Cyclophilin40 isomerase activity is regulated by a temperature-dependent allosteric interaction with Hsp90.
Cyclophilin 40 (Cyp40) comprises an N-terminal cyclophilin domain with peptidyl-prolyl isomerase (PPIase) activity and a C-terminal tetratricopeptide repeat (TPR) domain that binds to the C-terminal-EEVD sequence common to both heat shock protein 70 (Hsp70) and Hsp90. We show in the present study that binding of peptides containing the MEEVD motif reduces the PPIase activity by ∼30%. CD and fluorescence assays show that the TPR domain is less stable than the cyclophilin domain and is stabilized by peptide binding. Isothermal titration calorimetry (ITC) shows that the affinity for the-MEEVD peptide is temperature sensitive in the physiological temperature range. Results from these biophysical studies fit with the MD simulations of the apo and holo (peptide-bound) structures which show a significant reduction in root mean square (RMS) fluctuation in both TPR and cyclophilin domains when-MEEVD is bound. The MD simulations of the apo-protein also 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. Peptide binding leads to a distortion in the shape of the active site and a significant reduction in these strongly anti-correlated motions, providing an explanation for the allosteric effect of ligand binding and loss of PPIase activity. Together the experimental and MD results suggest that on heat shock, dissociation of Cyp40 from complexes mediated by the TPR domain leads to an increased pool of free Cyp40 capable of acting as an isomerase/chaperone in conditions of cellular stress.
Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
Elizabeth A
EA
Blackburn
Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
Martin A
MA
Wear
IGMM-Edinburgh Cancer Research Centre, University of Edinburgh, Crewe Road South, EH4 2XR, U.K.
Vivian
V
Landré
IGMM-Edinburgh Cancer Research Centre, University of Edinburgh, Crewe Road South, EH4 2XR, U.K.
Vikram
V
Narayan
Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
Jia
J
Ning
Chemical and Biological Engineering Department, Koc University, Istanbul 34415, Turkey.
Burak
B
Erman
IGMM-Edinburgh Cancer Research Centre, University of Edinburgh, Crewe Road South, EH4 2XR, U.K.
Kathryn L
KL
Ball
Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K. m.walkinshaw@ed.ac.uk.
Malcolm D
MD
Walkinshaw
D000494:Allosteric Regulation; D020816:Amino Acid Motifs; D020134:Catalytic Domain; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins / Q000737:chemistry / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D011489:Protein Denaturation; D060066:Protein Interaction Maps; D013696:Temperature
D000081406:Peptidyl-Prolyl Isomerase F; D018841:HSP90 Heat-Shock Proteins; D021983:Cyclophilins
allostery; heat-shock protein 90; immunophilin; molecular dynamics; peptidyl-prolyl isomerase; tetratricopeptide (TPR) domain