Structural evidence for a proline-specific glycopeptide recognition domain in an O-glycopeptidase

The glycosylation of proteins is typically considered as a stabilizing modification, including resistance to proteolysis. A class of peptidases, referred to as glycopeptidases or O-glycopeptidases, circumvent the protective effect of glycans against proteolysis by accommodating the glycans in their active sites as specific features of substrate recognition. IMPa from Pseudomonas aeruginosa is such an O-glycopeptidase that cleaves the peptide bond immediately preceding a site of O-glycosylation, and through this glycoprotein-degrading function contributes to the host-pathogen interaction. IMPa, however, is a relatively large multidomain protein and how its additional domains may contribute to its function remains unknown. Here, through the determination of a crystal structure of IMPa in complex with an O-glycopeptide, we reveal that the N-terminal domain of IMPa, which is classified in Pfam as IMPa_N_2, is a proline recognition domain that also shows the properties of recognizing an O-linked glycan on the serine/threonine residue following the proline. The proline is bound in the center of a bowl formed by four functionally conserved aromatic amino acid side chains while the glycan wraps around one of the tyrosine residues in the bowl to make classic aromatic ring-carbohydrate CH-pi. interactions. This structural evidence provides unprecedented insight into how the ancillary domains in glycoprotein-specific peptidases can noncatalytically recognize specific glycosylated motifs that are common in mucin and mucin-like molecules.

Automated summary

Protein glycosylation is usually considered as a stabilizing modification, but certain enzymes called glycopeptidases or O-glycopeptidases have the ability to degrade glycoproteins by recognizing specific glycan features of the substrate. The crystal structure of IMPa in complex with an O-glycopeptide shows that its N-terminal domain is a proline recognition domain that can also identify O-linked glycans, providing insights into how ancillary domains in glycoprotein-specific peptidases recognize glycosylated motifs.