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Structural basis for the recognition and cleavage of histone H3 by cathepsin L.

Adams-Cioaba MA, Krupa JC, Xu C, Mort JS, Min J - Nat Commun (2011)

Bottom Line: Canonical substrate-cathepsin L interactions are observed in the complex between the protease and the histone H3 peptide.Systematic analysis of the impact of posttranslational modifications at histone H3 on substrate selectivity suggests cathepsin L to be highly accommodating of all modified peptides.This is the first report of cathepsin L-histone H3 interaction and the first structural description of cathepsin L in complex with a substrate.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada.

ABSTRACT
Proteolysis of eukaryotic histone tails has emerged as an important factor in the modulation of cell-cycle progression and cellular differentiation. The recruitment of lysosomal cathepsin L to the nucleus where it mediates proteolysis of the mouse histone H3 tail has been described recently. Here, we report the three-dimensional crystal structures of a mature, inactive mutant of human cathepsin L alone and in complex with a peptide derived from histone H3. Canonical substrate-cathepsin L interactions are observed in the complex between the protease and the histone H3 peptide. Systematic analysis of the impact of posttranslational modifications at histone H3 on substrate selectivity suggests cathepsin L to be highly accommodating of all modified peptides. This is the first report of cathepsin L-histone H3 interaction and the first structural description of cathepsin L in complex with a substrate.

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Related in: MedlinePlus

Structural and sequence alignment of inhibitors and substrate sequences in the cathepsin L cleft.(a). Superimposition of the H319−33 (cyan) peptide with the cathepsin L inhibitors, AZ12878478 (yellow, PDB 3HHA), and S-benzyl-N-(biphenyl-4-ylacetyl)-L-cysteinyl-N′5′-(diaminomethyl)-D-ornithyl-N-(2-phenylethyl)-L-tyrosinamide (orange, PDB 3BC3). In addition to the AZ12878478, an acetate ion aligning with the catalytic residues and a polyethylene moiety in the prime region were also modelled. (b) Sequence alignments of selected cathepsin L substrates as identified by mass spectrometry. Each protein target is subject to multiple site of cleavage with both canonical and atypical substrate recognition by the S2 pocket, though consistently there is preference for large and bulky hydrophobic side chains at the S2 site.
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f2: Structural and sequence alignment of inhibitors and substrate sequences in the cathepsin L cleft.(a). Superimposition of the H319−33 (cyan) peptide with the cathepsin L inhibitors, AZ12878478 (yellow, PDB 3HHA), and S-benzyl-N-(biphenyl-4-ylacetyl)-L-cysteinyl-N′5′-(diaminomethyl)-D-ornithyl-N-(2-phenylethyl)-L-tyrosinamide (orange, PDB 3BC3). In addition to the AZ12878478, an acetate ion aligning with the catalytic residues and a polyethylene moiety in the prime region were also modelled. (b) Sequence alignments of selected cathepsin L substrates as identified by mass spectrometry. Each protein target is subject to multiple site of cleavage with both canonical and atypical substrate recognition by the S2 pocket, though consistently there is preference for large and bulky hydrophobic side chains at the S2 site.

Mentions: The papain-like cysteine proteases, including cathepsin L, possess substrate-binding pockets that can be divided into seven binding subsites, S4 to S3′, that interact with distinct amino acids of the target substrate and confer substrate specificity. Extensive investigations of cathepsin L substrate specificity have identified a strong preference for hydrophobic residues in the S2 subsite, with some preference for positively charged residues in the S1 subsite. Little substrate discrimination is typically observed for the S3 and S4 subsites. The binding of H319−33 by mC25A is in agreement both with the S2 subsite preference for hydrophobic residues (H3L20) and with the in vivo and in vitro cleavage data that identified the A21–T22 peptide bond as the primary site of H3 hydrolysis by cathepsin L16. The orientation of H3L20 is also consistent with the predicted mode of canonical substrate recognition derived from extensive studies of cathepsin L inhibitor (Fig. 2)18192122232425. The importance of leucine recognition by the S2 pocket is further highlighted by the loss of competitive inhibition observed for the H314−31L20A and H314−31Q19A/L20A mutant peptides in our assay. Leucine recognition by the S2 subsite may also underlie the substrate turnover observed when cathepsin L is presented with leucine-containing peptides derived from the histone H4 tail. Furthermore, the loss of competitive inhibition on mutation of L20A also suggests that alternative peptides originating from cleavage of other peptide bonds between A21 and S28 of the H3 tails16 arise from the intrinsic but secondary exopeptidase activity of cathepsin L and not from the primary cleavage of alternative sites. Hence, the initiation of histone H3 cleavage at the A21–T22 bond and subsequent removal of additional residues from the C-terminal peptide of the histone H3 generated the six histone H3 peptide populations that begin with T22, K23, A24, A25, K26 and S2816. Consistently, we have determined the structure of mC25A in complex with the H319−33 peptide from several different crystals, but have not captured alternative residues in the S2 subsite, thus supporting A21–T22 peptide bond as the primary and initial site of catalysis.


