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.     

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.     

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.     

Crystal structures of the chromosomal proteins Sso7d/Sac7d bound to DNA containing T-G mismatched base-pairs

Sso7d and Sac7d are two small chromatin proteins from the hyperthermophilic archaeabacterium Sulfolobus solfataricus and Sulfolobus acidocaldarius, respectively. The crystal structures of Sso7d-GTGATCGC, Sac7d-GTGATCGC and Sac7d-GTGATCAC have been determined and refined at 1.45 A, 2.2 A and 2.2 A, respectively, to investigate the DNA binding property of Sso7d/Sac7d in the presence of a T-G mismatch base-pair. Detailed structural analysis revealed that the intercalation site includes the T-G mismatch base-pair and Sso7d/Sac7d bind to that mismatch base-pair in a manner similar to regular DNA. In the Sso7d-GTGATCGC complex, a new inter-strand hydrogen bond between T2O4 and C14N4 is formed and well-order bridging water molecules are found. The results suggest that the less stable DNA stacking site involving a T-G mismatch may be a preferred site for protein side-chain intercalation.     

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.     

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.     

The effects of loop size on Sac7d-hairpin DNA interactions

Hairpin structure is a common feature of DNA molecules. They are located near functional loci, such as regulation and promotion sites, as well as in cruciform structures, and they provide potential binding sites for endogenous proteins. The effects of different hairpin loops that are composed of one to five thymidines, designated as L1-L5, and have a common self-complementary stem, CTATATAG, on the interactions with Sac7d were studied. In thermostability studies, Sac7d stabilized a tetra-loop hairpin DNA and hairpin DNA with GTTC tetra-loop regions better than it stabilized tri- and penta-loops. Circular dichroism (CD) spectra showed that hairpins retained primarily a B-type conformation upon Sac7d binding. Intermolecular interactions between hairpins were likely decreased, due to the Sac7d-induced kinks, as shown by an increase at 220nm in the CD spectra. Surface plasmon resonance (SPR) observations suggested that the rates of Sac7d binding to hairpin DNA depend on the loop size of the hairpin duplexes. At a fixed stem length, Sac7d binds to tetra-loop hairpin DNA duplexes with a higher association rate and lower dissociation rate, compared with their tri- and penta-loop counterparts. In addition, the tri-loop and GTC tri-loop hairpin DNA had lower affinity for Sac7d because of the smaller and tighter loop size. Our study indicates that Sac7d binding affinity to hairpin DNA is primarily determined by loop size and stem integrity, and the results presented here provide a model for studies concerning other minor groove DNA-binding proteins that kink hairpin DNA.     

Mth10b, a unique member of the Sac10b family, does not bind nucleic acid

The Sac10b protein family is regarded as a group of nucleic acid-binding proteins that are highly conserved and widely distributed within archaea. All reported members of this family are basic proteins that exist as homodimers in solution and bind to DNA and/or RNA without apparent sequence specificity in vitro. Here, we reported a unique member of the family, Mth10b from Methanobacterium thermoautotrophicum ΔH, whose amino acid sequence shares high homology with other Sac10b family proteins. However, unlike those proteins, Mth10b is an acidic protein; its potential isoelectric point is only 4.56, which is inconsistent with the characteristics of a nucleic acid-binding protein. In this study, Mth10b was expressed in Escherichia coli and purified using a three-column chromatography purification procedure. Biochemical characterization indicated that Mth10b should be similar to typical Sac10b family proteins with respect to its secondary and tertiary structure and in its preferred oligomeric forms. However, an electrophoretic mobility shift analysis (EMSA) showed that neither DNA nor RNA bound to Mth10b in vitro, indicating that either Mth10b likely has a physiological function that is distinct from those of other Sac10b family members or nucleic acid-binding ability may not be a fundamental factor to the actual function of the Sac10b family.     

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.     

