Michael Anthony Massiah

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 fifia-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 monoautoubiquitination 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 Bbox1 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 fifia-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.

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.

Protein ubiquitination is an essential cellular process that maintains protein homeostasis, regulates protein, and cell functions, and removes aggregated and misfolded protein. Disruption in function of any of the protein components of the ubiquitination pathway is associated with human diseases including cancers. An important member in the ubiquitination cascade is the very large E3 ligase family that directs substrate modification. The RING-type E3 ligases possess a cysteine/histidine-rich zinc-binding RING domain that confers ligase functionality. RING domains adopt a canonical ββα-fold. TRIM proteins represent a novel class of RING-type E3 ligase. TRIM proteins consist of an N-terminal RING domain followed by one or two B-box domains. The two types of B-box domains play essential roles in protein ubiquitination by contributing to substrate targeting, ligase activity enhancement, and redundancy of ligase activity. This review presents a general background of the B-box domains, a structural and functional comparison with RING domains, and a summary of recent work demonstrating their role in proteolysis. We discuss new findings that reveal B-box domains which are ubiquitous and are found in non-TRIM plant proteins without the adjacent RING domain, indicating that B-boxes are members of RING-class E3 ligases.

Mutations of human MID1 are associated with X‐linked Opitz G Syndrome (XLOS), which is characterized by midline birth defects. XLOS‐observed mutations within the MID1 B‐box1 domain are associated with cleft lip/palate, wide‐spaced eyes and hyperspadias. Three of the four XLOS‐observed mutations in the B‐box1 domain results in unfolding but the structural and functional effects of the P151L mutation is not characterized. Here, we demonstrate that the P151L mutation does not disrupt the overall tertiary structure of the B‐box1 domain and the adjacent domains. In fact, MID1 E3 ligase activity is slightly enhanced. However, the P151L mutation disrupted the ability of MID1 to catalyze the poly‐ubiquitination of alpha4, a novel regulator of PP2A. This observation is consistent with results observed with the other three structure‐destabilizing B‐box1 mutations in targeting alpha4 but not PP2A. Alpha4 is shown to bind and sequester the catalytic subunit of PP2A and protect it from MID1‐mediated ubiquitination and as a result, an increase in alpha4 can contribute to an increase in PP2A, playing a greater role in midline development during embryogenesis.

The human MID1 protein is required for the proper development during embryogenesis. Mutations of MID1 are associated with X‐linked Opitz G syndrome, characterized by midline anomalies. MID1 associates with the microtubules and functions as an ubiquitin E3 ligase, targeting protein phosphatase 2A for ubiquitin‐mediated regulation. The mechanism of microtubule association is not known. Recently, a 60‐amino acid region termed the C‐terminal subgroup One Signature (COS) box/domain was identified at the C‐terminal end of the coiled‐coil (CC) domain that facilitates microtubule localization. Insertion of the MID1 COS domain at the C‐terminal end of the CC domain of a nonmicrotubule‐associated TRIM protein confers microtubule localization. Here, we report the solution structure of the COS domain of MID1. The domain adopts a helix‐loop‐helix structure in which the N‐ and C‐terminal ends are in close proximity. Hydrophobic residues stabilizing the interaction of the two α‐helices form a central hydrophobic core. The loop separating the α‐helices is structured, with two of its hydrophobic residues making contact with the central core. On the outer surface, positively charged residues form a distinct basic patch near the termini that we postulate is important for microtubule binding. A model of the structure of the preceding coiled‐coil and COS domains (CC‐COS) show that the COS domain forms a helical bundle at the C‐terminal end of the CC domain similar to the spectrin‐like fold observed with some known microtubule‐binding proteins. Interestingly, the CC‐COS domains bind to microtubules, demonstrating for the first time that MID1 can directly associate with the microtubules.Database: Structural data are available in PDB database under the accession number 5IM8.

