Structure of human DPEP3 in complex with the SC-003 antibody Fab fragment reveals basis for lack of dipeptidase activity

https://doi.org/10.1016/j.jsb.2020.107512Get rights and content
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Highlights

  • Structure of DPEP3 free and in complex with Fab of antibody drug conjugate SC-003.

  • SC-003 binds the DPEP3 dimerization interface and does not alter its structure.

  • DPEP3 binds zinc with lower affinity compared to DPEP1 due to non-canonical Tyr.

  • DPEP3 lacks peptidase activity mostly due to substitution of catalytic Asp with Asn.

  • This study furthers understanding of dipeptidase activity and DPEP3 targeting efforts.

Abstract

Dipeptidase 3 (DPEP3) is one of three glycosylphosphatidylinositol-anchored metallopeptidases potentially involved in the hydrolytic metabolism of dipeptides. While its exact biological function is not clear, DPEP3 expression is normally limited to testis, but can be elevated in ovarian cancer. Antibody drug conjugates targeting DPEP3 have shown efficacy in preclinical models with a pyrrolobenzodiazepine conjugate, SC-003, dosed in a phase I clinical trial (NCT02539719). Here we reveal the novel atomic structure of DPEP3 alone and in complex with the SC-003 Fab fragment at 1.8 and 2.8 Å, respectively. The structure of DPEP3/SC-003 Fab complex reveals an eighteen-residue epitope across the DPEP3 dimerization interface distinct from the enzymatic active site.

DPEP1 and DPEP3 extracellular domains share a conserved, dimeric TIM (β/α)8-barrel fold, consistent with 49% sequence identity. However, DPEP3 diverges from DPEP1 and DPEP2 in key positions of its active site: a histidine to tyrosine variation at position 269 reduces affinity for the β zinc and may cause substrate steric hindrance, whereas an aspartate to asparagine change at position 359 abolishes activation of the nucleophilic water/hydroxide, resulting in no in vitro activity against a variety of dipeptides and biological substrates (imipenem, leukotriene D4 and cystinyl-bis-glycine). Hence DPEP3, unlike DPEP1 and DPEP2, may require an activating co-factor in vivo or may remain an inactive, degenerate enzyme.

This report sheds light on the structural discriminants between active and inactive membrane dipeptidases and provides a benchmark to characterize current and future DPEP3-targeted therapeutic approaches.

Keywords

Antibody drug conjugate
Metalloprotease
Epitope
Structure–function
Enzyme
Zinc

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