Solution structure, stability, and nucleic acid binding of the hyperthermophile protein Sso10b2

The Sso10b (or Alba) family of proteins is a conserved group of archaeal and eukaryotic proteins which are thought to play a role in both chromatin organization and RNA metabolism. We describe here the solution structure and properties of Sso10b2 from Sulfolobus solfataricus. NMR data including residual dipolar couplings and (15)N relaxation data demonstrated that the protein adopts a beta(1)alpha(1)beta(2)alpha(2)beta(3)beta(4) topology with an IF-3-like fold. The protein dimerizes in solution at 30 degrees C via a hydrophobic surface defined by the C-terminal alpha(2)beta(3)beta(4) elements with a structure similar to one of the putative dimers indicated by previous crystal structures. DSC and circular dichroism data demonstrated an unusual two-state structural transition near the growth temperature which led to an increase in beta-sheet content without dissociation of the dimer. The cooperativity of the transition exceeded that of a dimer at pH 7, demonstrating the presence of higher order oligomers near the growth temperature at pH 7. Reverse titrations of Sso10b2 with nucleic acid showed that the protein binds single-stranded DNA (K(d) of 3 x 10(-)(7) M) with higher affinity than RNA (1.3 x 10(-)(6) M) or double-stranded DNA (1.5 x 10(-)(5) M) in 10 mM KH(2)PO(4) (pH 7.0, 20 degrees C). NMR chemical shift perturbation data indicated that single-stranded DNA and RNA binding occurred across the same dimer interface and encompassed a surface defined by the C-terminal ends of the beta(1), beta(2), and beta(3) strands of each monomer.     

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.     

Dissociation of the tubulin dimer is extremely slow,thermodynamically very unfavorable,and reversible in the absence of an energy source

The finding that exchange of tubulin subunits between tubulin dimers (alpha-beta + alpha'beta' <--> alpha'beta + alphabeta') does not occur in the absence of protein cofactors and GTP hydrolysis conflicts with the assumption that pure tubulin dimer and monomer are in rapid equilibrium. This assumption underlies the many physical chemical measurements of the K(d) for dimer dissociation. To resolve this discrepancy we used surface plasmon resonance to determine the rate constant for dimer dissociation. The half-time for dissociation was approximately 9.6 h with tubulin-GTP, 2.4 h with tubulin-GDP, and 1.3 h in the absence of nucleotide. A Kd equal to 10(-11) M was calculated from the measured rate for dissociation and an estimated rate for association. Dimer dissociation was found to be reversible, and dimer formation does not require GTP hydrolysis or folding information from protein cofactors, because 0.2 microM tubulin-GDP incubated for 20 h was eluted as dimer when analyzed by size exclusion chromatography. Because 20 h corresponds to eight half-times for dissociation, only monomer would be present if dissociation were an irreversible reaction and if dimer formation required GTP or protein cofactors. Additional evidence for a 10(-11) M K(d) was obtained from gel exclusion chromatography studies of 0.02-2 nM tubulin-GDP. The slow dissociation of the tubulin dimer suggests that protein tubulin cofactors function to catalyze dimer dissociation, rather than dimer assembly. Assuming N-site-GTP dissociation is from monomer, our results agree with the 16-h half-time for N-site GTP in vitro and 33 h half-life for tubulin N-site-GTP in CHO cells.     

Affinity chromatography and properties of cGMP-dependent protein kinase from prawn tissues

Using affinity chromatography on 8-(2-aminoethyl)-amino-cAMP Sepharose, the cGMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from tissues of the prawn Palaemon adspersus was purified to homogeneity as demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The degree of enzyme purification was 11 200, recovery--6.5%; the isoelectric point for the enzyme lies at 5.5. Data from gel filtration and centrifugation in sucrose density gradient suggest that the dimer of cGMP-dependent protein kinase has a molecular weight of 157 000, sedimentation coefficient of 7.2S and a Stokes' radius of 50 A. An active form of the enzyme with Mr = 76 500 (4.5S, 39 A) which apparently represents a subunit of the cGMP-dependent protein kinase was discovered. The activity of the both enzyme forms are stimulated by low concentrations of cGMP (Ka = 1.10(-7) M). The monomer and dimer molecules appear as prolate ellipsoids with axial ratios close to 7. The native cGMP-dependent protein kinase is probably made up of two subunits each of which contains a regulatory and a catalytic sites.     

