The crystal structure of a Cys25 → Ala mutant of human procathepsin S elucidates enzyme–prosequence interactions

Abstract

The crystal structure of the active-site mutant Cys25 !Ala of glycosylated human procathepsin S is reported. It was determined by molecular replacement and refined to 2.1 Å resolution, with an R-factor of 0.198. The overall structure is very similar to other cathepsin L-like zymogens of the C1A clan. The peptidase unit comprises two globular domains, and a small third domain is formed by the N-terminal part of the prosequence. It is anchored to the prosegment binding loop of the enzyme. Prosegment residues beyond the prodomain dock to the substrate binding cleft in a nonproductive orientation. Structural comparison with published data for mature cathepsin S revealed that procathepsin S residues Phe146, Phe70, and Phe211 adopt different orientations. Being part of the S19 and S2 pockets, they may contribute to the selectivity of ligand binding. Regarding the prosequence, length, orientation and anchoring of helix a3p differ from related zymogens, thereby possibly contributing to the specificity of propeptide-enzyme interaction in the papain family. The discussion focuses on the functional importance of the most conserved residues in the prosequence for structural integrity, inhibition and folding assistance, considering scanning mutagenesis data published for procathepsin S and for its isolated propeptide.

Residue-Specific Annotation of Disorder-to-Order Transition and Cathepsin Inhibition of a Propeptide-Like Crammer from D. melanogaster

“…Given the sequence similarity (Figure 1), crammer and the propeptides seem to share similar binding modes. The complex structure of procathepsin S shows that the propeptide side chain of W58 is in contact with the side chain of W153 of the pro-segment binding loop (PBL) [48]. In procathepsin L, F56 of the propeptide makes close contacts with Y151 of the PBL [49] (Figure S7).…”

“…Multiple sequence alignment (Figure 1A) shows that this glutamic acid is highly conserved among crammer, CTLA-2α, CTLA-2β and propeptides of human cathepsins L, K, and S. The salt bridge between R28 and E67 of crammer is structurally equivalent to the salt bridges R31-E70, R3-E70, and R33-E72 of the propeptides of human cathepsins L [49], K [51], [52], and S [48], respectively. The R33-E72 salt bridge in human cathepsin S contributes to the proper orientation of the α-helix toward the active site cleft [48]. Therefore, this study proposes that the R28-E67 salt bridge, connecting α-helices 2 and 4 in crammer, is essential for the proper orientation of α-helix 4 with respect to the active site of CTSB.…”

“…Superimposition of the Cα atoms of crammer (red; PDB entry 2KTW) with those of the human cathepsin propeptides L (light grey; PDB entry 1CS8 [49], [58]), K (dark grey; PDB entry 1BY8 [52]) and S (black; PDB entry 2C0Y [48]) yields a moderate pair-wise positional root mean square deviation (RMSD) of 4.1 Å, 5.6 Å and 4.4 Å, respectively. The relatively large positional deviation is mainly due to the different orientations of the individual-helices.…”

“…3D coordinates for crammer (yellow), and human procathepsins K (blue) and S (red), are taken from the PDB (entries 2KTW [43], 1BY [52], and 2C0Y [48], respectively.) The Drosophila procathepsin B (light grey) structure is modeled using Modeller [59]–[62], based on the structure of human procathepsin B (PDB code: 3PBH [63]).…”

Cloning and characterization of a basic cysteine-like protease (cathepsin L1) expressed in the gut of larval Diaprepes abbreviatus L. (Coleoptera: Curculionidae)

“…Different models of Da-proCTSL1 were built from the X-ray coordinates of the procathepsin L1 from Fasciola hepatica (PDB accession number 2O6X) (Stack et al, 2008) the larval midgut procathepsin L3 of Tenebrio molitor (PDB accession number 3QT4) (Beton et al, 2012), the Cys25Ala mutant of human procathepsin S (PDB accession number 2C0Y) (Kaulmann et al, 2006), the human procathepsin L (PDB accession number 1CS8) (Coulombe et al, 1996), and the activated cathepsin L of Toxoplasma gondii in complex with the propeptide (PDB accession number 3F75) (Larson et al, 2009), used as templates, respectively. Finally, a hybrid model of Da-proCathL1 was built up from the five previous models.…”