Abstract
The Marasmius oreades agglutinin (MOA) is the holotype of an emerging family of fungal chimerolectins and an active Ca2+/Mn2+-dependent protease, which exhibits a unique papain-like fold with special active site features. Here we investigated the functional significance of the structural elements differentiating MOA from other papain-like cysteine proteases. X-ray crystal structures of MOA co-crystallized with two synthetic substrates reveal cleaved peptides bound to the catalytic site, corresponding to the final products of the proteolytic reaction. Anomalous diffraction data on crystals grown in the presence of calcium and manganese, cadmium or zinc resolve the calcium/manganese preference of MOA and elucidate the inhibitory roles of zinc and cadmium towards papain-like cysteine proteases in general. The reported structures, together with activity data of MOA active site variants, point to a conservation of the general proteolysis mechanism established for papain. Ultimately, the findings suggest that papain and the papain-like domain of MOA are the product of convergent evolution.
Highlights
Papain-like cysteine proteases (PLCPs, EC 3.4.22) form the cysteine protease superfamily with the largest number of members (Turk et al, 1997; Bro€mme, 2001; Lecaille et al, 2002)
Starting from an IPTG-inducible pT7 vector containing the cDNA for wild-type Marasmius oreades agglutinin (MOA) (MOApT7-LO, described by Cordara et al (2011), a mutation was introduced at different codons by site-directed mutagenesis using the QuikChange include a calcium/manganese (II) kit (Stratagene)
MOA is a chimerolectin/protease hybrid representative of a largely unexplored family of metal-dependent fungal enzymes (Cordara et al, 2017). This enzyme family carries a unique papain-like domain, which acts as dimerization domain
Summary
Papain-like cysteine proteases (PLCPs, EC 3.4.22) form the cysteine protease superfamily with the largest number of members (Turk et al, 1997; Bro€mme, 2001; Lecaille et al, 2002). Papain-like proteases are characterized by a conserved αþβ folding motif, commonly referred to as the “papain fold”. The papain fold comprises two subdomains, known as L(left)- and R(right)-domains (Drenth et al, 1968; Heinemann et al, 1982). The interface between the two subdomains forms a V-shaped groove, at the bottom of which lies the catalytic center (Drenth et al, 1968; Turk and Guncar, 2003) (Fig. 1a; see Fig. S1 for superimposition of structural motifs)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
