Elucidatiop of Tangle Formation Mechanism Based on 3D-structural Analysis of Tau Protein

点击翻译按钮获取中文摘要

1)X-ray crystal structure analysis of microtubule-binding domainTo elucidate 3D structure of tau protein, we attempted various crystallizations of tau and its fragments. Consequently, we succeeded in the crystallization of tau microtubule-binding domain (MBD) as GST fusion protein, which were prepared as recombinant by gene expression from E.coli. The crystal data are : tetragonal, space group P4_32_12, a=b=92.24Å, c=57.68Å. X-ray diffraction data up to 2Å resolution were collected by synchrotron of Spring 8. The crystal structure was solved by the molecular replacement method using the 3D structure of GST. After several structure refinement, the present R factor is 0.258. Although the MBDs formed dimeric structure, they showed large conformational flexibility and their atomic positions were not determined. This is in contrast with the rigid 3D structure of GST. Since the MBD constructs the core structure of tau PHF, the present result indicates the close relationship between the confo … More rmational flexibility of MBD and PHE formation. We are now in progress in analyzing the crystal structure more accurately.2)Analyses of solution structure of MBDMBD is composed of three-or four-repeated structure, where each repeat peptide (named R1 -R4) consists of 31 or 32 amino acid residues. To elucidate the conformational flexibility of each repeat peptide, the solution structures of R1 -R4 in TEE solution were analyzed by the combination of NMR and model building. Consequently, the the structures of R1, R2 and R4 peptides showed α-helical structure of amphipathic distribution of respective amino acid residues. In contrast, R3 took both of extended and α-helical structures of amphipathic behavior. This result suggest the molecular association of MBD through alternative hydrophilic and hydrophobic interactions.3)Investigation of MBD interactions and model of repeat-structure-dependent PHF formationThe association of neighboring MBDs were investigated by ThS fluorescence, light scattering and surface plasmon resonance analyses, and electromicroscopy. On the basis of these results, we proposed the first model of repeat structure-controlled step-by-step filament formation of 4RMBD. This model would be helpful upon considering rational approaches to prevent tau deposition in AD Less