Effect of Glycosylation on the Free Energy Landscape of the Catalytic Domain of Human Carbonic Anhydrase IX

Human carbonic anhydrase IX (HCA IX) is a protein whose catalytic function can be affected by N-linked glycosylation. However, the impact of glycosylation on the free-energy landscape of its catalytic domain has not been extensively studied. This study combines molecular dynamics simulations with kinetic transition network analysis to examine how glycosylation alters the conformational organization of HCA IX's catalytic domain, both in monomeric and dimeric forms.

The multidimensional conformational space is broken down into discrete free energy minima based on the distribution of reciprocal interatomic distances (DRID). The effective barriers between these minima are then estimated using the max flow-min cut formalism. These free energy landscapes are represented through disconnectivity graphs, which faithfully reflect the underlying kinetics.

By analyzing minimum free energy paths, mean first passage times, and frustration metrics, the study quantifies the effect of glycosylation on landscape topography.

Unglycosylated HCA IX systems reveal funnel-like landscapes with a limited number of metastable states near the native protein fold. However, the presence of glycosylation significantly increases landscape complexity, leading to a diverse array of relaxation timescales. Interestingly, the two glycan chains have distinct effects on landscape topography, despite having similar sequences.

Additionally, dimerization links the dynamics of the glycan chains, with transitions between critical metastable states involving coordinated motions in both chains. This research demonstrates that visualizing glycoprotein energy landscapes using disconnectivity graphs and transition networks can provide valuable insights into their organization.