Assessing the Direct Binding of Ark-Like E3 RING Ligases to Ubiquitin and Its Implication on Their Protein Interaction Network

Abstract

The ubiquitin pathway required for most proteins' targeted degradation involves three classes of enzymes: E1-activating enzyme, E2-conjugating enzyme, and E3-ligases. The human Ark2C is the single known E3 ligase that adopts an alternative, Ub-dependent mechanism for the activation of Ub transfer in the pathway. Its RING domain binds both E2-Ub and free Ub with high affinity, resulting in a catalytic active Ub_R-RING-E2-Ub_D complex formation. We examined potential changes in the conformational plasticity of the Ark2C RING domain and its ligands in their complexed form within the ubiquitin pathway through molecular dynamics (MD). Three molecular mechanics force fields compared to previous NMR relaxation studies of RING domain of Arkadia were used for effective and accurate assessment of MDs. Our results suggest the Ark2C Ub-RING docking site has a substantial impact on maintaining the conformational rigidity of E2-E3 assembly, necessary for the E3's catalytic activity. In the Ub_R-RING-E2-Ub_D catalytic complex, the Ub_R molecule was found to have greater mobility than the other Ub, bound to E2. Furthermore, network-based bioinformatics helped us identify E3 RING ligase candidates which potentially exhibit similar structural modules as Ark2C, along with predicted substrates targeted by the Ub-binding RING Ark2C. Our findings could trigger a further exploration of related unrevealed functions of various other E3 RING ligases.

Keywords: E3 RING ligases; PPI network; molecular dynamics; ubiquitin.

Figure 1

(A) Stabilization of the closed conformation of the E2-Ub thioester conjugation enzyme by E3 RING ligases. In the schematic interaction, E2 is shown to interact with: (i) Canonical dimeric E3 RING domain (e.g., cIAP2, RNF4), (ii) RING ligases with an additional N-terminal component (e.g., RNF38), (iii) Monomeric E3 RING ligases of the Arkadia type (e.g., RNF165). Arrows with dashed line signifies interactions favoring the activation of Ub for transfer to substrates (“closed conformation” or “E2~Ub”); (B) Active and inactive complexes (left panel) and 3D structure of active complex (right panel) of Ark2C with E2 enzyme and Ub.

Figure 2

(A) Structural fluctuations (RMSF of Ca atoms per residue) for the core of RING domain of the human Ark2C E3 ligase simulations (black) and in the complex with E2-Ub in the catalytic conformation (Ub–RING-E2-Ub, red); (B) Structural fluctuations (RMSF of Ca atoms per residue) for the core of RING domain of the human Ark2C E3 ligase simulations (black) and in the complex with E2-Ub in the non-catalytic conformation (RING-E2-Ub, red); (C) Structural fluctuations (RMSF of Ca atoms per residue) for the E2 enzyme in the non-catalytic conformation of the E3-E2-Ub_D complex (black) and in the catalytic conformation of the Ub_R-E3-E2-Ub_D complex (red); (D) Structural fluctuations (RMSF of Ca atoms per residue) for the Ub_R bound to RING domain (black), and Ub_D bound to E2 (red) in the catalytic conformation of the Ub_R-E3-E2-Ub_D complex.

Figure 3

Illustration of the ℓ = 3 network-based approach used to identify (A) potential E3 RING ligases with Ub docking sites and (B) putative Ark and Ark2C interactors. In each case, the element under question is presented in red.

Figure 4

Comparison between the order parameter of the NH bond of the Arkadia RING_927–994 domain predicted by employing the AMBER-99SB-STAR-ILDNP molecular mechanic’s force field and NMR relaxation data [27]. The top plane indicates observations regarding the secondary structure as obtained from the previous NMR-based study [27].