SlyD Accelerates -to- Prolyl Isomerization in a Mechanosignaling Protein under Load.

Prolyl isomerization is recognized as one of the key regulatory mechanisms, which plays a crucial role in cell signaling, ion channel gating, phage virus infection, and molecular timing. This isomerization is usually slow but often accelerated by an enzyme, called peptidyl-prolyl isomerase (PPIase). In the current project, we investigate using single-molecule force spectroscopy (SMFS) the impact of a bacterial PPIase, SlyD, on the isomerization of the proline 2225 (P2225) in an isolated 20th domain of a cytoskeletal mechanosensing protein filamin-A (FlnA20). To explore the FlnA20-PPIase interaction, we have used multiple SMFS modes, like constant velocity, constant distance, and jumping trap experiments. In our previous study, we reported the unique nature of the P2225, which is conserved in all naturally occurring filamins and can slowly (minutes) interconvert between isomers, in absence of any PPIase. Our current results show a staggering 25-fold acceleration of the -to- isomerization rate in the presence of saturating SlyD concentration (7.25 μM) compared to the unenzymatic condition. A SlyD concentration-dependent depletion of the isomeric lifetime was also observed. Additionally, we observed that SlyD stabilizes the -isomer in the native state of FlnA20 by ∼2 T. This is the first single-molecule observation of the - isomerization catalysis by a PPIase in a mechanosensing protein.