Structural insights into the interaction of the crenarchaeal chromatin protein Cren7 with DNA

Cren7, a newly found chromatin protein, is highly conserved in the Crenarchaeota. The protein shows higher affinity for double‐stranded DNA than for single‐stranded DNA, constrains negative DNA supercoils in vitro and is associated with genomic DNA in vivo. Here we report the crystal structures of the Cren7 protein from Sulfolobus solfataricus in complex with two DNA sequences. Cren7 binds in the minor groove of DNA and causes a single‐step sharp kink in DNA (～53°) through the intercalation of the hydrophobic side chain of Leu28. Loop β3‐β4 of Cren7 undergoes a significant conformational change upon binding of the protein to DNA, suggesting its critical role in the stabilization of the protein–DNA complex. The roles of DNA‐contacting amino acid residues in stabilizing the Cren7–DNA interaction were examined by mutational analysis. Structural comparison of Cren7‐DNA complexes with Sac7d‐DNA complexes reveals significant differences between the two proteins in DNA binding surface, suggesting that Cren7 and Sul7d serve distinct functions in chromosomal organization.     

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.     

Restriction fragment fingerprint and genome sizes of Staphylococcus species using pulsed-field gel electrophoresis and infrequent cleaving enzymes.

Large restriction fragments of genomic DNA from Staphylococcus species were separated by pulsed-field gel electrophoresis (PFGE). Five different strains of S. aureus (ISP8, SAU3A, PS96, ATCC 6538, ATCC 15564) and three representative strains of S. haemolyticus SM102, S. warneri MCS4, S. cohnii LK478 from human hosts, and one strain of S. aureus (ATCC 8432) from an avian host were used in this study. Since Staphylococcus is A + T rich (approximately 67%), restriction fragments were obtained by digesting chromosomal DNA with endonucleases that recognize GC-rich sequences. Five enzymes Csp I, Sma I, Ecl XI, Ksp I, or Sac II were used for generation of few (7 to 16) distinctly separated fragments, with average sizes in the range of 200-300 kb. The size distribution of restriction fragments for each enzyme for each strain produced a strain-identifying fingerprint, and the genome size of each strain was determined from such restriction fragments separated by PFGE.     

Ssh10b, a conserved thermophilic archaeal protein, binds RNA in vivo

Proteins of the Sac10b family, which is highly conserved among hyperthermophilic archaea, have been regarded as DNA-binding proteins. Based on their in vitro DNA-binding properties, these proteins are thought to be involved in chromosomal organization or DNA repair/recombination. We show that Ssh10b, a member of the Sac10b family from Sulfolobus shibatae, bound with similar affinities to double-stranded DNA, single-stranded DNA and RNA in vitro. However, the protein was exclusively bound to RNA in S. shibatae cells, as revealed by in vivo UV cross-linking and co-immunoprecipitation. Ribosomal RNAs were among the RNA species co-immunoprecipitated with Ssh10b. Consistent with this observation, Ssh10b was co-purified with ribosomes under low salt conditions. Furthermore, we demonstrate by UV-cross-linking hybridization that, when the cells were irradiated with UV, Ssh10b became cross-linked to 16S, 23S rRNAs and mRNAs. Our data indicate that RNA is the physiological binding target of the Sac10b family.     

Purification and subunit structure of hepatocyte growth factor from rat platelets

A hepatocyte growth factor (HGF) that stimulates DNA synthesis of adult rat hepatocytes in primary culture was purified as a homogeneous material from platelets of 1000 rats by a four-step procedure: stimulation of its release from platelets by thrombin, cation-exchanger fast protein liquid chromatography (FPLC) on a Mono S column, heparin-Sepharose CL-6B chromatography, and reverse-phase HPLC on a C4 column. The purified HGF stimulated DNA synthesis of adult rat hepatocytes in primary culture at 1 ng/ml and was maximally effective at 5 ng/ml, being about twice as potent as EGF at this concentration. HGF did not stimulate DNA synthesis of Swiss 3T3 cells. It was found to be a heat- and acid-labile protein that was inactivated by reduction with dithiothreitol. The purified HGF had a molecular mass of 82 kDa, as estimated by SDS-PAGE, and was found to be a heterodimer which dissociated into a large subunit of 69 kDa and a small one of 34 kDa by SDS-PAGE under reducing conditions. These biological and chemical properties showed that HGF was not identical with any known growth factors, including platelet-derived growth factor (PDGF).