Crystal structure of Streptococcus pyogenes EndoS, an immunomodulatory endoglycosidase specific for human IgG antibodies

Abstract

To evade host immune mechanisms, many bacteria secrete immunomodulatory enzymes. Streptococcus pyogenes, one of the most common human pathogens, secretes a large endoglycosidase, EndoS, which removes carbohydrates in a highly specific manner from IgG antibodies. This modification renders antibodies incapable of eliciting host effector functions through either complement or Fc γ receptors, providing the bacteria with a survival advantage. On account of this antibody-specific modifying activity, EndoS is being developed as a promising injectable therapeutic for autoimmune diseases that rely on autoantibodies. Additionally, EndoS is a key enzyme used in the chemoenzymatic synthesis of homogenously glycosylated antibodies with tailored Fc γ receptor-mediated effector functions. Despite the tremendous utility of this enzyme, the molecular basis of EndoS specificity for, and processing of, IgG antibodies has remained poorly understood. Here, we report the X-ray crystal structure of EndoS and provide a model of its encounter complex with its substrate, the IgG1 Fc domain. We show that EndoS is composed of five distinct protein domains, including glycosidase, leucine-rich repeat, hybrid Ig, carbohydrate binding module, and three-helix bundle domains, arranged in a distinctive V-shaped conformation. Our data suggest that the substrate enters the concave interior of the enzyme structure, is held in place by the carbohydrate binding module, and that concerted conformational changes in both enzyme and substrate are required for subsequent antibody deglycosylation. The EndoS structure presented here provides a framework from which novel endoglycosidases could be engineered for additional clinical and biotechnological applications.

Fig. 1.

EndoS structure, domain organization, and comparison with known protein structures. (A) Structure of EndoS_D233Q(98–995) viewed in two orientations, inclusive of the glycosidase (blue), leucine-rich repeat (green), hybrid Ig (magenta), carbohydrate binding module (cyan), and three-helix bundle (red) domains. The loop extending from the middle of the leucine-rich repeat is in orange. Superposition of the EndoS glycosidase domain to that of EndoF₃ (B), leucine-rich repeat domain to InternalinJ (C), hybrid Ig domain to the IL-4 receptor (D), carbohydrate binding module to CtCBM62 (E), and three-helix bundle domain to DnaK (F).

Fig. 2.

Structural features of the EndoS glycosidase, leucine-rich repeat, and hybrid Ig domains. (A) Superposition of the EndoS glycosidase domain (blue) with that of EndoF₃ (gray) with surfaces rendered. The glycan structure as resolved in the EndoF₃–glycan complex structure is in magenta. EndoS glycosidase domain loops that diverge most in structure from those in EndoF₃ are labeled. (B) Intramolecular contacts between residues in the EndoS glycosidase domain (blue) and the apex of the loop extending from the leucine-rich repeat domain (orange). Van der Waals interactions are shown as black dashed lines; hydrogen bond as a dashed red line. (C) Intramolecular engagement of residues from the leucine-rich repeat (green) and the inserted hybrid Ig subdomain (magenta). (C, Left) Interaction showing the entire hybrid Ig subdomain. (C, Right) Close-up view of contacts made by leucine-rich repeat residue Asp600 (green) with hybrid Ig subdomain residues. Hydrogen bonds are shown as dashed red lines.

Fig. 3.

Model of the EndoS/IgG1 Fc encounter complex. (A) Distribution of location of the IgG1 Fc on the EndoS structure from the top 500 initial models of the encounter complex. Colors of EndoS domains are as in previous figures. Each yellow ball represents the center of mass of a single IgG1 Fc dimer. (B) Side view of the final refined EndoS/IgG1 Fc encounter complex model. Monomers of the Fc homodimer are in gray (with corresponding glycan in black) and in yellow (with corresponding glycan in tan). (C) Top view of the encounter complex model with close-up of the EndoS glycosidase loop bisecting the Fc monomers in shown in the box on the right.