Complete thermodynamic and kinetic characterization of the isomer-specific interaction between Pin1-WW domain and the amyloid precursor protein cytoplasmic tail phosphorylated at threonine668

Peptidyl prolyl cis - trans isomerization acts as an effective molecular timer that plays significant roles in biological and pathological processes. Enzymes such as Pin1 catalyze cis - trans isomerization, accelerating the otherwise slow isomerization rate into timescales relevant for cellular signaling. Here we have combined NMR lineshape analysis, fluorescence spectroscopy, and isothermal titration calorimetry to determine the kinetic and thermodynamic parameters describing the trans -specific interaction between the binding domain of Pin1 (WW domain) and a key cis-trans molecular switch in the amyloid precursor protein cytoplasmic tail. A three-state model, in which the cis - trans isomerization equilibrium is coupled to the binding equilibrium through the trans isomer, was found to fit the data well. The trans isomer binds the WW domain with ~22 μM affinity via very fast association (approaching the diffusion limit) and dissociation rates. The common structural and electrostatic characteristics of Pin1 substrates, which contain a phosphorylated serine/threonine-proline motif, suggest that very rapid binding kinetics are a general feature of Pin1 interactions with other substrates. The fast binding kinetics of the WW domain allows rapid response of Pin1 to the dynamic events of phosphorylation and dephosphorylation in the cell that alter the relative populations of diverse Pin1 substrates. Furthermore, our results also highlight the vastly different rates at which slow uncatalyzed cis-trans isomerization and fast isomer-specific binding events occur. These results, along with the experimental methods presented herein, should guide future experiments aimed at the thermodynamic and kinetic characterization of cis - trans molecular switches and isomer-specific interactions involved in various biological processes.