Molecular dynamics simulations of site point mutations in the TPR domain of cyclophilin 40 identify conformational states with distinct dynamic and enzymatic properties.

Mert Gur,Malcolm D Walkinshaw,Burak Erman,Jia Ning,Kathryn L Ball,Vikram Narayan,Elizabeth A Blackburn

doi:10.1063/1.5019457

Abstract

Cyclophilin 40 (Cyp40) is a member of the immunophilin family that acts as a peptidyl-prolyl-isomerase enzyme and binds to the heat shock protein 90 (Hsp90). Its structure comprises an N-terminal cyclophilin domain and a C-terminal tetratricopeptide (TPR) domain. Cyp40 is overexpressed in prostate cancer and certain T-cell lymphomas. The groove for Hsp90 binding on the TPR domain includes residues Lys227 and Lys308, referred to as the carboxylate clamp, and is essential for Cyp40-Hsp90 binding. In this study, the effect of two mutations, K227A and K308A, and their combinative mutant was investigated by performing a total of 5.76 μs of all-atom molecular dynamics (MD) simulations in explicit solvent. All simulations, except the K308A mutant, were found to adopt two distinct (extended or compact) conformers defined by different cyclophilin-TPR interdomain distances. The K308A mutant was only observed in the extended form which is observed in the Cyp40 X-ray structure. The wild-type, K227A, and combined mutant also showed bimodal distributions. The experimental melting temperature, Tm, values of the mutants correlate with the degree of compactness with the K308A extended mutant having a marginally lower melting temperature. Another novel measure of compactness determined from the MD data, the “coordination shell volume,” also shows a direct correlation with Tm. In addition, the MD simulations show an allosteric effect with the mutations in the remote TPR domain having a pronounced effect on the molecular motions of the enzymatic cyclophilin domain which helps rationalise the experimentally observed increase in enzyme activity measured for all three mutations.

Highlights

The two domain structure of Cyclophilin 40 (Cyp40) (PDB code 1IHG)6 is shown in the lower panel of Fig. 1
Comparison of molecular dynamics (MD) simulations for the apo and a holo peptide (MEEVD) bound state of Cyp40 showed a significant reduction in root mean square (RMS) fluctuation in both TPR and cyclophilin domains when MEEVD is bound
Three mutants were generated starting from this wild type (WT) Cyp40 structure: (i) CY227, where residue Lys227 was mutated to alanine (K227A), (ii) CY308, where lysine 308 was mutated to alanine (K308A), and (iii) CYD, where both lysine 227 and 308 were mutated to alanine (K227A, K308A)

Summary

Introduction

The two domain structure of Cyp (PDB code 1IHG) is shown in the lower panel of Fig. 1. In the second TPR motif formed by helix R (D262-K285) and S (W289L300), helix R is again extended by an additional helical turn of four residues compared to a canonical TPR motif An insertion in this location is a feature that distinguishes the immunophilin TPR domains from TPR domains present in the larger group of chaperone proteins that compete for Hsp or Hsp with Cyp. The MD simulations of the apo protein highlight strong anti-correlated motions between residues around the PPIase-active site and a band of residues running across four of the seven helices in the TPR domain which were significantly reduced in the holo structure explaining the loss of PPIase activity upon peptide binding. The TPR domain protein carboxy-terminus of Hsc interacting protein (CHIP, PDB code 2C2L) is an E3 ligase that binds to the C-terminal EEVD motif in a very similar manner to that of the Cyp40-Hsp complex.

Methods

Results

Conclusion