Exploring the influence of hyperthermophilic protein Ssh10b on the stability and conformation of RNA by molecular dynamics simulation

The hyperthermophilic Ssh10b from Sulfolobus shibatae is a member of the Sac10b family, which binds RNA in vivo as a physiological substrate, and it has been postulated to play a key role in chromosomal organization in Archaea. Even though the crystal structure of Ssh10b‐RNA was resolved successively by X‐ray diffraction (Protein Data Bank [PDB] code: 3WBM), the detailed dynamic characteristics of Ssh10b‐RNA are still unclear. In this study, molecular dynamics (MDs) simulations at 6 temperatures (300, 350, 375, 400, 450, and 500 K) and molecular mechanics Generalized‐Born surface area (MM‐GB/SA) free energy calculations were performed to investigate the mechanism of how Ssh10b protects and stabilizes RNA. The simulation results indicate that RNA is stabilized by Ssh10b when the temperature rises up to 375 K. RNA is found to undergo conformational transition between A‐RNA and A′‐RNA when Ssh10b binds to RNA at 3 different temperatures (300, 350, and 375 K). Salt bridges, hydrogen bonds and hydrophobic interactions are observed, and some residues have significant impact on the structural stability of the complex. This study increases our understanding of the dynamics and interaction mechanism of hyperthermophilic proteins and RNA at the atomic level, and offers a model for studying the structural biology of hyperthermophilic proteins and RNA.     

Cloning, expression and purification of DNA-binding protein Mvo10b from Methanococcus voltae

Mvo10b from the mesophilic archaeon Methanococcus voltae is a member of the Sac10b family which may play an important role in the organization and accessibility of genetic information in Archaea. Since Mvo10b is a DNA-binding protein as the other member in the Sac10b family, to obtain a recombinant Mvo10b requires an efficient and inexpensive expression and purification system for producing the protein free of nucleic acid contamination. Previously, the hyperthermophilic archaeal Ssh10b of the Sac10b family was successfully purified. However, the protocol adopted to purify Ssh10b is not appropriate for purifying the mesophilic Mvo10b. This study describes the successful expression and purification of the recombinant Mvo10b. The expression of recombinant Mvo10b was carried out in Escherichia coli, and the target protein was expressed in the soluble form. The protein was purified by polyethyleneimine (PEI) precipitation followed by nickel ion metal affinity chromatography. The purity of Mvo10b was checked to insure being free of nucleic acid contamination. The final protein yield is about 30 mg/l of LB culture. The ensemble of NMR and far-UV CD data shows that the purified Mvo10b has abundant regular secondary structures and is correctly folded, which may have similar 3D structure as its hyperthermophilic counterpart [P62A]Ssh10b. The developed protocol has potential application in the production of the other thermophilic and mesophilic proteins in the Sac10b family.     

Ssh10b2与其同源蛋白Ssh10b在细胞含量及固定DNA超螺旋的能力上存在明显差异

极端嗜热古菌———芝田硫化叶菌(Sulfolobus shibatae)基因组含一对亲缘关系较远的同源基因,ssh10b和ssh10b2。这对同源基因编码的蛋白(Ssh10b和Ssh10b2)属于古菌Sac10b DNA结合蛋白家族。关于Ssh10b以及与其极为相似的硫矿硫化叶菌(S.solfataricus)Sso10b、嗜酸热硫化叶菌(S.acidocaldarius)Sac10b蛋白已有较多研究,推测这些蛋白可能在染色体组织和包装、DNA重组、基因表达调控等方面起作用。克隆并在大肠杆菌中表达了ssh10b2基因,纯化了重组Ssh10b2蛋白。免疫印迹定量分析表明,ssh10b2在芝田硫化叶菌中有表达,但其细胞含量仅相当于Ssh10b的约十分之一。重组Ssh10b2对双链DNA的亲和力低于Ssh10b。此外,Ssh10b2和Ssh10b在与双链DNA结合时表现出相似的凝胶阻滞模式。有意思的是,Ssh10b2固定DNA负超螺旋的能力明显低于Ssh10b。这些结果提示,Ssh10b和Ssh10b2可能具有不同的生理作用。     

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.     

