Hidden alternative structures of proline isomerase essential for catalysis

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Enzyme catalysis is intrinsically linked to protein dynamics, but understanding the atomic-level mechanisms of rare conformational substates remains a challenge.
• Existing methods like X-ray crystallography and NMR spectroscopy provide complementary but incomplete views of enzyme dynamics and catalytic states.

Purpose of the Study:

• To structurally characterize interconverting substates of human proline isomerase, cyclophilin A (PPIA), at an atomic level.
• To elucidate the role of collective protein motions in enzyme catalysis by linking conformational dynamics to catalytic rates.

Main Methods:

• Ambient-temperature X-ray crystallographic data collection combined with automated electron-density sampling to resolve rare protein conformations.
• Site-directed mutagenesis to stabilize a previously hidden minor conformation of cyclophilin A.
• NMR relaxation studies to analyze enzyme dynamics in solution.

Main Results:

• Dual crystallographic strategies successfully unraveled interconverting substates of cyclophilin A (PPIA).
• A conservative mutation outside the active site inverted the equilibrium between substates and stabilized the minor conformation.
• This mutation significantly reduced both conformational interconversion rates and the catalytic rate, demonstrating a direct link.

Conclusions:

• Crystallographic approaches can define functional minor protein conformations crucial for catalysis.
• Collective motions in cyclophilin A directly contribute to its catalytic power.
• The findings provide a new framework for understanding enzyme catalysis through protein dynamics.