An Unusual Member of the Papain Superfamily: Mapping the Catalytic Cleft of the Marasmius oreades agglutinin (MOA) with a Caspase Inhibitor

Abstract

Papain-like cysteine proteases (PLCPs) constitute the largest group of thiol-based protein degrading enzymes and are characterized by a highly conserved fold. They are found in bacteria, viruses, plants and animals and involved in a number of physiological and pathological processes, parasitic infections and host defense, making them interesting targets for drug design. The Marasmius oreades agglutinin (MOA) is a blood group B-specific fungal chimerolectin with calcium-dependent proteolytic activity. The proteolytic domain of MOA presents a unique structural arrangement, yet mimicking the main structural elements in known PLCPs. Here we present the X-ray crystal structure of MOA in complex with Z-VAD-fmk, an irreversible caspase inhibitor known to cross-react with PLCPs. The structural data allow modeling of the substrate binding geometry and mapping of the fundamental enzyme-substrate interactions. The new information consolidates MOA as a new, yet strongly atypical member of the papain superfamily. The reported complex is the first published structure of a PLCP in complex with the well characterized caspase inhibitor Z-VAD-fmk.

Fig 1. Comparison of MOA and papain PLCP domains.

(A) The dimerization domain of MOA is a good structural match of papain and papain-like cysteine proteases. (B,C) This is clearly visible from the structural superposition of MOA (PDB ID: 3EF2 [14]) and papain (PDB ID: 1CVZ [15]), aligned according to the standard view, first described by Heinemann et al. [16]. The catalytic Cys-His dyads are indicated. In the figure, the L(eft)- and R(ight)-domain of papain are represented in different colors. (D) The fold conservation between the two enzymes is partially lost in the L-domain, where most structural elements of papain are replaced by the MOA dimerization interface.

Fig 2. Z-VAD-fmk inhibitor and interaction with PLCP active site.

(A) PLCP active site, as mapped by Schechter & Berger [32] and revised by Turk et al. [4]; figure adapted from [33]. In a simplified representation of a generic PLCP substrate, the residues on the N-terminal side of the scissile bond are defined as P1-P4 moieties, counting outwards, while those on the C-terminal side are defined as P1’-P3’. Following this description, the scissile bond lies between positions P1 and P1’. The binding subsites on the enzyme are numbered S1-S4 (unprimed subsites) and S1’-S3’ (primed subsites), depending on the substrate position that they interact with. The S2 binding site is represented as a deeper well to highlight its character of substrate binding pocket. Subsites S3 and S2’ are drawn as dotted lines to represent their nature of “binding areas”. Sites S4 and S3’ are represented as shallow grooves to stress their very low conservation among enzymes of the PLCP superfamily. (B) The Z-VAD-fmk molecule is a substrate-mimetic Val-Ala-Asp tripeptide inhibitor carrying a thiol-reactive fluoromethylketone (fmk) on the carboxy-terminus and a capping benzoxyl carbonyl (Z) moiety on its N-terminus.

Fig 3. MOA Z-VAD-fmk complex and electron density of the caspase inhibitor.

(A) Structure of MOA (‘ZVAD-dual’) in complex with the Z-VAD-fmk inhibitor (cyan/magenta), three blood group B branched trisaccharide ligands (blue) and two calcium ions (dark magenta). The figure includes the symmetry-related protomer (dark green), shown to match the representation of the functional MOA dimer in Fig 1A. The inhibitor molecule was found in two different orientations (B, ‘ZVAD-direct’, cyan; C, ‘ZVAD-inverted’, magenta), interacting with the MOA L- or R-domains, respectively, or in both orientations, with different occupancies (D, ‘ZVAD-dual’); the arrow points towards the C-terminus of a natural PLCP substrate. For all three structures, the figure shows the final σ_A-weighted 2mFo-DFc map for Z-VAD-fmk, contoured at 1σ. For stereo figures and electron density before inclusion of the ligand, see S1 Fig.

Fig 4. Z-VAD-fmk bound to the MOA active site.

Stereographic representation of Z-VAD-fmk in (A) ‘direct’ (cyan) and (B) ‘inverted’ (magenta) orientations. Key interactions at the unprimed and primed subsites of the MOA catalytic cleft are depicted.

Fig 5. MOA-substrate interactions, derived according to the PLCP paradigm.

(A) Stereographic representation of a manually docked polyalanine substrate (purple) to the active site of MOA. The peptide follows the PLCP substrate orientation and takes advantage of the interactions identified by the Z-VAD-fmk-MOA complex. (B,C) Side-by-side schematic representation of the substrate interactions derived for MOA (B) and papain (C); adapted from [5]). The oxyanion hole and the scissile bond are marked in red. (D) Structural superimposition of MOA (PDB ID: 3EF2 [14]) and papain (PDB ID: 1PPN [50]), revealing the Gly66-calcium substitution and the Trp-Gln swap.

Fig 6. Position-dependent substrate preference of MOA.

The diagram was generated ex novo through the iceLogo server (http://iomics.ugent.be/icelogoserver/logo.html) [51], using a database of peptides derived from the LC-MS analysis of the MOA proteolytic digestion products published by Wohlschlager et al. [18]. The Z-VAD-fmk peptide group in both the direct and inverted orientations is reported underneath the iceLogo diagram for a direct comparison with the binding preferences at each occupied subsite.

Fig 7. Substrate specificity pockets of MOA and papain.

The left and center panels show the solvent-exposed surface at the specificity-determining S2 subsite of papain (A) or MOA (B). A structural alignment of the two proteins (panel C) shows a more shallow S2 binding pocket in MOA compared to papain, explaining the preference of MOA for small P2 residues.