Molecular Insights into Substrate Binding of the Outer Membrane Enzyme OmpT

The enzyme OmpT of the outer membrane of Escherichia coli shows proteolytic activity and cleaves peptides and proteins. Using molecular dynamics simulations in a fully hydrated lipid bilayer on a time scale of hundreds of nanoseconds, we draw a detailed atomic picture of substrate recognition in the OmpT-holo enzyme complex. Hydrogen bonds and salt bridges are essential for maintaining the integrity of the active site and play a central role for OmpT in recognizing its substrate. Electrostatic interactions are critical at all stages from approaching the substrate to docking at the active site. Computational alanine scanning based on the Molecular Mechanics Generalized Born Surface Area (MM-GBSA) approach confirms the importance of multiple residues in the active site that form salt bridges. The substrate fluctuates along the axis of the beta-barrel, which is associated with oscillations of the binding cleft formed by the residue pairs D210-H212 and D83-D85. Principal component analysis suggests that substrate and protein movements are correlated. We observe the transient presence of putative catalytic water molecules near the active site, which may be involved in the nucleophilic attack on the cleavable peptide bond of the substrate.

Automated summary

This study used molecular dynamics simulations to illustrate the substrate recognition process of the outer membrane enzyme OmpT of Escherichia coli in a hydrated lipid bilayer. Hydrogen bonds and salt bridges play a significant role in maintaining the integrity of the active site and substrate recognition by OmpT. Electrostatic interactions are crucial in all stages of substrate binding and docking at the active site. Computational alanine scanning confirmed the importance of multiple residues in the active site that form salt bridges. The substrate fluctuates along the axis of the beta-barrel and correlates with the oscillations of the binding cleft formed by specific residue pairs. Principal component analysis indicates a correlation between substrate and protein movements. Transient presence of putative catalytic water molecules near the active site suggests their involvement in the cleavage of the substrate's peptide bond.