as wild type. However, the interaction of dimeric Sac7d with DNA diverged from that of wild type, suggesting different DNA-binding modes for dimeric Sac7d. In addition, a large difference in extinction coefficient was observed in all dimer/DNA CD spectra, which was reminiscent of the spectrum of ψ-DNA. Conjugation of various chemical groups to mutant Sac7d is possible through the C-terminal thiol group. This offers a possible approach in the design of a thermostable biomolecule with novel functions.     

Investigations of Sso7d catalytic residues by NMR titration shifts and electrostatic calculations

Sso7d is a small basic protein consisting of 62 amino acids isolated from the thermoacidophilic archeobacterium Sulfolobus solfataricus. The protein is endowed with DNA binding properties, RNase activity, and the capability of rescuing aggregated proteins in the presence of ATP. In this study, the electrostatic properties of Sso7d are investigated by using the Poisson-Boltzmann calculation of the surface potential distribution and following by NMR spectroscopy the proton chemical shift pH titration of acidic residues. Although the details of the catalytic mechanism still have to be defined, the results from NMR experiments confirm the possible involvement of Glu35 as the proton acceptor in the catalytic reaction, as seen by its abnormally high pK(a) value. Poisson-Boltzmann calculations and NMR titration shifts suggest the presence of a possible hydrogen bond between Glu35 and Tyr33, with a consequent rather rigid arrangement at these positions. Comparison with RNase T1 suggests that Tyr7 may be a good candidate for acting as a proton donor in the active site of Sso7d as shown by its low phenolic pK(a) of approximately 9.3. Titration experiments performed with the UpA, a RNA dinucleotide model, showed that the protein residues affected by the interaction are mainly located in a different region with respect to the surface affected by DNA recognition, in good agreement with the surface potential distribution found with electrostatic calculations.     

Guanidine-induced unfolding of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus

The unfolding induced by guanidine hydrochloride of the small protein Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus has been investigated by means of circular dichroism and fluorescence measurements. At neutral pH and room temperature the midpoint of the transition occurred at 4M guanidine hydrochloride. Thermodynamic information was obtained by means of both the linear extrapolation model and the denaturant binding model, in the assumption of a two-state N<==>D transition. A comparison with thermodynamic data determined from the thermal unfolding of Sso7d indicated that the denaturant binding model has to be preferred. Finally, it is shown that Sso7d is the most stable against both temperature and guanidine hydrochloride among a set of globular proteins possessing a very similar 3D structure.     

Thermodynamics of DNA binding and distortion by the hyperthermophile chromatin protein Sac7d

Sac7d is a hyperthermophile chromatin protein which binds non-specifically to the minor groove of duplex DNA and induces a sharp kink of 66 degrees with intercalation of valine and methionine side-chains. We have utilized the thermal stability of Sac7d and the lack of sequence specificity to define the thermodynamics of DNA binding over a wide temperature range. The binding affinity for poly(dGdC) was moderate at 25 degrees C (Ka = 3.5(+/-1.6) x 10(6) M(-1)) and increased by nearly an order of magnitude from 10 degrees C to 80 degrees C. The enthalpy of binding was unfavorable at 25 degrees C, and decreased linearly from 5 degrees C to 60 degrees C. A positive binding heat at 25 degrees C is attributed in part to the energy of distorting DNA, and ensures that the temperature of maximal binding affinity (75.1+/-5.6 degrees C) is near the growth temperature of Sulfolobus acidocaldarius. Truncation of the two intercalating residues to alanine led to a decreased ability to bend and unwind DNA at 25 degrees C with a small decrease in binding affinity. The energy gained from intercalation is slightly greater than the free energy penalty of bending duplex DNA. Surprisingly, reduced distortion from the double alanine substitution did not lead to a significant decrease in the heat of binding at 25 degrees C. In addition, an anomalous positive DeltaCp of binding was observed for the double alanine mutant protein which could not be explained by the change in polar and apolar accessible surface areas. Both the larger than expected binding enthalpy and the positive heat capacity can be explained by a temperature dependent structural transition in the protein-DNA complex with a Tm of 15-20 degrees C and a DeltaH of 15 kcal/mol. Data are discussed which indicate that the endothermic transition in the complex is consistent with DNA distortion.     

Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold

We have engineered pH sensitive binding proteins for the Fc portion of human immunoglobulin G (hIgG) (hFc) using two different strategies – histidine scanning and random mutagenesis. We obtained an hFc-binding protein, Sso7d-hFc, through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus; Sso7d-hFc was isolated from a combinatorial library of Sso7d mutants using yeast surface display. Subsequently, we identified a pH sensitive mutant, Sso7d-his-hFc, through systematic evaluation of Sso7d-hFc mutants containing single histidine substitutions. In parallel, we also developed a yeast display screening strategy to isolate a different pH sensitive hFc binder, Sso7d-ev-hFc, from a library of mutants obtained by random mutagenesis of a pool of hFc binders. In contrast to Sso7d-hFc, both Sso7d-his-hFc and Sso7d-ev-hFc have a higher binding affinity for hFc at pH 7.4 than at pH 4.5. The Sso7d-mutant hFc binders can be recombinantly expressed at high yield in E. coli and are monomeric in solution. They bind an epitope in the CH3 domain of hFc that has high sequence homology in all four hIgG isotypes (hIgG_1–4), and recognize hIgG_1–4 as well as deglycosylated hIgG in western blotting assays. pH sensitive hFc binders are attractive candidates for use in chromatography, to achieve elution of IgG under milder pH conditions. However, the surface density of immobilized hFc binders, as well as the avidity effect arising from the multivalent interaction of dimeric hFc with the capture surface, influences the pH dependence of dissociation from the capture surface. Therefore, further studies are needed to evaluate if the Sso7d mutants identified in this study are indeed useful as affinity ligands in chromatography.