Structure and function of the Mind bomb E3 ligase in the context of Notch signal transduction

Abstract

The Notch signaling pathway has a critical role in cell fate determination and tissue homeostasis in a variety of different lineages. In the context of normal Notch signaling, the Notch receptor of the 'signal-receiving' cell is activated in trans by a Notch ligand from a neighboring 'signal-sending' cell. Genetic studies in several model organisms have established that ubiquitination of the Notch ligand, and its regulated endocytosis, is essential for transmission of this activation signal. In mammals, this ubiquitination step is dependent on the protein Mind bomb 1 (Mib1), a large multi-domain RING-type E3 ligase, and its direct interaction with the intracellular tails of Notch ligand molecules. Here, we discuss our current understanding of Mind bomb structure and mechanism in the context of Notch signaling and beyond.

Figure 1

Schematic of major components in the Notch signaling pathway and Mib domain organization. (a) Notch signaling initiates upon transcellular engagement of a Notch ligand with a Notch receptor, and relies on ubiquitin-dependent internalization of the ligand. In vertebrates, ubiquitination is dependent on the E3 ligase Mind bomb 1 (Mib1). Transcellular delivery of force is thought to expose a site (S2) within the Notch negative regulatory region (NRR) for cleavage by ADAM-family metalloproteases. S2 cleavage is followed by cleavage at site S3 by gamma secretase, leading to release and entry of of the intracellular Notch (ICN) domain into the nucleus. Within the nucleus, ICN forms a ternary transcriptional complex with CSL and MAML to regulate target gene expression. (b) Domain organization of Mib1 highlighting the MZM, REP, ANK, and RNG regions of the protein. The MZM domain contains two Mib-Herc2 domains (blue and yellow) flanking a ZZ Zinc finger (green). The REP domain contains two tandem “Mib repeat” elements (orange and red). The ANK domain is composed of nine ankyrin-type repeats (light green), and the C-terminal RNG domain is composed of three RING elements (cyan).

Figure 2

Potential architecture of Mib1 and comparison to a typical Cullin E3 ligase.

Figure 3

Structural features of the Mib1 MZM-REP region. (a) Ribbon representation of the MZM-REP structure, colored as in Figure 1. (b) Comparison of the Mib-Herc2 and Mib repeat domain folds to the SH3 domain of Abl. The variable linker connecting β strands 1 and 2 is highlighted in the color of the analogous domain within the larger MZM-REP fragment shown in panel (a). (c) Peptide binding mode for the Mib-Herc2 and SH3 domains. The Mib-Herc2 and SH3 domains are represented as a surface, and colored as in (b). Respective peptide epitopes are shown as sticks.

Figure 4

Bipartite ligand recognition of Mib1. (a) Co-crystal structure of MZM-REP and a Jag1 N-box peptide. Left panel: Mib1 represented as a surface and colored by conservation on a sliding scale from crimson (most conserved), to cyan (least conserved). The N-box peptide is represented as sticks (yellow, colored by atom type). Middle panel: close-up view of the Mib-Herc2:peptide binding interface. Right panel: sequence alignment of N-box epitopes from human (h) and Drosophila (d) Notch ligands. (b) Structure of the isolated Mib repeat region and intermolecular crystal contacts. Mib repeats 1 and 2 are shown in the same orientation and colored by surface charge (red: acidic, blue: basic). Peptides from the N-terminus and C-terminus of neighboring molecules are shown as sticks (yellow, and colored by atom). (c) Model of Nrf2 recognition by a Keap1 homodimer, and (d) schematic of ligand recognition by Mib1 in cis (left) and in trans (right).