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

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.

Figure 1. Crystal structures of apo-mC25A and the mC25A and histone H3₁₉₋₃₃ peptide complex.

(a) The crystal structure of the apo form of the mature, inactive cathepsin L mutant, mC25A. (b) Alignment of the apo-C25A structure with other cathepsin structures of PDB codes 1NPZ, 1VSN and 3BC3 reveals no perturbation in global structure or on the conformation of residues comprising the active-site cleft on mutation of the catalytic cysteine. (c) The standard nomenclature designating the peptide-binding subsites of the cathepsin L active-site-binding cleft. The substrate residues apparent in the crystal structure are indicated in bold. (d) The crystal structure of the mC25A cathepsin L mutant in complex with a peptide derived from the human H3 tail corresponding to residues 19–33 of histone H3 (QLATKAARKSAPATG). Only residues Q19, L20 and A21 could be placed. (e) The Q19–A21 segment of the substrate peptide was fitted into unprimed subsites. The electrostatic surface of mC25A is shown with regions of negative charge indicated in red and positive charges in blue. (f) The Fo–Fc difference density at 3σ contour. The placement of L20 in the S2 subsite was unambiguous. (g) The H3L20 residue occupies the S2 subsite, where it makes a variety of van der Waals contacts with the indicated mC25A side chains. As predicted from biological data, H3A21 occupies the S1 subsite where the S1–S1′ peptide bond would be oriented for nucleophilic cleavage.

Figure 2. 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.

Figure 3. Sequence and structural conservation among human cathepsins L and V and murine cathepsin L.

(a) Structure-based sequence alignment of cathepsins L and V. White residues on red background indicate identical residues, red residues on white represent similar residues and the other residues are coloured in black. (b) Surface representation of human cathepsins L, V and murine cathepsin L. The structure of murine cathepsin L is modelled based on human cathepsin L. The histone H3 peptide in human cathepsin L complex structure is superimposed to human cathepsin V and murine cathepsin L structures for comparison.