Structure,stability And Phosphorylated Protein Binding Of Pin1 As Studied By NMR

Pin1,a 163-residue peptidyl-prolyl cis/trans isomerase,contains an N-terminal WW domain and a C-terminal PPIase domain.Pin1 WW domain specifically recognizes pSer/Thr-Pro motif and PPIase catalyzes the cis/trans isomerization of this protein.The action of Pin1 controls a series of protein function in the cell process,such as cell cycle and cell growth.Pin1 combines with pSmad 2/3、pTau,moreover impacts the formation of enzyme in nervous,which plays an important role in the treatment of neurological diseases.Pin1 regulates the activity of protein Tau which correlates to microtubule in the tangled nervenfasser of Alzheimer’s disease.Pin1 recognizes and binds to the PEpTPPP structure of Smad3,inhibits the transduction of TGF-β by inducing the degradation of ubiquitin-proteasome.The synergistic effect of Pin1 and protein kinases improve the transcriptional activity of cyclin D1 promoter by the adjustment of phosphorylated c-Jun in the breast cancer tissue.700 MHz high-field solution NMR equipped with a QCI cryogenic probe was used to study the structure,stability of Pin1 WW domain and its interaction with some phosphorylated protein involving in human diseases,like pTau and pSmad3.The skeleton and branched chain signals were confirmed by 2D1H-15 N HSQC and 3D HNCO,HN(CO)CA,HNCA,HNCACB and CBCA(CO)NH.Moreover,the changes of hydrogen bonds in Pin1 WW domain were obtained by a set of variation temperature experiments from 5 ℃ to 25 ℃.The binding sites of Pin1 WW domain and phosphopeptides were confirmed by titration experiments.Pin1 WW S16 R and G20 D,which were selected according to natural evolution,were compared to Pin1 WW WT on the changes of structure,stability and the interaction with pTau.BSA was used to simulate cellular environment,meanwhile influence on structure,stability of Pin1 WW domains and the interaction with pSmad3 in molecular crowding environment were researched.For the first time,we revealed the details of binding between Pin1 and phosphorylated substrate at atomic resolution level by mimicking molecular crowding.Though BSA coundn’t simulate the complex cellular environment as a single macromolecule,that is important to the following study of real cell.A primary model of multiple-phosphorylated c-Jun protein binding with Pin1 was established based on experimental results,which lays foundations for the following study of Pin1 in breast cancer.