Molecular basis of peptide recognition in metallopeptidase Dug1p from Saccharomyces cerevisiae.

Appu Kumar Singh, Mirage Singh, Vaibhav Kumar Pandya, Balasubramani G L, Vijay Singh, Mary Krishna Ekka, Monica Mittal, S Kumaran

Biochemistry 2014 Dec 23

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Dug1p, a M20 family metallopeptidase and human orthologue of carnosinase, hydrolyzes Cys-Gly dipeptide, the last step of glutathione (GSH) degradation in Saccharomyces cerevisiae. Molecular bases of peptide recognition by Dug1p and other M20 family peptidases remain unclear in the absence of structural information about enzyme-peptide complexes. We report the crystal structure of Dug1p at 2.55 Å resolution in complex with a Gly-Cys dipeptide and two Zn(2+) ions. The dipeptide is trapped in the tunnel-like active site; its C-terminus is held by residues at the S1' binding pocket, whereas the S1 pocket coordinates Zn(2+) ions and the N-terminus of the peptide. Superposition with the carnosinase structure shows that peptide mimics the inhibitor bestatin, but active site features are altered upon peptide binding. The space occupied by the N-terminus of bestatin is left unoccupied in the Dug1p structure, suggesting that tripeptides could bind. Modeling of tripeptides into the Dug1p active site showed tripeptides fit well. Guided by the structure and modeling, we examined the ability of Dug1p to hydrolyze tripeptides, and results show that Dug1p hydrolyzes tripeptides selectively. Point mutations of catalytic residues do not abolish the peptide binding but abolish the hydrolytic activity, suggesting a noncooperative mode in peptide recognition. In summary, results reveal that peptides are recognized primarily through their amino and carboxyl termini, but hydrolysis depends on the properties of peptide substrates, dictated by their respective sequences. Structural similarity between the Dug1p-peptide complex and the bestatin-bound complex of CN2 suggests that the Dug1p-peptide structure can be used as a template for designing natural peptide inhibitors.

Citation

Appu Kumar Singh, Mirage Singh, Vaibhav Kumar Pandya, Balasubramani G L, Vijay Singh, Mary Krishna Ekka, Monica Mittal, S Kumaran. Molecular basis of peptide recognition in metallopeptidase Dug1p from Saccharomyces cerevisiae. Biochemistry. 2014 Dec 23;53(50):7870-83