The zinc-binding Bbox1 domain in protein MID1, a member of the TRIM family of proteins, facilitates the ubiquitination of the catalytic subunit of protein phosphatase 2A and alpha4, a protein regulator of PP2A. The natural mutation of residue A130 to a valine or threonine disrupts substrate recognition and catalysis. While NMR data revealed the A130T mutant Bbox1 domain failed to coordinate both structurally essential zinc ions and resulted in an unfolded structure, the unfolding mechanism is unknown. Principle component analysis revealed that residue A130 served as a hinge point between the structured β-strand-turn-β-strand (β-turn-β) and the lasso-like loop sub-structures that constitute loop1 of the ββα-RING fold that the Bbox1 domain adopts. Backbone RMSD data indicate significant flexibility and departure from the native structure within the first 5 ns of the molecular dynamics (MD) simulation for the A130V mutant (>6 Å) and after 30 ns for A130T mutant (>6 Å). Overall RMSF values were higher for the mutant structures and showed increased flexibility around residues 125 and 155, regions with zinc-coordinating residues. Simulated pKa values of the sulfhydryl group of C142 located near A130 suggested an increased in value to ~9.0, paralleling the increase in the apparent dielectric constants for the small cavity near residue A130. Protonation of the sulfhydryl group would disrupt zinc-coordination, directly contributing to unfolding of the Bbox1. Together, the increased motion of residues of loop 1, which contains four of the six zinc-binding cysteine residues, and the increased pKa of C142 could destabilize the structure of the zinc-coordinating residues and contribute to the unfolding.

MID1 catalyzes the ubiquitination of the protein alpha4 and the catalytic subunit of protein phosphatase 2A. Mutations within the MID1 Bbox1 domain are associated with X-linked Opitz G syndrome (XLOS). Our functional assays have shown that mutations of Ala130 to Val or Thr, Cys142 to Ser and Cys145 to Thr completely disrupt the polyubiquitination of alpha4. Using NMR spectroscopy, we characterize the effect of these mutations on the tertiary structure of the Bbox1 domain by itself and in tandem with the Bbox2 domain. The mutation of either Cys142 or Cys145, each of which is involved in coordinating one of the two zinc ions, results in the collapse of signal dispersion in the HSQC spectrum of the Bbox1 domain indicating that the mutant protein structure is unfolded. Each mutation caused the coordination of both zinc ions, which are ∼13 Å apart, to be lost. Although Ala130 is not involved in the coordination of a zinc ion, the Ala130Thr mutant Bbox1 domain yields a poorly dispersed HSQC spectrum similar to those of the Cys142Ser and Cys145Thr mutants. Interestingly, neither cysteine mutation affects the structure of the adjacent Bbox2 domain when the two Bbox domains are engineered in their native tandem Bbox1-Bbox2 protein construct. Dynamic light scattering measurements suggest that the mutant Bbox1 domain has an increased propensity to form aggregates compared to the wild type Bbox1 domain. These studies provide insight into the mechanism by which mutations observed in XLOS affect the structure and function of the MID1 Bbox1 domain.

MID1 is a microtubule-associated protein that belongs to the TRIM family. MID1 functions as an ubiquitin E3 ligase, and recently was shown to catalyze the polyubiquitination of, alpha4, a protein regulator of protein phosphatase 2A (PP2A). It has been hypothesized that MID1 regulates PP2A, requiring the intermediary interaction with alpha4. Here we report that MID1 catalyzes the in vitro ubiquitination of the catalytic subunit of PP2A (PP2Ac) in the absence of alpha4. In the presence of alpha4, the level of PP2Ac ubiquitination is reduced. Using the MID1 RING-Bbox1-Bbox2 (RB1B2) construct containing the E3 ligase domains, we investigate the functional effects of mutations within the Bbox domains that are identified in patients with X-linked Opitz G syndrome (XLOS). The RB1B2 proteins harboring the C142S, C145T, A130V/T mutations within the Bbox1 domain and C195F mutation within the Bbox2 domain maintain auto-polyubiquitination activity. Qualitatively, the RB1B2 proteins containing these mutations are able to catalyze the ubiquitination of PP2Ac. In contrast, the RB1B2 proteins with mutations within the Bbox1 domain are unable to catalyze the polyubiquitination of alpha4. These results suggest that unregulated alpha4 may be the direct consequence of these natural mutations in the Bbox1 domain of MID1, and hence alpha4 could play a greater role to account for the increased amount of PP2A observed in XLOS-derived fibroblasts.