Post-translational Modifications of Cyclophilin D Fine-Tune Its Conformational Dynamics and Activity: Implications for Its Mitochondrial Function.

Abstract

Mitochondria are the powerhouse of a cell, whose disruption due to mitochondrial pore opening can cause cell death, leading to necrosis and many other diseases. The peptidyl-prolyl cis-trans isomerase cyclophilin D (CypD) is a key player in the regulation of the mitochondrial pore. The activity of CypD can be modulated by the post-translational modification (PTM). However, the detailed mechanism of this functional modulation is not well understood. Here, we investigate the catalytic mechanism of unmodified and modified CypD by calculating the reaction free energy profiles and characterizing the function-related conformational dynamics using molecular dynamics simulations and associated analyses. Our results show that unmodified and modified CypD considerably lower the isomerization free energy barrier compared to a free peptide substrate, supporting the catalytic activity of CypD in the simulation systems. The unmodified CypD reduces the free energy difference between the cis and trans states of the peptide substrate, suggesting a stronger binding affinity of CypD toward cis, consistent with experiments. In contrast, phosphorylated CypD further stabilizes trans, leading to a lower catalytic rate in the trans-to-cis direction. The differential catalytic activities of the unmodified and phosphorylated CypD are due to a significant shift of the conformational ensemble upon phosphorylation under different functional states. Interestingly, the local flexibility is both reduced and enhanced at distinct regions by phosphorylation, which is explained by a "seesaw" model of flexibility modulation. The allosteric pathway between the phosphorylation site and a distal site displaying substantial conformational changes upon phosphorylation is also identified, which is influenced by the presence of the substrate or the substrate conformation. Similar conclusions are obtained for the acetylation of CypD using the same peptide substrate and the influence of substrate sequence is also examined. Our work may serve as the basis for the understanding of other PTMs and PTM-initiated allosteric regulations in CypD.