Structural basis of mammalian mucin processing by the human gut O-glycopeptidase OgpA from Akkermansia muciniphila

Abstract

Akkermansia muciniphila is a mucin-degrading bacterium commonly found in the human gut that promotes a beneficial effect on health, likely based on the regulation of mucus thickness and gut barrier integrity, but also on the modulation of the immune system. In this work, we focus in OgpA from A. muciniphila, an O-glycopeptidase that exclusively hydrolyzes the peptide bond N-terminal to serine or threonine residues substituted with an O-glycan. We determine the high-resolution X-ray crystal structures of the unliganded form of OgpA, the complex with the glycodrosocin O-glycopeptide substrate and its product, providing a comprehensive set of snapshots of the enzyme along the catalytic cycle. In combination with O-glycopeptide chemistry, enzyme kinetics, and computational methods we unveil the molecular mechanism of O-glycan recognition and specificity for OgpA. The data also contribute to understanding how A. muciniphila processes mucins in the gut, as well as analysis of post-translational O-glycosylation events in proteins.

Fig. 1. The overall structure of OgpA.

a Schematic representation of OpeRATOR (OgpA) and b GlycOCATCH activities on O-glycopeptides. c Two cartoon representations showing the general fold and secondary structure organization of OgpA_WT1, including the catalytic (orange) and β-sandwich (yellow) domains. We have decided to show the OgpA_WT1 structure because the density map explains the architecture of all loops that decorate the active site d Upper panel. Close up view of the active site of OgpA shown as cartoon/stick representation of the superimposed OgpA_WT1 (yellow and orange) and OgpA_WT2 (gray) structures. Lower panel. Close up view of the active site of OgpA shown as electrostatic surface representation. e Two electrostatic surface representations of OgpA_WT1 showing the location of the putative O-glycopeptide binding site and the catalytic site.

Fig. 2. The O-glycopeptide substrate GD binding site.

a Surface representation of the OgpA_H205A/E206A-GD-SUB crystal structure, with annotated domains and loops. b Cartoon representation of the OgpA_H205A/E206A-GD-SUB crystal structure. c Surface representation of OgpA showing the location of the GD O-glycopeptide substrate into the active site. d Electron density map of the GD substrate shown at 1.0 σ r.m.s.d. e–g Three different cartoon representations of OgpA showing the location of the GD O-glycopeptide into the active site, the main residues and secondary structure elements. The key hydrogen bond interactions between OgpA and the GD-SUB are shown in dotted lines. The full list of interactions is reported in Supplementary Fig. 6.

Fig. 3. The O-glycopeptide GD product binding site.

a Surface representation of the OgpA_WT-GD-PRO crystal structure, with annotated domains and loops. b Cartoon representation of the OgpA_WT-GD-PRO crystal structure. c Surface representation of OgpA showing the location of the GD O-glycopeptide product into the active site. d Electron density map of the GD product shown at 1.0 σ r.m.s deviation. e, f Two different cartoon representations of OgpA showing the location of the GD O-glycopeptide product into the active site, the main residues and secondary structure elements. g Two views of the structural superposition of GD O-glycopeptide substrate (gray) and product (blue and yellow) in the OgpA_WT-GD-SUB and OgpA_WT-GD-PRO crystal structures, respectively. The key hydrogen bond interactions between OgpA and the GD-PRO are shown in dotted lines. The full list of interactions is reported in Supplementary Fig. 6.

Fig. 4. Structural basis of OgpA specificity for O-glycopeptides.

a O-glycopeptides used to measure OgpA activity by reverse-phase HPLC. b X-ray crystal structure of OgpA_WT-GD-PRO with C1 glycan. Docking calculations of OgpA with different O-glycans, including c Tn, d 3SC1, e 6SC1, f 3S6SC1, g C2 and h C3. The predicted clashes are shown as dotted circles.

Fig. 5. Structural basis of O-glycopeptide processing peptidases.

Structural comparison of OgpA and a BT4244 from B. thetaiotaomicron (PDB code 5KD8), b ZmpB from C. perfringens (strain ATCC 13124) (PDB code 5KDU) c IMPa from P. aeruginosa (PDB code 5KDX), and d StcE from the enterohemorrhagic E. coli (PDB code 3UJZ). The α-helices, β-strands and loops that interact with the O-glycans are represented in orange for OgpA, and in gray for the other enzymes. The O-glycopeptide product found in the crystal structure of OgpA is colored in yellow and blue. The O-glycopeptide product found in the crystal structures of BT4244, ZmpB, IMPa and StcE, is colored in green. e Superposition of the O-glycan found in the X-ray crystal structure of OgpA (orange), BT4244 (blue), ZmpB (green) and IMPa (yellow) f The α-helix length in each OgpA (orange), BT4244 (blue), ZmpB (green) and IMPa (yellow)and StcE (red) O-glycan endopeptidases complexes. .

Fig. 6. Subsite nomenclature for OgpA.

a The proposed subsite nomenclature for OgpA substrate recognition. Amino acids that are solvent exposed and not interacting with OgpA are colored in gray; amino acids of the N-terminal with respect of the scissile bond that interact with OgpA subsites are colored in green; amino acids of the C-terminal with respect of the scissile bond that interact with OgpA substites are colored in blue. Gal and GalNac residues are colored in yellow. b Surface representation of the active site of the OgpA_H205A/E206A-GD-SUB crystal structure with annotated subsites involved in substrate recognition.