NMR To Explore The Interaction Mechanism Of Phos-Ubiquitin With Its Ligand And Kinase

Protein act as the executors of life activities,so understanding the structure and function of protein is of great significance to the exploration of human physiological activities.The structures of protein determine functions,the analysis of protein structure provides a solid foundation for protein signal pathway research,protein-protein interaction research and drug design.However,the structure of the protein in the cell is not static.As we all know,the protein structure in the PDB database is actually an ensemble of all conformations continuously detected over a period of time.If the different ensemble structures are sorted by timeline,a complete dynamic process of the protein is formed.Protein participates in the upstream and downstream signaling pathways through constant "movement" and protein-protein interaction,thereby exerting its physiological functions.This paper mainly focuses on the following protein systems as the research objects to discuss the interaction mechanism and the dynamic characteristics of proteins:1.Study on the relationship between ubiquitin and ubiquitin-related proteins before and after phosphorylation modification;2.Study on different ubiquitin Chains and phosphorylated Ubiquitin Kinase PINK1.This paper mainly uses liquid nuclear magnetic resonance as a research method to explore the mechanism of protein interaction and its dynamic characteristics at different time scales.Firstly,we will introduce many NMR techniques and methods used in this paper before expounding on ubiquitin-related work.And then,based on the experience of this research group in ubiquitin research for many years,we elaborate the technical route of preparing ubiquitin chains of different types,different chain lengths and different topological structures.In addition,we also elaborated on the method to design a Ub chain with one of the subunits selectively labeled or modified.For example,phosphorylation at Ub residue S65 allowed us to study the interplay between phosphorylation and modification.The site-specific attachment of a prosthetic probe allowed us to perform NMR and FRET measurements.In short,the preparation of ubiquitin samples introduced in this paper not only provides a solid foundation for the following research work,but also provide reference and help for scientific researchers participating in ubiquitin research.Next,this paper explores the interaction of phosphorylated ubiquitin(phosphorylation at position S65,hereinafter referred to as pUb)specifically with the Ub-associated domain(UBA)in the two proteasome shuttle factors,the latter being involved in the target Physiological behavior towards proteasome or self-assembly.Ub is phosphorylated by PINK1 and then presents two main conformations,named relaxed-pUb(hereinafter referred to as pUbRL)and retracted-pUb(hereinafter referred to as pUbRT).Using NMR spectroscopy,we show that pUbRL but not pUbRT preferentially interacts with the UBA from two proteasomal shuttle factors Ubqln2 and Rad23A.Yet discriminatorily,Ubqln2-UBA binds to pUb more tightly than Rad23A does and selectively enriches pUbRL upon complex formation.Further,we determine the solution structure of the complex between Ubqln2-UBA and pUbRL,and uncover the thermodynamic basis for the stronger interaction.NMR kinetics analysis at different timescales further suggests an indued-fit binding mechanism for pUb-UBA interaction.Notably,at a relatively low saturation level,the dissociation rate of the UBA-pUbRL complex is comparable with the exchange rate between pUbRL and pUbRT.Thus,a kinetic constraint would dictate the interaction between Ub and UBA,thus fine-tuning the functional state of the proteasomal shuttle factors.Finally,this paper used K63-diUb,K48-diUb and Ml-diUb as ubiquitin chain models to study how ubiquitin multimerization affects PINK 1-mediated phosphorylation.Based on these three important ubiquitin systems,we conducted research on the basic unit of the ubiquitin chain—dimeric ubiquitin.We found that the proximal subunit of M1-diUb has a phosphorylation rate about 5 times faster than that of the distal subunit.For K63-diUb and K48-diUb,the distal subunit is faster than the proximal subunit.Subsequently,we explored the mechanism of this difference in phosphorylation rate through CEST,RDC,molecular dynamics simulation and structural analysis.Exploring the mechanism of the difference in phosphorylation rate of different chain types is of great significance for understanding the phosphorylation modification events of different chain types and chain lengths in cells.