Novel Protease Inhibitors via Computational Redesign of Subtilisin BPN′ Propeptide

Daugherty, Ashley B.; Muthu, Pravin; Lutz, Stefan

doi:10.1021/bi300832v

Cited by 5 publications

(1 citation statement)

References 43 publications

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“…This suggests that the linkage between the conformational and proteolytic stability of subtilase propeptides is not definite. Accordingly, Daugherty et al ( 49 ) designed thermostable propeptide variants that acquired secondary structure independently but were weaker subtilisin inhibitors than the wild-type propeptide. It could be argued that the inhibitory potency and proteolytic stability of a propeptide are determined by its C-terminal tail, which binds into the active site cleft of the subtilase domain in a product-like manner ( 23 , 24 ).…”

Section: Discussionmentioning

confidence: 99%

High selectivity of the hyperthermophilic subtilase propeptide domain toward inhibition of its cognate protease

Bahun,

Poklar Ulrih

2023

Microbiol Spectr

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Microbial extracellular subtilases are highly active proteolytic enzymes commonly used in commercial applications. These subtilases are synthesized in their inactive proform, which matures into the active protease under the control of the propeptide domain. In mesophilic bacterial prosubtilases, the propeptide functions as both an obligatory chaperone and an inhibitor of the subtilase catalytic domain. In contrast, the propeptides of hyperthermophilic archaeal prosubtilases act mainly as tight inhibitors and are not essential for subtilase folding. It is unclear whether this stronger inhibitory activity of hyperthermophilic propeptides results in their higher selectivity toward their cognate subtilases, in contrast to promiscuous mesophilic propeptides. Here, we showed that the propeptide of pernisine, a hyperthermostable archaeal subtilase, strongly interacts with and inhibits pernisine, but not the homologous subtilisin Carlsberg and proteinase K. Instead, the pernisine propeptide was readily degraded by subtilisin Carlsberg and proteinase K. In addition, the catalytic domain of unprocessed propernisine was also susceptible to degradation but became proteolytically stable after autoprocessing of propernisine into the inactive, noncovalent complex propeptide:pernisine. This allowed efficient transactivation of the autoprocessed complex propeptide:pernisine through degradation of pernisine propeptide by subtilisin Carlsberg and proteinase K at mesophilic temperature. Moreover, we demonstrated that active pernisine molecules are inhibited by the propeptide that is released after pernisine-catalyzed degradation of the unprocessed propernisine catalytic domain. This highlights the high inhibitory potency of the hyperthermophilic propeptide toward its cognate subtilase and its importance in regulating subtilase maturation, to prevent the degradation of the unprocessed subtilase precursors by the prematurely activated molecules. IMPORTANCE Many microorganisms secrete proteases into their environment to degrade protein substrates for their growth. The important group of these extracellular enzymes are subtilases, which are also widely used in practical applications. These subtilases are inhibited by their propeptide domain, which is degraded during the prosubtilase maturation process. Here, we showed that the propeptide of pernisine, a prion-degrading subtilase from the hyperthermophilic archaeon, strongly inhibits pernisine with extraordinarily high binding affinity. This interaction proved to be highly selective, as pernisine propeptide was rapidly degraded by mesophilic pernisine homologs. This in turn allowed rapid transactivation of propernisine by mesophilic subtilases at lower temperatures, which might simplify the procedures for preparation of active pernisine for commercial use. The results reported in this study suggest that the hyperthermophilic subtilase propeptide evolved to function as tight and selective regulator of maturation of the associated prosubtilase to prevent its premature activation under high temperatures.

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Section: Discussionmentioning

confidence: 99%

High selectivity of the hyperthermophilic subtilase propeptide domain toward inhibition of its cognate protease

Bahun,

Poklar Ulrih

2023

Microbiol Spectr

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Computational Library Design for Increasing Haloalkane Dehalogenase Stability

et al. 2014

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We explored the use of a computational design framework for the stabilization of the haloalkane dehalogenase LinB. Energy calculations, disulfide bond design, molecular dynamics simulations, and rational inspection of mutant structures predicted many stabilizing mutations. Screening of these in small mutant libraries led to the discovery of seventeen point mutations and one disulfide bond that enhanced thermostability. Mutations located in or contacting flexible regions of the protein had a larger stabilizing effect than mutations outside such regions. The combined introduction of twelve stabilizing mutations resulted in a LinB mutant with a 23 °C increase in apparent melting temperature (Tm,app , 72.5 °C) and an over 200-fold longer half-life at 60 °C. The most stable LinB variants also displayed increased compatibility with co-solvents, thus allowing substrate conversion and kinetic resolution at much higher concentrations than with the wild-type enzyme.

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A propeptide-based biosensor for the selective detection of Vibrio cholerae using an environment-sensitive fluorophore

DeColli

Koolik

Seminara

et al. 2022

Cell Chemical Biology

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Novel Protease Inhibitors via Computational Redesign of Subtilisin BPN′ Propeptide

Cited by 5 publications

References 43 publications

High selectivity of the hyperthermophilic subtilase propeptide domain toward inhibition of its cognate protease

High selectivity of the hyperthermophilic subtilase propeptide domain toward inhibition of its cognate protease

Computational Library Design for Increasing Haloalkane Dehalogenase Stability

A propeptide-based biosensor for the selective detection of Vibrio cholerae using an environment-sensitive fluorophore

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