NEDD8 Deamidation Inhibits Cullin RING Ligase Dynamics

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).

Figure 1

Role of NEDD8 in the activation of Cullin RING ligases. Schematic illustration showing the mechanism of Cullin RING ligase activation by NEDDylation. Conjugation of NEDD8 (N8) to the WHB^SD (W) triggers a change in its orientation which frees the Rbx^RING domain from inhibitory interactions.

Figure 2

Crystal conformations of Cul5^CTD closed and open states. (A) Crystal structures of Cul5^CTD-Rbx1 in the closed/open state were used to initiate independent MD simulations. Dotted black lines connect the Cα atoms (spheres) of S567 and R714 in Cul5^CTD. ECTD in both complexes refers to the extreme C-terminal domain, which comprises aa:726-780. (B) Non-covalent interactions between WHB^SD and 4HB/αβ^SD in the open conformation of NEDD8~Cul5^CTD-Rbx1 are shown in (A). The black circle represents the R452/E705 salt bridge, while black lines correspond to S539/E697 and E629/T695 hydrogen bonds. The RING domain of Rbx1 (blue) is omitted for the sake of clarity.

Figure 3

Dynamic ensembles of Cul5^CTD closed, and open states were observed from simulations. (A) Radius of gyration for Cul5^CTD-Rbx1 variants and S567-R714 separation in Cul5^CTD calculated from MD simulations. Mean ± std deviation for each variant is calculated over eight independent MD trajectories performed for 200 ns. (B) Probability distributions for the radius of gyration (Rg) of closed/open states of Cul5^CTD-Rbx1 calculated from the 1.6 μs macro trajectory for each state. The macro trajectory was obtained by combining eight independent 200 ns runs. (C) Probability distribution of S567-R714 distance in Cul5^CTD calculated from the 1.6 μs macro trajectory for each state.

Figure 4

NEDDylation biases the conformational landscape of Cullin^CTD-Rbx1 towards an ensemble of open conformations which minimize RING/ECTD interaction. (A) 2D-free energy landscapes calculated for the open state of Cul5^CTD-Rbx1 with and without NEDD8 from 1.6 μs macro trajectories obtained by combining eight independent 200 ns trajectories for each state. Upon removal of NEDD8, ECTD/RING interaction is more favorable. (B) Mean + SEM for several ECTD/Rbx1^RING and 4HB/αβ^SD-NEDD8 contacts across all Cul5^CTD-Rbx1 trajectories initiated from the open state with and without NEDD8. Contact cutoff was chosen to be 0.4 nm. (C) A representative conformation of NEDD8~Cul5^CTD-Rbx1 showing NEDD8 interacting with 4HB/αβ^SD. Interacting atoms are shown as sticks. A black dotted circle indicates a salt bridge between NEDD8 (K48) and 4HB^SD (E407). The red dotted circle indicates van der Waals interactions between NEDD8 (M1/M62) and αβ^SD (N655/S656).

Figure 5

Formation of an intramolecular salt-bridge in dNEDD8. (A) Salt-bridge stability as a function of time in a 500 ns MD simulation of dNEDD8. In the bottom plot, salt-bridge occupancy is shown as either 1 or 0 to indicate the presence or absence of a salt-bridge, respectively. Salt-bridge occupancy was calculated using a 0.5 nm cutoff. The occupancy of the salt-bridge was calculated to be 44.2%. (B) MD snapshot showing the formation of R74-E40 salt-bridge in dNEDD8. (C) The NMR CSP plot showing the effect of Q40E substitution in NEDD8. The CSP = [(δH_NEDD8 - δH_dNEDD8)2 + (δN_NEDD8 - δN_dNEDD8)2/25]1/2, where δH_NEDD8 and δH_dNEDD8 are amide proton chemical shifts of residue in wt-NEDD8 and dNEDD8, respectively. The dashed yellow line and red line denote Mean+SD and Mean+2*SD, respectively. (D) The residues with high CSPs are mapped onto the NEDD8 structure. The residues with CSP above Mean+SD are colored yellow, and the residues with CSP above Mean+2*SD are colored orange.

Figure 6

Deamidation of NEDD8 enhances its dissociation from WHB^SD. (A) R74-mediated interactions in the Cul5-WHB^SD~NEDD8 complex (PDB: 3DQV). Orange lines indicate interactions of R74 with and K764 (backbone) and Y765 (sidechain). (B) Mean force-time profile for the dissociation of NEDD8-wt and its mutants from WHB^SD obtained from steered MD (SMD) simulations. Twelve independent SMD runs were performed for 20 ns in the case of each complex. (C) F_max and unbinding work (W) determined from average profiles obtained by steered MD. One standard error of mean is indicated in ( ). (D) I44-mediated hydrophobic interactions in the Cul5-WHB^SD~NEDD8 complex. (E) Mean occupancy of the R74-K764 hydrogen bond from NEDD8-wt, dNEDD8, and NEDD8-I44A SMD runs.

Figure 7

R74-E40 salt-bridge may hinder NEDD8/WHB^SD association by disallowing compact conformations of the NEDD8 C-terminal tail. (A) Mean± one standard error of R74/E760 hydrogen bond occupancy for NEDD8-wt and dNEDD8 conjugates. (B) Snapshot from trajectory 1 of Cul1-WHB^SD~NEDD8 showing an intramolecular salt-bridge involving R74/E40. The Cα atom positions of A72/G76 are shown as pink spheres. (C) Combined probability distribution of the C-terminal tail conformation obtained from five 300 ns trajectories for each conjugate. The A72-G76 distance varies from 0.74 to 0.9 nm in crystal complexes of Cul1/5-WHB^SD~NEDD8. (D) Mean ± one standard error of the C-terminal tail conformation across all five trajectories for each conjugate.

Figure 8

An intermolecular salt-bridge involving E40 destabilizes the Cul1-WHB^SD~NEDD8 complex. (A) Relative positions of R717 and Q40 in the Cul1-WHB^SD~NEDD8. The red line connects the Nη1 atom of R717 to the Oϵ atom of Q40 and has a length of 0.41 nm. The distance between the atoms drops below 0.32 nm in NEDD8-wt simulations, indicating a hydrogen bond formation. (B) Contact occupancies of R717-Q40 hydrogen bond, and R717-E40 salt-bridge were calculated using cutoffs of 0.32 and 0.5 nm, respectively. Mean ± std. Error is calculated for over three independent 200 ns trajectories. (C) Mean ± std error of the number of contacts formed between NEDD8 (I36/L71) and Cul1-WHB^SD (helix-29) sidechains using a cutoff of 0.45 nm. (D) Average RMSDs of Cul1-WHB^SD~NEDD8 complexes over three independent 200 ns trajectories. ± One std. Error is indicated in ( ).