Novel Protease Inhibitors via Computational Redesign of Subtilisin BPN′ Propeptide

Citations Over Time

Abstract

The propeptide domain of subtilisin BPN' functions as a molecular chaperone for its cognate protease yet quickly assumes a predominantly unfolded structure following cleavage by the mature protease. In contrast, structural stabilization of the propeptide domain has been proposed to competitively inhibit protease self-cleavage, suggesting the possibility for the generation of novel proteinaceous subtilisin inhibitors. Using a Rosetta fixed backbone design, we have redesigned the subtilisin BPN' propeptide structure to generate synthetic peptide sequences with increased and tunable structural stability. Molecular dynamics simulations provide supporting evidence that the artificial sequences retain structure without its protease cognate unlike the inherently disordered wild-type propeptide. Experimental evaluation of two designer domains by spectroscopic methods verified their structural integrity. Furthermore, the novel propeptide domains were shown to possess significantly enhanced thermostability. Nevertheless, their modest functional performance as protease inhibitors raises doubt that propeptide stability alone is sufficient for effective inhibitor design.

Related Papers

• → Acylation of subtilisin with long fatty acyl residues affects its activity and thermostability in aqueous medium(1994)12 cited
• → Requirement of Left-Handed Glycine Residue for High Stability of the Tk-Subtilisin Propeptide as Revealed by Mutational and Crystallographic Analyses(2007)32 cited
• → Accelerated maturation of Tk‐subtilisin by a Leu→Promutation at the C‐terminus of the propeptide, which reduces the binding of the propeptide to Tk‐subtilisin(2012)20 cited
• → Functional analysis of the propeptides of subtilisin E and aqualysin I as intramolecular chaperones(2001)23 cited
• → The role of tryptophan residues in the autoprocessing of prosubtilisin E(2005)8 cited