Molecular Mechanism Of Sirt5-catalyzed Histone Desuccinylation And Structural And Functional Studies Of Staphylococcus Aureus Surface Protein SdrE

Post-translational modification(PTM)of histone proteins is an important method of epigenetic regulation.These modifications,including methylation,acetylation,phosphorylation and so on,are located mainly in flexible N-terminal region of histones.Histone modifications and the combinaiton of various modifications constitute the so-called "histone code" that contains specific biological information.Histone modifications can be sepecifically read out by their "reader" or"effector" molecules,triggering a series of downstream biological events to regulate various biological processes.At the same time,histone modifications are dynamicly regulated by their "wirters" and "erasers",which are enzeymes responsible for adding and removing the modifications,respectively.Histone acetylation is a kind of acylated modifications that has been studied in depth.In addition,with the development of mass spectrometry technology in recent years,some novel acylation modifications,such as propionylation,butyrylation,succinylation,malonylation,glutarylation,hydroxyisobutylation,β-hydroxybutyrylation,and crotonylation,have gradually been identified.Histone acylation is regulated by acyltransferases and deacylation enzymes.Sirtuins protein family,as an important class of deacylation enzymes,play an important part in the removal of histone acylation.Sirt5 is a memer of Sirtuin family which mainly localizes in mitochondria.Compared with Sirtl,2,3 and 6,Sirt5 exhibits relatively weak deacetylation activity,but can more efficiently remove negatively charged modifications,such as malonylation,glutarylation,and especially succinylation,from lysine residues.In the first part,we studied molecular mechanism of Sirt5-catalyzed histone desuccinylation.By using photo-crosslinking pull down and label-free mass spectrometry,we identified Sirt5 potentially bind to the H3K122 succinylated histone peptide(H3K122su).Further biochemical experiments showed that Sirt5 can catalyze the removal of succinyl group from H3K122su peptide.Additionally,H3K9su was also reported to be a substrate of Sirt5,indicating a hisotne desuccinylase activity of Sirt5.To investigate whether human Sirt5 is a ubiquitous desuccinylase for histone succinylation sites,13 histone succinyl peptides peptides corresponding to previously identified succinylation sites on histones were synthesized for enzymatic assays.Although H3K9su was not verified by MS/MS,since the structure of Sirt5 in complex with H3K9su peptide has been reported,we also synthesized the H3K9su peptide for our experiments.Enzymatic studies revealed that Sirt5 catalyzes the desuccinylation of all peptides except H4K31su,indicating that Sirt5 functions as a ubiquitous,sequence-selective desuccinylase against histone succinyl peptides.To investigate the mechanisms associated with substrate recognition and catalysis,we solved the crystal structures of human Sirt5 in complex with H3K122su,H2AK95su,H2BK120su,and H4K91su peptides,revealing that the main chain hydrogen bonds formed between Sirt5 and the succinyl lysine-2,+1,and +3 sites of the histone peptides are highly conserved among these structures.Additionally,enzymatic assays reveal that a proline residue at +1 site disrupts recognition of the histone succinyl substrate by affecting hydrogen bond formation.Our results illustrate the molecular basis underlying the sequence-selective histone-desuccinylase activity of Sirt5 and provided new insights into Sirt5 function.The second part is the structural and functional studies of Staphylococcus aureus surface protein SdrE.SdrE was previously reported to bind human complement factor H(FH)as an immune-evasion tactic.we identified the shortest region at FH’s C-terminus(FH1206-1226),which binds SdrE N2 and N3 domains(SdrEN2N3)with high affinity,and determined the crystal structures of apo-SdrEN2N3 and the SdrEN2N3-FH 1206-1226 complex.Structural analysis in combination with mutageneis studies reveal a novel ’close,dock,lock and latch’(CDLL)mechanism for SdrE to recognize its ligand.Our findings imply that SdrE functions as a ’clamp’ to capture FH’s C-terminal tail via a unique CDLL mechanism and sequesters FH on the surface of S.aureus for complement evasion.The third part is the structural and functional studies of S.aureus endonuclease RNase HII.RNase HII is capable of cleaving DNA-RNA hybrid strands and is involved in various biological processes such as RNA mismatching,removal of Okazaki fragments,nucleic acid metabolism,and DNA damage repair.We determined the crystal structure of S.aureus RNase HII(Sa-RNase HII),which displays a novel dimer conformation.Both small-angle X-ray scattering and gel-filtration analysis confirmed that Sa-RNase HII exists as a homodimer in solution.Enzymatic analysis revealed that the dimeric form is catalytically active,suggesting that its aggregation state has special physiological significance.