Structural And Functional Insights Into Type ? Secretion System Effector-Immunity Proteins From Pseudomonas Aeruginosa And Wss1 From Saccharomyces Cerevisiae

This dissertation contains two parts:Structural and Functional insights into type VI secretion system Effector-Immunity proteins from Pseudomonas aeruginosa and Wssl from Saccharomyces cerevisiae.Pseudomonas aeruginosa has antibacterial activity and pathogenicity,It is equipped with a powerful secretory system.Type VI secretion system(T6SS)play an important role in it.Effectors are the protein secreted by T6SS.It ensures that the bacteria can interact with other bacteria and environment or Eukaryotic host.Meanwhile,T6SS can express immunity proteins to protect itself or sister cells from toxicity.It has been an attractive topic to investigate the structural and virulence of T6SS effector and its related immunity protein.This dissertation chose the PldA(Tle5)and PldB(TIe5B)and their respective immunity protein from Pseudomonas aeruginosa,PldA and PldB are two phospholipase effector protein secreted by T6SS of Pseudomonas aeruginosa.They can facilitate the bacterial internalization process into human epithelial cells.PA3488(Tli5)and PA5086-PA5088 are cognate immunity proteins of PldA and PldB respectively.They can interact with their cognate effector proteins to suppress their virulence.We carried out the research of their structure and their antagonistic mechanism of their respective immunity.Finally,we obtained the structure of PA3488 and PA5088,PA5087.The structure of PA3888 takes the form of ctab claw,we successfully fit the structure of PA3488 into the Small angle X-ray scattering(SAXS)model of the complete PldA-PA3488 complex.We identified two important motifs in PA3488 through sequence and structural analysis.One is a conserved loop-(3-hairpin motif that exists in the PldA immunity homologs,the other is long and sharp a-a that directly interacts with PldA according to the SAXS data.PA5088 and PA5087 form super-ring folds.The divergence electrostatic potential surfaces in the PA5088 and PA5087 may be the key factor resistance against toxins from different species.Our docking experiments have shed light on the putitive interaction mechanism of their function as phospholipase D inhibitors.The cluster of aromic residues in PA5088,PA5087 and PA5086 may interact with these key residues of Streptomyces PLD through aromatic stacking.Furthermore,Our work provided insights into a novel inhibition mechanism that may inhance our understanding of phospholipase D family proteins.In living creature,DPCs originate from either the endogenic factors or from exogenous factors,DPCs are particularly toxic because they interfere with essential DNA transactions.For example,DPCs inhibit unwinding of the DNA duplex by replicative helicases,thus prohibiting replication completion and consequently cell division.Wssl is a metallprotease which can degrade the protein part of DPCs.As a result,Wssl from Saccharomyces cerevisiae was chosen for structural investigation in order to be more informative to explain how the structure determines the function.In our experiment,we purified the ScWss1 and its active site mutant ScWss1116E-Q and ScWss1-DNA complex,all used for crystallization.Finally,we obtained the truncate structure of ScWss1 and ScWss1 116E-Q(ScWss121-148 and ScWss117-151 116E-Q).Therefore,the full-length protein structure was futher studied by SAXS.The most conserved part of the Wss1 family protein is the protease domain,which is responsible for degradation of the component of DPCs.The protease domain contains a short Zinc-binding consensus sequence HEXXH(X donates any residue)at the catalytic center,and it was proved to be the most structurally conserved part of the protein.We also distinguished the structure of SpWssl from ScWssl,we speculate the highly unconservative electronic surface distribution together with the absence of an obvious substrate-binding cleft could explain the promiscuity of Wssl protease with respect to the substrate identity.Finally,another intriguing feature drawn from the crystal structure of the proabylysin protein which has been proved lacking of hydrolytic.Combined all these structures and SAXS model,we speculated a mechanism of how the Sc Wssl enzymatic modulate " on-off " switch based on DNA.When ScWssl is free of DNA,some part at the C-terminal of the protein interact with the zinc ion and blockes the catalytic center to contact other substrates,while ScWss1 is in combination with DNA,the flexible region linking the protease domain and the C-terminal part of the protein may be deformed and the obstacle at the catalytic center can be removed,thus the enzyme is activated to be capable of acntact and hydrolyse protein substrates.