Solution conformation of a cyclophilin-bound proline isomerase substrate

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Pharmacology

Background:

• Cyclophilin (CyP) is a key cellular receptor for the immunosuppressant drug cyclosporin A (CsA).
• CyP functions as a peptidyl prolyl cis-trans isomerase (PPIase), catalyzing the isomerization of proline-containing peptide bonds.
• Understanding the PPIase mechanism is critical for drug development and cellular signaling research.

Purpose of the Study:

• To elucidate the conformational state of a standard model substrate when bound to Cyclophilin.
• To gain insights into the molecular mechanism of Cyclophilin's peptidyl prolyl cis-trans isomerase activity.
• To investigate the role of substrate conformation in PPIase function.

Main Methods:

• Transferred Nuclear Overhauser Effect (TRNOE) measurements using two-dimensional 1H Nuclear Magnetic Resonance (2D 1H NMR).
• Utilized the standard model substrate suc-AAPF-pNA for conformational analysis.
• NMR spectroscopy was employed to determine the bound conformation of the substrate.

Main Results:

• The CyP-bound suc-AAPF-pNA substrate adopts a predominantly cis-like conformation.
• The critical A-P peptide bond deviates by no more than 40 degrees from planarity.
• This specific conformation is indicative of the active state during PPIase catalysis.

Conclusions:

• Cyclophilin binds its substrate in a conformation that favors the cis isomer.
• The determined conformation provides direct evidence for the PPIase mechanism.
• These findings contribute to understanding how CyP facilitates proline isomerization.