Studies On The Structure And Function Of Key Proteins In Regulated Intramembrane Proteolysis(RIP) Pathway Of Pseudomonas Aeruginosa

Extracellular functionσfactor(σ^ECF)Alg U in P.aeruginosa is a key transcriptional element for stress signal transduction and amplification in envelope stress responses（ESRs）system.The activation of Alg U is achieved by the step-by-step degradation of Muc A by proteases AlgW,Muc P and Clp XP in regulated intramembrane proteolysis（RIP）pathway.Muc A and Muc B are critical negative modulators of sigma factor Alg U and regulate physiological process like alginate synthesis,mucoid conversion and virulence factor secretion of P.aeruginosa.In non-mucoid strains,Muc B and Muc A form a stable complex to protect Muc A against the cleavage of Site-1 protease AlgW.However,signal molecules such as misfolded outer membrane proteins（OMPs）and the mis-located lipopolysaccharide（LPS）accumulated in the periplasm will activate Muc A cascade proteolysis（Muc A-RIP）and lead to mucoid conversion.At present,it has been confirmed that OMPs initiates the Muc A-RIP by activating protease AlgW to cleave the periplasmic region of Muc A,but the specific signal transduction mechanism of LPS activating Muc A-RIP is rarely reported.In addition,the structural basis and molecular mechanism of repressor Muc A,Muc B and membrane anchored protease AlgW are not completely clear.Therefore,this study aims to explore the structural basis of Muc A,Muc B and AlgW,and to reveal the molecular mechanism of LPS signal activating Muc A-RIP pathway,which will contribute to understanding the mechanism of ESRs stress signal transduction in P.aeruginosa and providing therapeutic strategies for P.aeruginosa infectious diseases.Based on the techniques of structural biology and molecular biology,the first part of this project successfully analyzed the complex structure of Muc A^peri-Muc B,which is an important element related to alginate biosynthesis and biofilm formation.The results also revealed the molecular mechanism of LPS activating Muc A-RIP.The research results are as follows:（1）The crystal structure of Muc B in complex with the periplasmic domain of Muc A was successfully solved.Based on the structural analysis,it was found that salt bridges,hydrogen bond and hydrophobic were the main interactions between Muc A^peri and Muc B.Pull-Down experiments in vitro further confirmed that W158,L162and H170 were the key residues in the binding of Muc A to Muc B.（2）The structural comparison between Muc A^peri-Muc B and Escherichia coli functional equivalent Rse A^peri-Rse B showed that although the assembly mode of their structures is the same,there are great differences in local conformation,especially in the region of Muc B-Loop_92-113,and the Muc A^peri-Muc B complex presents a relatively loose structural state in which the Muc A degradation site was exposed in the solvent.Further analysis showed that the conformational change was caused by the binding of a PEG molecule to the hydrophobic cavity of Muc B,which triggered the Loop_92-113inversion and exposed the degradation site of Muc A to the solvent.These conformational changes will be beneficial to the subsequent recognition and degradation of Muc A by AlgW.（3）The PEG molecules bound in the hydrophobic pocket of Muc B can be simulated as various intercellular signal molecules,such as Lipid-A or fatty acids with polar groups and aliphatic side chains.In vitro,proteolysis and structural analysis showed that signal molecules with polar group and aliphatic side chain bind to the hydrophobic cavity of Muc B,which was the minimum effector molecule that initiated the conformational change of Muc B and exposed the Muc A degradation site.The mutation of L31W,the key residue in the hydrophobic cavity of Muc B,will reduce the degree of Muc A degradation caused by signal molecules.（4）The detection of biochemical indexes such as biofilm,virulence factor,infection and alginate in muc A,muc B gene knockout strains,complementary strains and mutant strains showed that the significant increasement in alginate synthesis and biofilm formation and decrement in corresponding virulence factors such as pyocyanin and Pyoverdine followed the knockout of muc A and muc B gene and key sites mutation.These in-vivo experiments showed that muc A and muc B play an important role in regulating the physiological function of P.aeruginosa.The first part of the experimental data demonstrated the structural basis of Muc A-Muc B and the conformational regulation mode of Muc B response to lipophilic effectors,indicating that Muc A/Muc B regulatory elements have the ability to capture a variety of periplasmic signals.In addition,the regulatory mechanisms of muc A and muc B genes on the physiological processes such as alginate synthesis and secretion,mucoid conversion,biofilm formation,adaptability and drug resistance ensure that P.aeruginosa can occupy the survival advantage under changing environmental conditions.The second part of the project is to explore the protein structure of membrane-anchored protease AlgW and reveal the enzymatic mechanism of AlgW in RIP pathway.The results are as follows:（1）Based on the technology of structural biology,we reported the structures of AlgW binding to different effector molecules（tripeptide,decapeptide,both decapeptide and signal molecules）.All of these structures have significant differences in structural integrity,assembly mode and Loop-A orientation.（2）Structural analysis and proteolysis experiments showed that the binding of tripeptide,decapeptide and decapeptide+signal molecules could increase the angle between PDZ and Protease domain and enhance the activity of AlgW,which revealed that the effector molecule could abrogate the inhibition of PDZ on Protease domain by increasing the angle between PDZ and Protease,thus realizing the enhancement of AlgW activity.（3）Enzyme kinetic results showed that the PDZ domain and Loop-A were very important for AlgW activity,and amino acid sites W281,L282,M342 and R374 were crucial for OMPs recognition.（4）Lipid-A and OMPs have synergistic effect on the enhancement of AlgW protease activity,and they are very important for activating AlgW to degrade Muc A^peri and Muc B.The second part of the experimental data pointed out the importance of PDZ and Loop-A to the enzyme activity of AlgW,and revealed that signal molecules such as Lipid-A and OMPs enhance AlgW protease activity by increasing the angle between PDZ domain and Protease domain.These results provide a scientific foundation for understanding the enzymatic mechanism of Htr A-family protease.In sum,we explored the structural basis and molecular mechanism of Muc A-Muc B and protease AlgW in P.aeruginosa RIP pathway.Through the structure analysis,we revealed the mechanism of how damaged LPS（e.g.Lipid-A）activates Muc A-RIP pathway.This study contributes to the understanding of physiological phenotypes of alginate secretion,mucoid conversion and biofilm formation in P.aeruginosa,and provides a theoretical basis for the screening novel drug molecules against P.aeruginosa.