Structural basis for the recognition and cleavage of histone H3 by cathepsin L.

Adams-Cioaba MA, Krupa JC, Xu C, Mort JS, Min J - Nat Commun (2011)

Structural and sequence alignment of inhibitors and substrate sequences in the cathepsin L cleft.(a). Superimposition of the H319−33 (cyan) peptide with the cathepsin L inhibitors, AZ12878478 (yellow, PDB 3HHA), and S-benzyl-N-(biphenyl-4-ylacetyl)-L-cysteinyl-N′5′-(diaminomethyl)-D-ornithyl-N-(2-phenylethyl)-L-tyrosinamide (orange, PDB 3BC3). In addition to the AZ12878478, an acetate ion aligning with the catalytic residues and a polyethylene moiety in the prime region were also modelled. (b) Sequence alignments of selected cathepsin L substrates as identified by mass spectrometry. Each protein target is subject to multiple site of cleavage with both canonical and atypical substrate recognition by the S2 pocket, though consistently there is preference for large and bulky hydrophobic side chains at the S2 site.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105313&req=5

f2: Structural and sequence alignment of inhibitors and substrate sequences in the cathepsin L cleft.(a). Superimposition of the H319−33 (cyan) peptide with the cathepsin L inhibitors, AZ12878478 (yellow, PDB 3HHA), and S-benzyl-N-(biphenyl-4-ylacetyl)-L-cysteinyl-N′5′-(diaminomethyl)-D-ornithyl-N-(2-phenylethyl)-L-tyrosinamide (orange, PDB 3BC3). In addition to the AZ12878478, an acetate ion aligning with the catalytic residues and a polyethylene moiety in the prime region were also modelled. (b) Sequence alignments of selected cathepsin L substrates as identified by mass spectrometry. Each protein target is subject to multiple site of cleavage with both canonical and atypical substrate recognition by the S2 pocket, though consistently there is preference for large and bulky hydrophobic side chains at the S2 site.
Mentions: The papain-like cysteine proteases, including cathepsin L, possess substrate-binding pockets that can be divided into seven binding subsites, S4 to S3′, that interact with distinct amino acids of the target substrate and confer substrate specificity. Extensive investigations of cathepsin L substrate specificity have identified a strong preference for hydrophobic residues in the S2 subsite, with some preference for positively charged residues in the S1 subsite. Little substrate discrimination is typically observed for the S3 and S4 subsites. The binding of H319−33 by mC25A is in agreement both with the S2 subsite preference for hydrophobic residues (H3L20) and with the in vivo and in vitro cleavage data that identified the A21–T22 peptide bond as the primary site of H3 hydrolysis by cathepsin L16. The orientation of H3L20 is also consistent with the predicted mode of canonical substrate recognition derived from extensive studies of cathepsin L inhibitor (Fig. 2)18192122232425. The importance of leucine recognition by the S2 pocket is further highlighted by the loss of competitive inhibition observed for the H314−31L20A and H314−31Q19A/L20A mutant peptides in our assay. Leucine recognition by the S2 subsite may also underlie the substrate turnover observed when cathepsin L is presented with leucine-containing peptides derived from the histone H4 tail. Furthermore, the loss of competitive inhibition on mutation of L20A also suggests that alternative peptides originating from cleavage of other peptide bonds between A21 and S28 of the H3 tails16 arise from the intrinsic but secondary exopeptidase activity of cathepsin L and not from the primary cleavage of alternative sites. Hence, the initiation of histone H3 cleavage at the A21–T22 bond and subsequent removal of additional residues from the C-terminal peptide of the histone H3 generated the six histone H3 peptide populations that begin with T22, K23, A24, A25, K26 and S2816. Consistently, we have determined the structure of mC25A in complex with the H319−33 peptide from several different crystals, but have not captured alternative residues in the S2 subsite, thus supporting A21–T22 peptide bond as the primary and initial site of catalysis.

Bottom Line: Canonical substrate-cathepsin L interactions are observed in the complex between the protease and the histone H3 peptide.Systematic analysis of the impact of posttranslational modifications at histone H3 on substrate selectivity suggests cathepsin L to be highly accommodating of all modified peptides.This is the first report of cathepsin L-histone H3 interaction and the first structural description of cathepsin L in complex with a substrate.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada.

ABSTRACT
Proteolysis of eukaryotic histone tails has emerged as an important factor in the modulation of cell-cycle progression and cellular differentiation. The recruitment of lysosomal cathepsin L to the nucleus where it mediates proteolysis of the mouse histone H3 tail has been described recently. Here, we report the three-dimensional crystal structures of a mature, inactive mutant of human cathepsin L alone and in complex with a peptide derived from histone H3. Canonical substrate-cathepsin L interactions are observed in the complex between the protease and the histone H3 peptide. Systematic analysis of the impact of posttranslational modifications at histone H3 on substrate selectivity suggests cathepsin L to be highly accommodating of all modified peptides. This is the first report of cathepsin L-histone H3 interaction and the first structural description of cathepsin L in complex with a substrate.

Show MeSH
Related in: MedlinePlus