Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8.
Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates. To probe this issue in the proteolytic pathways leading to apoptosis we have examined the P(4), P(1) and P(1)' subsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quenched fluorescent peptide substrates containing o-aminobenzoyl (also known as anthranilic acid) and 3-nitro-tyrosine. Previous work has demonstrated the importance of the S(4) subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S(1) and S(1)' subsites that flank the scissile peptide bond. The S(1) subsite, the major specificity-determining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid over glutamic acid at this position. Thus caspases are among the most selective of known endopeptidases. We find that the caspases show an unexpected degree of discrimination in the P(1)' position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent. Large aromatic residues are also surprisingly well-tolerated, but charged residues are prohibited. While this describes the general order of P(1)' subsite preferences within the caspase family, there are some differences in individual profiles, with caspase-3 being particularly promiscuous. Overall, the subsite preferences can be used to predict natural substrates, but in certain cases the cleavage site within a presumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites. In the latter case we conclude that second-site interactions may overcome otherwise sub-optimal cleavage sequences.
Stennicke
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HR
Program for Apoptosis & Cell Death Research, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
Renatus
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M
Meldal
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Salvesen
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GS
D000409:Alanine / Q000737:chemistry; D017209:Apoptosis; D001665:Binding Sites; D020170:Caspase 1 / Q000737:chemistry*; D053148:Caspase 3; D053178:Caspase 6; D053179:Caspase 7; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000737:chemistry*; D002384:Catalysis; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D005998:Glycine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D062367:ortho-Aminobenzoates / Q000494:pharmacology
D005456:Fluorescent Dyes; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D012694:Serine; C505407:CASP3 protein, human; C505514:CASP6 protein, human; C505520:CASP7 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053178:Caspase 6; D053179:Caspase 7; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D020170:Caspase 1; D000409:Alanine; D005998:Glycine