Ubiquitination can occur on a single monoubiquitination
Ubiquitination can occur on a single (monoubiquitination) or several (multiubiquitination) lysine residues, or the same lysine residue can be subjected to several rounds of Ub conjugation (polyubiquitination). Ub bonds within polyUb chains are formed through 1 of 7 lysine residues in Ub, and the types of chains that form will dictate how the protein will be processed by the cell. For example, polyUb chains predominantly composed of Ub K48- or K29-links will target a protein for proteasomal degradation, whereas other Ub chains (K63-linked Ub chains, for example) regulate kinase activation, DNA damage, signal transduction, and endocytosis , .
E2–E3 enzyme combinations differentially affect ubiquitination outcomes . Moreover, E3 enzymes have defined activities and bind selectively to substrates. Indeed, deregulation of E3 ligases can affect cell growth signals, DNA repair and result in various malignancies . Among the various E3 ligases identified, RING finger-type E3 ligases are the most abundant. The E3 RING domain requires the action of a specific E2 to transfer Ub to the substrate or other Ub molecules (free polyUb chains); our study adds new insight into how one of these RING-type E3 ligases selectively catalyzes the transfer of Ub to the appropriate substrate or Ub itself.
The LNX (ligand of numb protein-X) or PDZRN (PDZ and RING) family of proteins consists of five members (LNX1–LNX5), the first four of which act as E3‐ubiquitin ligases in various roles, as reviewed elsewhere . LNX proteins comprise a RING domain and two to four PDZ (PSD-95, DlgA, ZO-1) domains . The LNX1 protein was first identified as a binding partner to the mNumb tyrosine-binding domain  and, later, its over-expression was shown to increase the proliferation—not differentiation—of neuroepithelial amikacin sulfate . Nie et al.  were the first to show that LNX1 functions as an E3‐ubiquitin ligase and degrades Numb in a Ub-dependent manner. LNX1 also interacts with and ubiquitinates c-Src kinase  and facilitates the endocytosis of junction adhesion molecule-4 , a cell adhesion molecule participating in tight junctions. LNX1 also ubiquitinates and regulates ErbB2 receptors in perisynaptic Schwann cells , and acts as a transcriptional regulator of activator protein 1 by interacting with and impeding the function of RhoC via its PDZ domain .
Despite extensive knowledge on the functional implications of LNX1, no structural analyses have been undertaken to explore the E3 ligase activity of this protein. As a continuation of our efforts to understand the actions of E3 ligases , , , , , here we report the structural and biochemical studies of LNX1. We identified Ubc13 as a new E2 for LNX1 and solved the complex structure of LNX1 with Ubc13 conjugated to Ub. This structure uncovers a new interface between LNX1 and Ub, and unravels the mechanism by which Ub-loaded Ubc13 is recruited for Ub transfer via the E3 ligase activity of LNX1.
Discussion LNX1 was first identified as a binding partner of Numb and interacts with c-Src in a PDZ-dependent manner, triggering its ubiquitination . Until recently, no structure-based mechanism had been established for the LNX family of proteins. Here, we identified the ubiquitination domain and mechanism of action of LNX1. Although, both LNX1 and LNX2 possess a RING domain flanked by two Zn-finger motifs (aligned with an rmsd of 0.31Å for 95 Cα atoms; sequence identity 65%) (Fig. 8c), only LNX1 requires both Zn-finger motifs for its activity. Moreover, similar to LNX2, LNX1 also shares structural homology to TRAF6 (rmsd of 1.47Å for 75 Cα atoms; sequence identity 27%), which extends to the RING and C-terminal Zn finger motif only (Fig. 8d). To our knowledge, LNX1 and LNX2 are the only E3 ligases with such domain architecture; LNX3 and LNX4 only harbor the C-terminal zinc finger motif (Supplementary Fig. 8a).