Study On The Interaction Of Protein Disulfide Isomerase With Small Molecule Inhibitor And Client Proteins By NMR Spectroscopy

Molecular chaperones are vital to the proper folding of protein in cells.Protein Disulfide Isomerase(PDI),a typical folding enzyme that mainly locates in the endoplasmic reticulum(ER)which is responsible for the pairing of disulfide bonds of nascent proteins and the formation of proper conformation for misfolded protein.PDI is also a kind of chaperone found abundantly cellular protein.Crystal structure shows that PDI adapts a U-shaped domain arrangement with abb’xa’c,where domain a and a’catalyst the rearrangement of disulfide bonds in redox reaction,whereas domain b’mainly provides substrate binding sites.PDI is involved in a variety of major human diseases such as metabolic diseases,neurodegenerative Alzheimer’s and Parkinson’s diseases and many cancers by interacting with related proteins.Therefore,the interaction between PDI and substrate protein is of great significance to reveal the molecular chaperone function of PDI.How PDI recognizes substrate proteins and performs correct function in the complex cellular environment requires detailed research from the level of molecular mechanism.Less knows about the complex structure of PDI interacting with substrate on account of high dynamics of the major domains abb’a’ in PDI with the x linker and c tail in solution.There is also no similarly research from the perspective of solution structure to elucidate the specific molecular mechanisms of PDI interacting with substrates.In this paper,the mechanisms of PDI interacting with substrates including small molecules Rutin,native protein asyn,multi-configuration protein Drk SH3,and structural protein CTA are investigated by Nuclear Magnetic Resonance(NMR)and other analytical tools.This research explains the binding modes of PDI interacting with small molecule and reveals the main mechanism of asyn aggregation inhibiting by chaperone PDI and elaborates the similar binding modes of PDI interacting with different types of client proteins.This thesis provides a reference for PDI binding to other types of substrates.There are three main aspects in this thesis as follows.1.Firstly,this research studies the backbone resanonce assignment of PDI-c using "divide and conquer" strategy,which is essential for a further study of PDI by NMR.Primarily,we obtain the stable PDI ab and PDI b’xa’ construsts,and the backbone resonance NH signals of PDI ab and PDI b’xa’ are assigned by triple resonance experiments,and the assignment data shows that the secondary structure of PDI ab and PDI b’xa’ construsts in solution were consistent with the crystal structure of PDI.The backbone resonance assignment of the stable structure of PDI-c is then obtained using the superposition of the PDI ab and PDI b’xa’ spectra and the HNCA spectrum of PDI-c.Based on the assignment data of the PDI-c,we identify the binding affinity(KD for 4-5 μM)between PDI and rutin(thrombus-related inhibitor)by NMR and isothermal calorimetric titration.The structural model of PDI b’xa’-Rutin and PDI-c-Rutin complex is obtained by combining the inter-and intramolecular NOE data detected by NMR and the X-crystal structure of PDI.The structure model of PDI combined with Rutin can provide the structural reference for future design of PDI-targeted drugs.2.Abnormally misfolded and aggregated a-synuclein(asyn)is the hallmark of Parkinson’s disease.PDI and other molecular chaperone has been shown to interact with asyn and inhibit its aggregation in vitro,but the mechanism for the recognition of asyn and inhibition of aggregation by PDI is still unclear.In this research,we subsequently investigate the interaction of PDI with asyn by NMR spectroscopy.It is found that both PDI and PDI b’xa’ bound with the N-terminal S1 and S2 regions of asyn.The binding affinity of S1 region within PDI is significantly enhanced after the truncation of asyn C-terminus,whereas Y39 phosphorylation abolishes the interaction of S2 region with PDI.Furthermore,ThT fluorescence assay shows that the S1 region binding with PDI promotes the aggregation of asyn,while S2 binding with PDI inhibits the fibrillation of asyn.ThT experiments also show that PDI b’xa’significantly inhibits the aggregation of asyn.Lastly,the mechanism of how PDI inhibits asyn aggregation is proposed.These findings provide experimental evidences for understanding the inhibitory role of PDI in asyn aggregation.3.We further investigate the interaction of PDI with the multi-configuration model protein Drk SH3 and the structural protein CTA.SH3 protein has distinct Folded and Unfolded states on the NMR time-scale,which is an ideal model for protein folding.CTA is the main active subunit for Vibrio cholerae infested organisms and is unfolded by PDI in ER.The binding mode of interaction of PDI with SH3 and CTA is still controversial.In this paper,the interaction pattern of PDI with SH3 and CTA are investigated by NMR.Results indicate that PDI specifically recognizes the unfolded state of SH3 and modulates the equilibrium between folded and unfolded state in SH3.PDI binds the unfolded state of SH3 depending on the b’xa’ domain,which can also apply to PDI unfolde CTA and PDI bind with αsyn.NMR titration is introduced to identify that PDI binds to client proteins(asyn,SH3 and CTA)mainly through its hydrophobic pocket of the b’domain.Finally,the docking models of PDI binding with asyn and CTA are established using NMR data.In summary,we obtain the backbone assignments of PDI-c by NMR,the interaction patterns of different types of substrate with PDI are investigated by NMR titration and other techniques.A logical structural model of PDI-c binding to Rutin is obtained.The mechanism of PDI inhibits asyn aggregation by binding to the S2 region is proposed,and the similar patterns of PDI binding to different structural types of client proteins are revealed.These findings provide experimental reference for further study on the interaction between PDI and other substrates.