Dynamic And Functional Studies Of Human Peptidyl-Prolyl Cis-Trans Isomerase Pin1

The integrity of protein backbone is maintained by peptide bond,which mainly adopts trans conformation due to its much lower free energy than that of the cis conformation.Because of the steric effects of a unique five-membered ring,proline can sample the cis conformer with a considerable population depending on the surrounding environment.The cis-trans isomerization of proline residues is important for biological functions,however,the isomerization rate is extremely low due to high activation barrier separating the cis and trans conformers.Fortunately,peptidyl-prolyl isomerase(PPIase)can accelerate the reaction by several orders of magnitude.Pinl(Peptidyl-proyl cis-trans isomerase NIMA-interacting 1)belongs to the parvulins family,which catalyzes the cis-trans isomerization of the substrates in a phosphorylation-dependent way.Pinl plays key roles in abundant biological processes such as mitosis,ubiquitination,gene expression and amyloid precursor proteins processing and is highly associated with neurodegenerative diseases and tumorigenesis.Therefore,structural,dynamic and functional studies of Pin1 are critical to uncover the molecular mechanisms underlying catalysis and the regulation of its isomerase activities.By combining chemical shift correlation analysis with small angle X-ray scattering studies,we determined two-state populations of Pin1 with unpreceded precisions and found that Pinl samples the compact state with a population equal to 29%.Two gauge the two-state populations,we proposed an effective method to stabilize the compact state of Pin1,which can also be applied to other multidomain proteins for further structural and dynamic studies.Protein dynamics are essential for exerting their biological functions.Hence,the method developed here for protein dynamics regulation paves avenues for modulating protein functions.In addition,we varied the length of interdomain linker of the Pinl,which is not conserved among different species,to modulate ensemble distribution of Pinl.Surprisingly,we found that enhanced interdomain contact can also result in higher isomerase activities and the relationships of the extent of interdomain contact and enzymatic activities are not that straightforward and should not be simply correlated.Considering that the substrates binding can lead to weakened and enhanced interdomain contact of Pinl,and in both cases it can promote catalysis in the PPIase domain,we concluded that there is a cooperativity between substrates binding in the WW domain and isomerization in the PPIase domain.Protein excited states are essential for biological functions.However,they may escape detection by other biophysical techniques other than solution nuclear magnetic resonance due to much lower population than the ground state.We found that the WW domain exhibits millisecond conformational exchange in solution by CPMG relaxation dispersion experiments.Significantly,a higher population of the excited state leads to higher substrate binding affinity.Therefore,we suggest a conformational selection mechanism for WW-substrate recognition.Taken together,we have investigated conformational dynamics of Pin1 spanning a range of spatial and time scale,including interdomain dynamics,millisecond chemical exchange of active sites and excited states of WW domain,which are functionally relevant.Our results in this study provide important information for developing allosteric drugs targeting the interdomain interface to regulate the isomerase activity of Pin1.