Networks of ion-pairs are responsible for the large differences in the thermal stability of two structurally similar aminopeptidases.

Abstract

Listen

Translate article

Aminopeptidases are an important class of enzymes for protein metabolism. Leucyl aminopeptidase (PepL) preferably removes leucine from the N-terminus of small peptides. PepL of Lacticaseibacillus casei was observed to be thermally unstable, while a structurally similar aminopeptidase T (AmpT) of Thermus thermophilus is highly stable. To understand the molecular interaction responsible for the large difference in their stability, molecular dynamics simulations were carried out to study the thermal stability of PepL and AmpT at 300 K to 450 K temperature range over 100 ns. PepL sampled a larger conformational space with a rugged free-energy landscape, while AmpT navigated a smoother energy landscape to reach the global minimum. The RMSD, RMSF, radius of gyration and principal component analysis suggested large movements in PepL than in AmpT with an increase in temperature. Analysis of residue-interaction network revealed AmpT possessing a greater number of low, medium and high energy contacts in comparison to PepL. AmpT showed a higher abundance of ion-pair clusters and ionic residues per cluster compared to PepL. Moreover, AmpT retained a greater number of high-energy contacts at elevated temperatures. These findings showed that the inherently lower stability of PepL originates from a comparatively smaller number of contacts and can be pivotal in engineering PepL for higher stability.