Molecular mechanism underlying the interaction of typical Sac10b family proteins with DNA

The Sac10b protein family is regarded as a family of DNA-binding proteins that is highly conserved and widely distributed within the archaea. Sac10b family members are typically small basic dimeric proteins that bind to DNA with cooperativity and no sequence specificity and are capable of constraining DNA negative supercoils, protecting DNA from Dnase I digestion, and do not compact DNA obviously. However, a detailed understanding of the structural basis of the interaction of Sac10b family proteins with DNA is still lacking. Here, we determined the crystal structure of Mth10b, an atypical member of the Sac10b family from Methanobacterium thermoautotrophicum ΔH, at 2.2 Å. Unlike typical Sac10b family proteins, Mth10b is an acidic protein and binds to neither DNA nor RNA. The overall structure of Mth10b displays high similarity to its homologs, but three pairs of conserved positively charged residues located at the presumed DNA-binding surface are substituted by non-charged residues in Mth10b. Through amino acids interchanges, the DNA-binding ability of Mth10b was restored successfully, whereas the DNA-binding ability of Sso10b, a typical Sac10b family member, was weakened greatly. Based on these results, we propose a model describing the molecular mechanism underlying the interactions of typical Sac10b family proteins with DNA that explains all the characteristics of the interactions between typical Sac10b family members and DNA.     

Refolding of the hyperthermophilic protein Ssh10b involves a kinetic dimeric intermediate

The α/β-mixed dimeric protein Ssh10b from the hyperthermophile Sulfolobus shibatae is a member of the Sac10b family that is thought to be involved in chromosomal organization or DNA repair/recombination. The equilibrium unfolding/refolding of Ssh10b induced by denaturants and heat was fully reversible, suggesting that Ssh10b could serve as a good model for folding/unfolding studies of protein dimers. Here, we investigate the folding/unfolding kinetics of Ssh10b in detail by stopped-flow circular dichroism (SF-CD) and using GdnHCl as denaturant. In unfolding reactions, the native Ssh10b turned rapidly into fully unfolded monomers within the stopped-flow dead time with no detectable kinetic intermediate, agreeing well with the results of equilibrium unfolding experiments. In refolding reactions, two unfolded monomers associate in the burst phase to form a dimeric intermediate that undergoes a further, slower, first-order folding process to form the native dimer. Our results demonstrate that the dimerization is essential for maintaining the native tertiary interactions of the protein Ssh10b. In addition, folding mechanisms of Ssh10b and several other α/β-mixed or pure β-sheet proteins are compared.     

Ssh10b2 differs from its paralogue Ssh10b in cellular abundance and the ability to constrain DNA supercoils]

The ssh10b and ssh10b2 genes, a pair of distantly related paralogues in Sulfolobus shibatae, encode members of the Sac10b DNA binding protein family in thermophilic archaea. It has been shown previously that Ssh10b exists in abundance in S. shibatae and is capable of constraining negative DNA supercoils, properties that are consistent with a speculated architectural role for the protein in chromosomal organization. In this study, the ssh10b2 gene was cloned and expressed in Escherichia coli, and the recombinant Ssh10b2 protein was purified to apparent homogeneity. Immunoblotting analysis using a specific anti - Ssh10b2 antibody showed that ssh10b2 was expressed in S. shibatae, but the cellular level of Ssh10b2 was only - 10% of that of Ssh10b. Recombinant Ssh10b2 was capable of interacting with both double-stranded and single-stranded DNA. The affinity of the protein for double-stranded DNA was higher than that reported for Ssh10b. The Ssh10b2 and Ssh10b proteins appeared to generate similar gel shift patterns on duplex DNA fragments. However, unlike Ssh10b, Ssh10b2 was unable to constrain DNA supercoils. These data suggest that Ssh10b2 does not serve as a general architectural factor in DNA compaction and organization in S. shibatae.     

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.     

Two conformations of archaeal Ssh10b. The origin of its temperature-dependent interaction with DNA

The DNA-binding protein Ssh10b from the hyperthermophilic archaeon Sulfolobus shibatae is a member of the Sac10b family, which has been speculated to be involved in the organization of the chromosomal DNA in Archaea. Ssh10b affects the DNA topology in a temperature dependent fashion that has not been reported for any other DNA-binding proteins. Heteronuclear NMR and site-directed mutagenesis were used to analyze the structural basis of the temperature-dependent Ssh10b-DNA interaction. The data analysis indicates that two forms of Ssh10b homodimers co-exist in solution, and the slow cis-trans isomerization of the Leu61-Pro62 peptide bond is the key factor responsible for the conformational heterogeneity of the Ssh10b homodimer. The T-form dimer, with the Leu61-Pro62 bond in the trans conformation, dominates at higher temperature, whereas population of the C-form dimer, with the bond in the cis conformation, increases on decreasing the temperature. The two forms of the Ssh10b dimer show the same DNA binding site but have different conformational features that are responsible for the temperature-dependent nature of the Ssh10b-DNA interaction.     

