Highly active and selective endopeptidases with programmed substrate specificities

Abstract

A family of engineered endopeptidases has been created that is capable of cleaving a diverse array of peptide sequences with high selectivity and catalytic efficiency (k cat /K M > 10 4 M −1 s −1 ). By screening libraries with a selection-counterselection substrate method, protease variants were programmed to recognize amino acids having altered charge, size and hydrophobicity properties adjacent to the scissile bond of the substrate, including Glu↓Arg, a specificity that to our knowledge has not been observed among natural proteases. Members of this artificial protease family resulted from a relatively small number of amino acid substitutions that (at least in one case) proved to be epistatic.Around 2% of the mammalian genome encodes for enzymes involved in protein degradation, underscoring the fundamental role of proteolysis in living organisms 1 . Unregulated proteolysis in vivo is lethal, and hence there is a critical requirement for precise sequence specificity as well as temporal and spatial control over protease activity 2,3 . The generalizable ability to engineer a protease to cleave any desired peptide sequence in an exquisitely selective manner and with high catalytic efficiency is of substantial interest for analytical, biotechnological and even therapeutic applications [4][5][6][7] . Although the utility of structure-guided mutagenesis to swap substrate preferences between homologous proteases has been previously demonstrated8 , 9, the systematic engineering of protease specificity to accommodate a diverse set of substrate sequences while maintaining high catalytic activity has remained elusive. Furthermore, typical directed evolution efforts to modify substrate preferences have given rise to enzymes exhibiting either relaxed selectivity or lower turnover for the new substrate10 ,11 . Herein we report the surprisingly general ability to program a family of highly selective and active proteases with new substrate specificities at both the P1 and P1′ positions, including sequences not recognized by the wild-type (WT) enzyme. Using a dual-substrate selection-counterselection flow cytometric assay to screen libraries derived from various diversification methods, protease variants were engineered to be specific for scissile bonds comprised of amino acids having altered charge, size, and NIH Public Access RESULTS Selection and counterselection strategiesThe Escherichia coli surface endopeptidase OmpT likely plays multiple roles in virulence 12 and exhibits a strong preference for cleavage between pairs of the basic amino acids lysine and arginine (especially the latter), cleaving these pairs with high catalytic efficiency 13 . Importantly, OmpT is not active until incorporated into the E. coli outer membrane, minimizing host lethality 14 . A quantitative, single cell-based assay optimized for dynamic range and sensitivity was developed to isolate only those OmpT variants capable of cleaving a desired selection peptide substrate (SelSub), but not a counterselection substrate (CtsSub) (Fig. 1a, ref...

Reprogramming the specificity of sortase enzymes

“…Unlike this earlier report, however, here we demonstrate that SrtA may be further reprogrammed to possess fully altered, not merely broadened specificity. This capability is somewhat surprising given the mechanistic similarity between sortases and cysteine proteases (16) and the well-appreciated difficulty of the successful engineering of proteases with altered substrate specificities (32,33). We hypothesize a number of possible explanations for this success.…”

Basis for substrate recognition and distinction by matrix metalloproteinases

“…We believe that this and similar knowledge may contribute to the redesign of MMPs to exhibit unconventional substrate specificity, which could have therapeutic, diagnostic, or other biotechnology applications (56,57). The substrate specificity of several proteases distinct from MMPs has been manipulated (58)(59)(60), but this was thought to be difficult to achieve with MMPs because of their presumed overlapping substrate specificity. MMPs not only offer a different set of potential target sequences but also have a longer substrate binding cleft than most serine proteases.…”

Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates

“…Although successful, the enrichment conferred by these methods is generally based on binding rather than catalysis. Georgiou and coworkers circumvented this potential problem by developing an interesting system that actually enables function-based isolation of novel protease variants from large libraries (34,42,43). However, their methodology is dependent on the use of cell surface-displayed proteases, which is not applicable to all proteases and therefore may limit its usefulness.…”