(739h) Exploring the Effect of Mutations on Thermodynamic and Enzymatic Properties of Cyclophilin 40

Cyclophilin 40 (also known as Cyp40 or PPID) is a member of the immunophilin family. It acts as a peptidyl-prolyl-isomerase (PPIase) enzyme speeding up rotation around the peptidyl prolyl amide bond and resulting in more efficient folding of peptide chains. Cyp40 is a potential therapeutic agent in protein misfolding diseases, and it is overexpressed in prostate cancer and certain T-cell lymphomas. Cyp40 is a two-domain protein. Its structure comprises an N-terminal cyclophilin domain and a C-terminal TPR domain. The TPR domain anchors the protein to heat shock protein 90 (Hsp90). The groove for Hsp90 binding on the TPR domain includes residues Lys227 and Lys308 which are essential for Cyp40-Hsp90 binding. Effects of mutating those two lysine into to alanine were investigated via Molecular Dynamics (MD) simulations and Thermodynamics. Three mutants were constructed: (i) K227A, (ii) K308A, and (iii) their combinative mutant K227A+K308A. All-atom MD simulations of 5.76 µs in total in explicit solvent were performed for these three mutants and the wild type Cyp40. Using MD trajectories specific heats were calculated and free energy surfaces were constructed. Except the K308A mutant, two distinct (extended or compact) conformations were observed in all simulations. A close correlation between experimental melting temperature, and the degree of compactness and coordination shell volume, both obtained by MD simulations, were observed. MD simulations also showed an allosteric effect between the mutations in the remote TPR domain and having the molecular motions of the enzymatic cyclophilin domain. Hence, helping to rationalize the experimentally observed increase in enzyme activity measured for all three mutations. In addition to specific heats, thermal expansion and compressibility were evaluated for WT Cyp40 and Cyp40 mutants using MD trajectories. Effects of single point and combined mutations on those thermodynamic properties were investigated in detail.