A stabilizing alpha/beta-hydrophobic core greatly contributes to hyperthermostability of archaeal [P62A]Ssh10b

The hyperthermophilic Ssh10b from Sulfolobus shibatae is a member of the Sac10b family, which has been postulated to play a role in chromosomal organization in Archaea. Ssh10b is capable of significantly constraining negative DNA supercoils at elevated temperatures. In this study, the solution structure of the dimeric P62A mutant Ssh10b ([P62A]Ssh10b) was determined by multidimensional NMR spectroscopy. The backbone 15N dynamics, H/D exchange with and without the denaturant GdmSCN, and chemical and thermal denaturation experiments were performed to investigate the molecular basis of high thermostability of [P62A]Ssh10b. Data analysis has revealed an alpha/beta-hydrophobic core consisting of two alpha-helices and one beta-sheet which are stabilized by cooperative hydrophobic and hydrogen-bonding interactions. This stabilizing alpha/beta-hydrophobic core of [P62A]Ssh10b exhibiting highly restricted internal motions is composed of residues having highly protected amide protons which exchange with solvent mostly by means of a global unfolding process. The K40N mutation greatly destabilizes the mutant [P62A]Ssh10b because this mutation disturbs the packing of alpha-helix against the beta-sheet reducing the stability of the alpha/beta-hydrophobic core in the mutant protein. In comparison with homologous mesophilic and thermophilic proteins, it can be presumed that the stabilizing alpha/beta-hydrophobic core in the [P62A]Ssh10b structure greatly contributes to the high thermostability of the protein.     

An abundant DNA binding protein from the hyperthermophilic archaeon Sulfolobus shibatae affects DNA supercoiling in a temperature-dependent fashion

The DNA binding protein Ssh10b, a member of the Sac10b family, has been purified from the hyperthermophilic archaeon Sulfolobus shibatae. Ssh10b constitutes about 4% of the cellular protein. Electrophoretic mobility shift assays showed that Ssh10b first bound a double-stranded DNA fragment with an estimated binding size of approximately approximately 12 bp, forming distinct shifts, until the DNA was coated with the protein. Binding of more Ssh10b resulted in the formation of smears of lower mobilities. The migration pattern of the smearing Ssh10b-DNA complexes was affected by temperature, whereas that of complexes associated with the distinct shifts was not. Interestingly, Ssh10b was capable of constraining negative DNA supercoils in a temperature-dependent fashion. While the ability of the protein to constrain supercoils was weak at 25 degrees C, it was enhanced substantially at 45 degrees C or higher temperatures (up to 80 degrees C). Taken together, our data suggest that archaeal proteins of the Sac10b family may affect the topology of chromosomal DNA in thermophilic archaea at their growth temperatures.     

极端嗜热古菌———芝田硫化叶菌DNA 连接酶的生化性质

极端嗜热古菌———芝田硫化叶菌 DNA 连接酶 (Ssh 连接酶 ) 的最适辅因子为 ATP ,在 dATP 存在时,该酶也能表现出较弱的连接活性 . ATP 或 dATP 都能够使该酶发生腺苷化,腺苷化的 Ssh 连接酶能够将腺苷基团转移至含切刻的 DNA 上 . 电泳迁移率改变实验表明, Ssh 连接酶能够结合双链 DNA ,且与含切刻及不含切刻的 DNA 结合的亲和力相同,但不结合单链 DNA. 酵母双杂交实验显示,硫磺矿硫化叶菌 ( 与芝田硫化叶菌亲缘关系很近 ) 的 DNA 连接酶,与该菌所含的 3 个增殖细胞核抗原 (PCNA) 同源蛋白中的一个 (PCNA-1) 有相互作用,而与另外 2 个同源蛋白 (PCNA-like 和 PCNA-2) 则无相互作用 . 在古菌中高度保守的 Sac10b 蛋白家族成员 Ssh10b 能够激活 Ssh 连接酶的活性,而硫化叶菌中的主要染色体蛋白——— 7 ku DNA 结合蛋白 (Ssh7) 则对该酶活性没有影响 .     

Favorable contribution of the C-terminal residue K97 to the stability of a hyperthermophilic archaeal [P62A]Ssh10b

The role of residue K97 at the C-terminal end of archaeal [P62A]Ssh10b in the hyperthermostability of the protein is investigated using three K97-mutant variants: K97E-, K97A-, and ΔK97-mutant [P62A]Ssh10b. The thermal- and GdmHCl-induced denaturation of the three mutant variants has been monitored by circular dichroism. The results reveal that the K97E mutation leads to a stronger destabilization effect than the K97A mutation by disturbing the electrostatic interaction of the salt-bridge D63-K97 and drawing an unfavorable charge-charge repulsive interaction into the structure. However, ΔK97-[P62A]Ssh10b shows much lower stability than K97E- and K97A-mutant [P62A]Ssh10b. Analysis suggests that residue K97 at the C-terminal end makes the favorable contributions to the stability of hyperthermophilic [P62A]Ssh10b not only by the favorable electrostatic interactions with residues in close vicinity but also through maintaining the side chain packing of the surrounding residues in the C-terminal area of the protein.     

