# Structures of proteinase 3 and the CD177 receptor complex reveal a major autoantibody epitope

## TL;DR

The study reveals how proteinase 3 interacts with CD177 and identifies a key site targeted by harmful antibodies in a type of vasculitis.

## Contribution

The paper identifies the CD177-binding site on PR3 as a major autoantibody epitope using structural and functional analyses.

## Key findings

- The CD177-binding site on PR3 overlaps with a hydrophobic patch and is targeted by PR3-ANCA autoantibodies.
- Structure-guided mutation of the CD177-binding site on PR3 reduces PR3-ANCA binding in ELISA assays.
- The N-terminal subdomain of CD177 (LU1 + LU2) is responsible for binding to PR3.

## Abstract

Granulomatosis with polyangiitis is a life-threatening systemic vasculitis, characterised by anti-neutrophil cytoplasmic autoantibodies (ANCA) most commonly against proteinase 3 (PR3), a protease expressed intracellularly and on the surface of neutrophils. Most cell surface PR3 is bound to the receptor CD177; however, the molecular mechanism of the interactions is not well understood. Here, we present crystal structures of CD177 in complex with PR3 and unliganded CD177. We describe a mainly hydrophobic binding interface between PR3 and CD177, involving the first two Ly6/uPAR (LU) domains of CD177. These form a globular structure which is connected to downstream domains via a flexible linker. Using a panel of PR3-ANCA-positive patient samples, we show that a significant proportion of ANCAs target the CD177-binding site of PR3 in these samples. Structure-guided mutation of the CD177-binding site on PR3 is effective in reducing PR3-ANCA binding. The results demonstrate that the CD177-binding surface of PR3 harbours a major PR3-ANCA epitope, and that the extent of binding to this surface varies between different patients.

Crystal structures of the CD177 ectodomain and a CD177-PR3 complex reveal binding between the first two Ly6/uPAR domains of CD177 and a hydrophobic surface on PR3. Binding competes with a subset of PR3-ANCA vasculitis patient autoantibodies.

Recombinant proteinase 3 (PR3) with an artificial N-linked glycan on I221N is effective in ELISA-based PR3-ANCA assays.The CD177 ectodomain folds into two subdomains, of which the N-terminal subdomain (LU1 + LU2) binds to PR3.The PR3-CD177 binding interface overlaps with the hydrophobic patch of PR3 and can be mutated to abolish binding.ELISA assays suggest that PR3-ANCA binding to PR3 is reduced when the CD177-binding site is masked or modified.

Recombinant proteinase 3 (PR3) with an artificial N-linked glycan on I221N is effective in ELISA-based PR3-ANCA assays.

The CD177 ectodomain folds into two subdomains, of which the N-terminal subdomain (LU1 + LU2) binds to PR3.

The PR3-CD177 binding interface overlaps with the hydrophobic patch of PR3 and can be mutated to abolish binding.

ELISA assays suggest that PR3-ANCA binding to PR3 is reduced when the CD177-binding site is masked or modified.

Crystal structures of the CD177 ectodomain and a CD177-PR3 complex reveal binding between the first two Ly6/uPAR domains of CD177 and a hydrophobic surface on PR3. Binding competes with a subset of PR3-ANCA vasculitis patient autoantibodies.