Protein ubiquitination is a post-translational modification that controls essential biological processes through its regulation of protein concentration, function, and cellular location. RING E3 ligases are a critical component of a three-enzyme cascade that facilitates the ubiquitination of proteins. RING-type E3 ligases represent one class of E3 ligases that function by binding the substrate protein and ubiquitin-conjugating enzymes (E2s). Proteins exhibiting RING-type E3 ligase activities do so via a domain that adopts a ββα-RING fold and coordinates two zinc ions. To date, structural studies show that the RING domain interacts with the catalytic domain of the E2 enzyme. The catalytic domain is approximately 150 amino acids and adopts a canonical structure consisting of four α-helices and 3–4 β-strands. Structural analyses of RING–E2 complexes reveal that RING domains interact on a similar surface of the E2 enzyme. We postulate that the mechanism of interaction between an E2 enzyme and its cognate RING E3 domain may contribute to the extent of substrate modification. In this review, we compare the primary and secondary structures of human E2 enzymes and examine their quaternary structure with RING domains. Our analyses reveal the interactions appear to be relatively conserved with similar types of amino acids involved.

The MID1 TRIM protein is important for ventral midline development in vertebrates, and mutations of its B-box1 domain result in several birth defects. The B-box1 domain of the human MID1 protein binds two zinc atoms and adopt a similar ββα-RING structure. This domain is required for the efficient ubiquitination of protein phosphatase 2A, alpha4, and fused kinase. Considering the structural similarity, the MID1 B-box1 domain exhibits mono-autoubiquitination activity, in contrast to poly-autoubiquitination observed for RING E3 ligases. To understand its mechanism of action, the interaction of the B-box1 domain with Ube2D1 (UbcH5a, E2), a preferred E2 ligase, is investigated. Using isothermal titration calorimetry, the MID1 RING and B-box1 domains were observed to have similar binding affinities with the Ube2D1 protein. However, NMR 15N–1H Heteronuclear Single Quantum Coherence titration, 15N relaxation data, and High Ambiguity Driven protein–protein DOCKing (HADDOCK) calculations show the B-box1 domain binding on a surface distinct from where RING domains bind. The novel binding interaction shows the B-box1 domain partially overlapping the noncovalent Ube2D1 and a ubiquitin binding site that is necessary for poly-autoubiquitination activity. The B-box1 domain also displaces the ubiquitin from the Ube2D1 protein. These studies reveal a novel binding interaction between the zinc-binding ββα-fold B-box1 domain and the Ube2D enzyme family and that this difference in binding, compared to RING E3 ligases, provides a rationale for its auto-monoubiquitination E3 ligase activity.