NEDD8 Deamidation Inhibits Cullin RING Ligase Dynamics

Front Immunol. 2021 Aug 17;12:695331. doi: 10.3389/fimmu.2021.695331. eCollection 2021.

Abstract

Cullin-RING ligases (CRLs) are a significant subset of Ubiquitin E3 ligases that regulate multiple cellular substrates involved in innate immunity, cytoskeleton modeling, and cell cycle. The glutamine deamidase Cycle inhibitory factor (Cif) from enteric bacteria inactivates CRLs to modulate these processes in the host cell. The covalent attachment of a Ubiquitin-like protein NEDD8 catalytically activates CRLs by driving conformational changes in the Cullin C-terminal domain (CTD). NEDDylation results in a shift from a compact to an open CTD conformation through non-covalent interactions between NEDD8 and the WHB subdomain of CTD, eliminating the latter's inhibitory interactions with the RING E3 ligase-Rbx1/2. It is unknown whether the non-covalent interactions are sufficient to stabilize Cullin CTD's catalytic conformation. We studied the dynamics of Cullin-CTD in the presence and absence of NEDD8 using atomistic molecular dynamics (MD) simulations. We uncovered that NEDD8 engages in non-covalent interactions with 4HB/αβ subdomains in Cullin-CTD to promote open conformations. Cif deamidates glutamine 40 in NEDD8 to inhibit the conformational change in CRLs by an unknown mechanism. We investigated the effect of glutamine deamidation on NEDD8 and its interaction with the WHB subdomain post-NEDDylation using MD simulations and NMR spectroscopy. Our results suggest that deamidation creates a new intramolecular salt bridge in NEDD8 to destabilize the NEDD8/WHB complex and reduce CRL activity.

Keywords: Cullin RING E3 ligases; NMR spectroscopy; bacterial effector; cycle inhibitory factor; deamidation; enteropathogenic E. coli; protein dynamics (molecular dynamics).

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Cullin Proteins / chemistry
  • Cullin Proteins / metabolism*
  • Kinetics
  • Molecular Dynamics Simulation
  • NEDD8 Protein / chemistry
  • NEDD8 Protein / genetics
  • NEDD8 Protein / metabolism*
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • Protein Processing, Post-Translational*
  • Structure-Activity Relationship

Substances

  • Cullin Proteins
  • NEDD8 Protein