Crystal structure of archaeal chromatin protein Alba2-double-stranded DNA complex from Aeropyrum pernix K1

All thermophilic and hyperthermophilic archaea encode homologs of dimeric Alba (Sac10b) proteins that bind cooperatively at high density to DNA. Here, we report the 2.0 Å resolution crystal structure of an Alba2 (Ape10b2)-dsDNA complex from Aeropyrum pernix K1. A rectangular tube-like structure encompassing duplex DNA reveals the positively charged residues in the monomer-monomer interface of each dimer packing on either side of the bound dsDNA in successive minor grooves. The extended hairpin loop connecting strands β3 and β4 undergoes significant conformational changes upon DNA binding to accommodate the other Alba2 dimer during oligomerization. Mutational analysis of key interacting residues confirmed the specificity of Alba2-dsDNA interactions.     

Crystal structure of an archaeal specific DNA-binding protein (Ape10b2) from Aeropyrum pernix K1

DNA binding proteins are essential in all organisms, and they play important roles in both compacting and regulating the genetic material. All thermophilic and hyperthermophilic archaea encode one or more copies of Alba or Sso10b, which is a small, abundant, basic protein that binds DNA. Here, we present the crystal structure of Ape10b2 from Aeropyrum pernix K1 at 1.70 A. Although the overall structure resembles the known Alba protein fold, a significant conformational change was observed in the loop regions. Specifically, the L5 loop is slightly longer, as compared to those of other known proteins, and the flexibility of this loop may facilitate the interaction with double stranded DNA. In addition, we showed that Ape10b2 binds to 16 and 39 bp duplex DNAs with high affinity. On the basis of our analyses, we have created a putative protein-DNA complex model.     

Evolutionary conservation and DNA binding properties of the Ssh7 proteins from Sulfolobus shibatae

The thermoacidophilic archaeon Sulfolobus shibatae synthesizes a large amount of the 7-ku DMA binding proteins known as Ssh7. Our hybridization experiments showed that two Ssh7-encoding genes existed in the genome of S. shibatae. These two genes, designated ssh7a and ssh7b, have been cloned, sequenced and expressed in Escherichia coli. The two Ssh7 proteins differ only at three amino acid positions. In addition, the cis-regulatory sequences of the ssh7a and ssh7b genes are highly conserved. These results suggest the presence of a selective pressure to maintain not only the sequence but also the expression of the two genes. We have also found that there are two genes encoding the 7-ku protein in Sulfolobus solfataricus. Based on this and other studies, we suggest that the gene encoding the 7-ku protein underwent duplication before the separation of Sulfolobus species. Binding of native Ssh7 and recombinant (r)Ssh7 to short duplex DNA fragments was analyzed by electrophoretic mobility shift assays. Both n     

Evolutionary conservation and DNA binding properties of the Ssh7 proteins fromSulfolobus shibatae

The thermoacidophilic archaeonSulfolobus shibatae synthesizes a large amount of the 7-ku DNA binding proteins known as Ssh7. Our hybridization experiments showed that two Ssh7-encoding genes existed in the genome of S.shibatae. These two genes, designatedssh7a andssh7b, have been cloned, sequenced and expressed inEscherichia coli. The two Ssh7 proteins differ only at three amino acid positions. In addition, thecis-regulatory sequences of thessh7a andssh7b genes are highly conserved. These results suggest the presence of a selective pressure to maintain not only the sequence but also the expression of the two genes. We have also found that there are two genes encoding the 7-ku protein inSulfolobus solfataricus. Based on this and other studies, we suggest that the gene encoding the 7-ku protein underwent duplication before the separation ofSulfolobus species. Binding of native Ssh7 and recombinant (r)Ssh7 to short duplex DNA fragments was analyzed by electrophoretic mobility shift assays. Both native and recombinant forms of the protein behaved in a similar fashion in the assays, suggesting that the interaction of Ssh7 with DNA is not affected either by specific lysine methylation found in the native Ssh7 proteins or by the difference between the two Ssh7 isomers in amino acid sequence. Our data show that Ssh7 binds duplex DNA fragments with a binding size of ∼ 6.6 base pairs and an apparent dissociation constant of (0.7–1.0) × 10^-7 mol/L under the assay conditions employed in the present study. In addition, Ssh7 binds more tightly to negatively supercoiled DNA than to linear or relaxed DNA.