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Crystal structure of MOA in complex with a peptide fragment: A protease caught in flagranti.

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.

SUBMITTER: Manna D

PROVIDER: S-EPMC8244254 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Signal Regulatory Protein ? (SIRP?) is a member of a closely related family of three cell surface receptors implicated in modulating immune/inflammatory responses. SIRP? is expressed on T lymphocytes where it appears to be involved in the integrin-independent adhesion of lymphocytes to antigen-presenting cells. Here we describe the first full length structure of the extracellular region of human SIRP?. RESULTS:We obtained crystals of SIRP? by making a complex of the protein with the Fab fragment of the anti-SIRP antibody, OX117, which also binds to SIRP? and SIRP?. We show that the epitope for FabOX117 is formed at the interface of the first and second domains of SIRP? and comprises residues which are conserved between all three SIRPs. The FabOX117 binding site is distinct from the region in domain 1 which interacts with CD47, the physiological ligand for both SIRP? and SIRP? but not SIRP?. Comparison of the three domain structures of SIRP? and SIRP? showed that these receptors can adopt different overall conformations due to the flexibility of the linker between the first two domains. SIRP? in complex with FabOX117 forms a dimer in the crystal. Binding to the Fab fixes the position of domain 1 relative to domains 2/3 exposing a surface which favours formation of a homotypic dimer. However, the interaction appears to be relatively weak since only monomers of SIRP? were observed in sedimentation velocity analytical ultracentrifugation of the protein alone. Studies of complex formation by equilibrium ultracentrifugation showed that only a 1:1 complex of SIRP?: FabOX117 was formed with a dissociation constant in the low micromolar range (Kd?=?1.2 +/- 0.3 ?M). CONCLUSION:The three-domain extracellular regions of SIRPs are structurally conserved but show conformational flexibility in the disposition of the amino terminal ligand-binding Ig domain relative to the two membrane proximal Ig domains. Binding of a cross-reactive anti-SIRP Fab fragment to SIRP? stabilises a conformation that favours SIRP dimer formation in the crystal structure, though this interaction does not appear sufficiently stable to be observed in solution.

Project description:UnlabelledCoronaviruses (CoVs) can cause highly prevalent diseases in humans and animals. Feline infectious peritonitis virus (FIPV) belongs to the genus Alphacoronavirus, resulting in a lethal systemic granulomatous disease called feline infectious peritonitis (FIP), which is one of the most important fatal infectious diseases of cats worldwide. No specific vaccines or drugs have been approved to treat FIP. CoV main proteases (M(pro)s) play a pivotal role in viral transcription and replication, making them an ideal target for drug development. Here, we report the crystal structure of FIPV M(pro) in complex with dual inhibitors, a zinc ion and a Michael acceptor. The complex structure elaborates a unique mechanism of two distinct inhibitors synergizing to inactivate the protease, providing a structural basis to design novel antivirals and suggesting the potential to take advantage of zinc as an adjunct therapy against CoV-associated diseases.ImportanceCoronaviruses (CoVs) have the largest genome size among all RNA viruses. CoV infection causes various diseases in humans and animals, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). No approved specific drugs or vaccinations are available to treat their infections. Here, we report a novel dual inhibition mechanism targeting CoV main protease (M(pro)) from feline infectious peritonitis virus (FIPV), which leads to lethal systemic granulomatous disease in cats. M(pro), conserved across all CoV genomes, is essential for viral replication and transcription. We demonstrated that zinc ion and a Michael acceptor-based peptidomimetic inhibitor synergistically inactivate FIPV M(pro). We also solved the structure of FIPV M(pro) complexed with two inhibitors, delineating the structural view of a dual inhibition mechanism. Our study provides new insight into the pharmaceutical strategy against CoV M(pro) through using zinc as an adjuvant therapy to enhance the efficacy of an irreversible peptidomimetic inhibitor.

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Crystal structure of MOA in complex with a peptide fragment: A protease caught in flagranti.

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