Biochemical Characterization And Functional Analysis Of PINA And Its Methylation In Hyperthermophilic Archaea

In our study,we discovered and identified a novel ATPase from Sulfolobus islandicus REY15A,named SisPINA,which can drive Holliday junction DNA?HJ?migration in vitro,and interact physically and functionally with HJ-specific endonuclease Hjc and coordinate HJ processing.Structural analysis indicated that ATP binding and hydrolysis causes a conformational change in SisPINA and promoted HJ branch migration.However,it is not clear how SisPINA works synergistically with other proteins in HJ processing and its specific process of homologous recombination repair in archaea.Therefore,more biochemical,structural and functional studies are needed to fully understand the nature of protein and reveal the mechanism of homologous recombination repair in archaea.In order to further reveal the function of SisPINA,we first tested protein-protein interaction and found that SisPINA and SisHjm have strong physical interaction by gel filtration and pull-down analysis.At the same time,the results showed that the carboxy terminal II-KH domain of SisPINA plays an important role in the interaction with other proteins,and the fifth domain of SisHjm is not the site of its interaction with SisPINA.There have been many reports on the methylation modification of archaeal proteins,mainly lysine methylation modification,for example,ferredoxin in Sulfolobus acidocaldarius,glutamate dehydrogenase in S.solfataricus,?-Glycosidases,ribosomal protein L11,aspartate aminotransferase and RFC large and small subunits.Cren7 and Sso7d in S.sulphate have different degrees of methylation modification.Methylation modification is widespread in archaea,suggesting that methylation modification is important in archaea.We sought to identify post-translational modifications of SisPINA in cells.The results of mass spectrometry showed that SisPINA was phosphorylated and methylated in the cells under natural physiological conditions.Methylation mainly occurs at the carboxy-terminal lysine?K474,K479,K498,and K500?,and it was previously demonstrated that the carboxy terminus is involved in the interaction between proteins.We tested whether the modification of this region regulates the interaction with other proteins and other features.The growth curve of the lysine site mutant strain showed that the methylation modification did not affect the growth rate of the protein.We found that the methylation modification of SisPINA protein did not affect the thermal stability of the protein by differential scanning calorimetry?DSC?experiments.Quantitative determination of the binding of the simulated methylated SisPINA protein to other proteins by isothermal titration calorimetry?ITC?revealed that the titration curve was very flat,possibly due to low protein concentration,but when we increased the protein concentration,we found the protein precipitation will occur,so we need other methods to quantify the binding between proteins.Mass spectrometry identified in vitro methylation found that methyltransferase aKMT protein can methylate SisPINA protein,but when in vitro interaction was examined with pull-down and gel filtration analysis,it was found that the two proteins did not interact with each other.It may be that the interaction occurs only in vivo.Using the pull-down assay to detect the interaction between SisPINA wild-type protein and methylation mimic protein and Hjm?RFCs?,we found that SisPINA methylation mimic protein loses its interaction w-ith RFCs and Hjm compared to wild-type SisPINA protein.The primary results indicated that methylation modification inhibits the interaction between proteins.Then we mutate these four lysine sites to alanine.Pull-down experiments showed that SisPINA?K474AK479AK498AK500A?and Hjm?RFCs?also did not interact with each other,indicating that these four lysine sites are important sites for protein-protein interactions.In order to further determine the exact interaction sites,we purified the lysine single mutant proteins separately and verified by pull-down experiments.We revealed that single mutants did not affect protein-protein interactions,suggesting that four lysine sites synergistically affect protein-protein interactions.Whether the methylation modification is essential for the function of SisPINA in the cell,and the effect of methylation modification on the intracellular function of SisPINA requires further study.When we analyzed the sequence of SisPINA homolog in the KOD1 genome of Thermococcus kodakaraensis,we found that the sequence encoding PINA protein in T.koda.karaensis is located near the CRISPR loci,and the CRISPR sequence can form HJ structure.Therefore,we guessed TkoPINA?Tko₀953?may be involved in CRISPR related functions.It was found that TkoPINA has strong binding activity to CRISPR sequences,but also has strong binding activity to single-stranded DNA?double-stranded DNA and HJ.Next,we examined interaction between TkoPINA and the neighboring proteins and found that TkoPINA protein has strong interaction with GTPase?Tko₀951?,but its effect on TkoPINA function is unknown.TkoPINA has showed no interaction with the site-determination protein MinD?Tko₀952?and the membrane formation inhibitory protein Maf?Tko₀954??both related to cell division?.In vitro biochemical experiments reavead that MinD has endonuclease activity,and the endonuclease activity of MinD was inhibited by Tko_PINA.The mechanism of this inhibition is not known.