Aspartic protease-pepstatin A interactions: Structural insights on the thermal inactivation mechanism

• Published in: Biochimie Vol. 189; pp. 26 - 39
• Main Authors: Purushothaman, Kavya, Bhat, Sagar Krishna, Siddappa, Shiva, Singh, Sridevi Annapurna, Subbaiah, Roopashree, Marathe, Gopal Kedihithlu, Rao G Appu Rao, Appu
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.10.2021
• Subjects: Aspartic protease; Molecular dynamic simulations; Pepstatin A; Thermal stability; Molecular dynamic simulations; CD; Pepstatin A; HIC; RMSF; Aspartic protease; MDS; Thermal stability
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2021.06.002

Aspartic proteases are the targets for structure-based drug design for their role in physiological processes and pharmaceutical applications. Structural insights into the thermal inactivation mechanism of an aspartic protease in presence and absence of bound pepstatin A have been obtained by kinetics of thermal inactivation, CD, fluorescence spectroscopy and molecular dynamic simulations. The irreversible thermal inactivation of the aspartic protease comprised of loss of tertiary and secondary structures succeeded by the loss of activity, autolysis and aggregation The enthalpy and entropy of thermal inactivation of the enzyme in presence of pepstatin A increased from 81.2 to 148.5 kcal mol−1, and from 179 to 359 kcal mol−1 K−1 respectively. Pepstatin A shifted the mid-point of thermal inactivation of the protease from 58 °C to 77 °C. The association constant (K) for pepstatin A with aspartic protease was 2.5 ± 0.3 × 10 5 M−1 and ΔGo value was −8.3 kcal mol−1. Molecular dynamic simulation studies were able to delineate the role of pepstatin A in stabilizing backbone conformation and side chain interactions. In the Cα-backbone, the short helical segments and the conserved glycines were part of the most unstable segments of the protein. Understanding the mechanism of thermal inactivation has the potential to develop re-engineered thermostable proteases. •The thermal stability of an aspartic protease from A. niger was enhanced by pepstatin A.•Inactivation mechanism of protease was followed by biophysical techniques and MDS.•Pepstatin A prevented autolysis and delayed the unfolding of the aspartic protease.•Thermal stability of aspartic protease in presence of pepstatin A was due to enhanced side chain interactions.