Studies On The Molecular Mechanism Of Transmembrane Domain Interactions Of Transmembrane Protein CD36

Transmembrane protein CD36 is a Class B scavenger receptor located on the cell surface of many types, which has an important role on the innate immunity, thrombosis, cell adhesion, and lip id transport. For a more comprehensive understanding of CD36-mediated ligand binding and signal transduction mechanisms, we studied on the structure and the interactional molecular mechanism of CD36 transmembrane domain.CD36 has two transmembrane domains. Our previous experimental results indicated that the second transmembrane domain of CD36 could not obviously dimerize. We found that the first transmembrane domain (TMl) of CD36 contained a number of conserved glycine residues by querying the NCBI database. These glycines may be involved in affecting homo-dimerization in form of GxxxG motif. Therefore, this paper mainly studies the first transmembrane domain of CD36. TOXCAT is a widely used assay method that quantitatively monitors self-association of TM helices in the real membrane environment of the bacteria E.coli. Firstly TOXCAT detection was used to measure CD36 transmembrane domain interactions. Experiments with the CAT activity of GpA which forms the strongest dimerization as 100%, the results showed that the CAT activity of CD36 wild type (WT) was 87.5%, which indicated that it could form relatively strong homo-dimerization. Mutating residues G12, G16 and A20 to isoleucine(I) resulted in significantly reducing dimerization. The overall results show that G12xxxG16xxxA20 motif plays a critical role in mediating the dimerization of TM1 domain.Using computer simulation to study the interactional conformation of the transmembrane peptides, mutation G16I only occurs -35° right-handed conformation during self-association and N terminal distance is about 18 angstroms.While mutation G23I only appeared -20° right-handed conformation during dimerization and N terminal distance is about 10 angstroms. In order to study the conformation, we respectively synthesized N-terminal attached a tryptophan (W) and a cysteine (C) to four peptides G 16I-W, G16I-C, G23I-W, G23I-C by Fmoc solid phase synthesis. Select a specific thiol reactive fluorophores mBBr (monobromobimane) to label G16I-C and G23I-C. Use tryptophan-induced fluorescence quenching method to measure the conformation of transmembrane peptides during self-association. F0 and FW represent the fluorescence intensity without and with the presence of the tryptophan residue. The fluorescence intensity data(F0/FW) depends on Ca-Ca distances between mBBr and tryptophan,which reflects the interactional conformation of two peptides. The results showed that Ca-Ca distance of G16I transmembrane peptides was greater than 15 A and Ca-Ca distance of G23I transmembrane peptides was 11 A, which was consistent with computer simulations.In this thesis, based on studies on the interacting molecular mechanism and conformation of the first transmembrane domain (TM1) of CD36,it is helpful to further explore the better understanding for relationship between structure and function of transmembrane protein in vivo and provide a theoretical basis for the subsequent CD36 targeting drug design and development.