B-box1 Domain of MID1 Interacts with the Ube2D1 E2 Enzyme Differently Than RING E3 Ligases

Abstract

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.

Absence of the RING domain inMID1results in patterning defects in the developing human brain

“…OS is a rare congenital malformation syndrome characterized by a multitude of symptoms including developmental delay and brain anomalies such as hypoplasia of the cerebellar vermis and the corpus callosum (Baldini et al, 2020). Biochemical dissection of the MID1 protein structure uncovered three zincbinding domains with the E3 polyubiquitin ligase activity provided by the RING (really interesting new gene) domain, whereas the BBox1 and BBox2 domains were shown to convey auto-monoubiquitination E3 ligase activity (Han et al, 2011;Kaur et al, 2023). Besides MID1, disruption of several other proteins containing E3 ubiquitin ligase activity has been associated with neurodevelopmental disorders (Upadhyay et al, 2017;Ebstein et al, 2021), for example, HUWE1 (Froyen et al, 2008) and UBE3A (Kishino et al, 1997), underscoring their significance for brain development.…”