Revealing the Disordered Inter-Domain Dynamics of Peptidyl-Prolyl Cis/Trans Isomerase Pin1 by Single Molecule FRET Measurements

Abstract

Pin1 is a peptidyl-prolyl cis/trans isomerase that isomerizes phospho-Serine/Threonine-Proline motifs. It plays an essential role in regulating protein functions and its deregulation is linked to various human diseases, including cancer and Alzheimer’s disease. Pin1 has an intrinsically disordered region (IDR) that constitutes a flexible linker connecting two distinguishable domains: WW and PPIase domains. WW domain recognizes and binds the phosphorylated peptide while PPIase domain has the enzymatic activity to isomerize Proline’s peptide bond. Given that WW domain has higher affinity than PPIase domain to the target peptide, it may inhibit the access of PPIase domain to the peptide unless the domains are properly coordinated. Thus, we hypothesize that the flexible IDR plays a crucial role in regulating the cooperation between two domains. Recent evidences from NMR studies have proposed the interdomain migration in this bivalent protein yet the mechanism remains elusive. We employ single molecule fluorescence resonance energy transfer (smFRET) technique to visualize the real-time inter-domain dynamics of Pin1 in interaction with target peptides. We show that the disordered dynamics of Pin1 extends to the order of milliseconds and leads to broad but distinct populations of closed and open forms of the protein. The exchange between the distinct groups of conformation is modulated by the interaction with target peptides. The real-time correlation between the peptide interaction and the conformational dynamics of Pin1 was further measured to identify the mechanistic features of the inter-domain cooperation. Our approach provides new insights into the role of IDR in coordinating multi-domain protein functions.