Specific Recognition Mechanism Of Human Vav2 SH2 Domain With PIP2/PIP3 And Juxtamembrane Region Of Receptor Tyrosine Kinase EphA2

We have found and confirmed that the SH2 domain of Vav2 can recognize membrane phospholipids.Vav2 is a guanine nucleotide exchange factor(GEF)of the Rho family of small GTPases.Vav2 distributes in the cytoplasm and is recruited to the plasma membrane through its SH2 domain to participate in cell signal transduction.We found that Vav2 SH2 domain can bind PI(4,5)P2 or PI(3,4,5)P3.Through biochemical and NMR experiments,we revealed the molecular basis of specific interaction between Vav2 SH2 domain and diC4-PI(4,5)P2/PI(3,4,5)P3 or the phosphorylated juxtamembrane region of EphA2(EphA2-JM-pY594).We further developed peptide-nanodisc(a nickel-lipid containing peptide-based nanodisc system,Ni-NTA-nanodisc)to study the binding of Vav2 SH2 domain to EphA2-JM-pY594 on native-like lipid membrane.In addition,we also made a preliminary exploration of the membrane recognition mechanism of UbiG.In chapter 1,there is a background introduction to membrane phospholipid,nanodisc,SH2 domains and Vav2.The phospholipids and plasma membrane play an important role in regulating protein structure and function.Nanodisc is an ideal membrane mimic for studies of membrane protein structure and membrane recognition.SH2 domains can mediate protein-protein interaction in cell signaling through tyrosine phosphorylation and the Vav2 is very important for cell life.In chapter 2,we showed that Vav2 SH2 domain is a lipid-binding module that can recognize PI(4,5)P2 and PI(3,4,5)P3 lipids weakly but specifically.The specific lipid-binding site in Vav2 SH2 domain was identified by NMR titration experiments using the head groups of PI(4,5)P2 and PI(3,4,5)P3,both of which bind to Vav2 SH2 domain with millimolar binding affinities.Previous studies suggested that Y594 is one of the tyrosine phosphorylation sites in EphA2 juxtamembrane region and a potential binding site for Vav2 SH2.Through ITC and NMR experiments,we obtained the information about the binding interface between the Vav2 SH2 domain and EphA2-JM-pY594,as well as the dissociation constants and thermodynamic parameters.Furthermore,by using Ni-NTA-nanodisc,we studied the binding of Vav2 SH2 domain to EphA2-JM-pY594 on lipid membrane and uncovered a role of membrane environment in modulating this kind of protein-protein recognition.The third part of the dissertation is a part of my other work during my doctoral period.CoQ is an essential lipid that plays key roles in the cellular respiratory chain.It also severs as a natural antioxidant.UbiG belongs to the SAM-dependent methyltransferase family.It is a rate-limiting enzyme that catalyzes both O-methylation steps in CoQ biosynthesis.Recently,UbiG was defined as a membrane binding protein and is capable of binding cardiolipin(CL)/PG.However,the structural basis that governs the specific CL/PG-binding of UbiG is unclear.Our preliminary results proved that UbiG binding to PG depends on membrane environment,and follow-up work is ongoing.