Studies On The Molecular Mechanism About Transmembrane Helix Associations Of Transmembrane Protein PSGL-1

P-selectin glycoprotein ligand-1(PSGL-1),expressed on leukocytes,is a homodimeric transmembrane protein that mediates the earliest adhesive event during an inflammatory response through binding to P-selectin on endothelial cell and platelets.It has been confirmed that the dimerization mediated by the transmembrane domain of PSGL-1 is essential for the implementation of biochemical function.But the undergoing mechanism of the transmembrane association that determines PSGL-1 TMD dimerization is still not clear.In this dissertation,we used the TOXCAT system to design seven chimeras with different sequence lengths and proved the length of the inserted TM helix is important in determining the CAT expression.We found that a 24-amino acid peptide including the residues 320-343 of PSGL-1 yielded the maximum CAT activity.Thus we used it for subsequent experiments and signed as PSGL-WT.The signal induced by the PSGL-WT was approximately 1.5 times as strong as that generated by GpA,which is well known to have stable dimerization,indicating that the PSGL-1 TMD was eazy to conform dimers to a certain extent.Then we probed the PSGL-1 TMD association by Leu-and Ala-scanning mutagenesis,and found that the mutation of residues 1324,A331 and T338 occupied the a position and residues C320,L327,F334 and L341 occupied the d position in a(abcdefg)n leucine heptad repeat motif prevented the dimer formation,which indicated that these residues might be the key influence factor in the oligomeric assembly.Molecular dynamics(MD)simulations were used to further identify the dimeric interface.In CG-MD models for the wild-type dimeric,the distances between the backbones of two monomers structure were flat with a distance about 7.5 A and the distribution of the crossing angles formed by the two monomers had a narrow peak at about-27°,suggesting that the dimeric assembly of PSGL-1 TMD was a stable right-handed conformation.In addition,the changes of crossing angles between the wild-type PSGL-1 TMD and its a,d positions mutants revealed the MD simulation results were consistent with the TOXCAT results.All the results demonstrated that a leucine zipper motif(the a,d positions)contributes to the dimeric conformation of PSGL-1 TMD.