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  • The LNX ligand of numb protein X or PDZRN

    2021-04-29

    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 [14]. LNX proteins comprise a RING domain and two to four PDZ (PSD-95, DlgA, ZO-1) domains [14]. The LNX1 protein was first identified as a binding partner to the mNumb tyrosine-binding domain [15] and, later, its over-expression was shown to increase the proliferation—not differentiation—of neuroepithelial Sotalol [16]. Nie et al. [17] 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 [18] and facilitates the endocytosis of junction adhesion molecule-4 [19], a cell adhesion molecule participating in tight junctions. LNX1 also ubiquitinates and regulates ErbB2 receptors in perisynaptic Schwann cells [20], and acts as a transcriptional regulator of activator protein 1 by interacting with and impeding the function of RhoC via its PDZ domain [21]. 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 [22], [23], [24], [25], [26], 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.
    Results
    Discussion LNX1 was first identified as a binding partner of Numb and interacts with c-Src in a PDZ-dependent manner, triggering its ubiquitination [18]. 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). We also identified Ubc13 as a functional E2 for LNX1 and determined the complex structure of LNX1–Ubc13~Ub, which depicts the first step of the ubiquitination process (Fig. 3a.). Comparing structural alignment of the RNF4 RING: Ubc13~Ub: Ube2V2 complex (PDB code 5AIU) [33] and the RNF4:Ubc13~Ub complex (PDB code 5AIT) [33], we noted that there is no conformational change in the RNF4: Ubc13~Ub interface, as Ube2V2 binds only to Ubc13 and faces away from the interface. In our complex structure, although Ube2V2 is absent, we speculate that, similar to RNF4, the binding of the second E2 might not change the interface between LNX1 and Ubc13~Ub. RING-type E3 ligases tend to form homodimers and heterodimers for their activity [30]. The complex structure of the LNX1–Ubc13~Ub further explains this need for dimerization, as the dimer stabilizes a “closed” E2~Ub conformation, which enhances the rate of Ub transfer, as reported earlier [31], [32], [37]. We show that LNX1 makes additional contacts with Ub to impose this “closed” conformation, with N-terminal loop residues Asp36, Asp38, and Lys100 of LNX1A contacting the Lys11 of UbA (Fig. 5b). A recent report noted the formation of a similar contact between glutamate (Glu10) of TRIM25 and Lys11 of Ub [37]. However, we note that both Asp36 and Asp38 of LNX1 interact with Lys100 of the other protomer to maintain dimerization and simultaneously interact with Lys11 of Ub (Fig. 5b). Moreover, mutating Asp36 or Asp38 disrupts the activity of LNX1 (Fig. 5c, d). This is a unique feature in LNX1, where the same set of amino acids is required for dimerization and also to interact with the Ub moiety to keep E2–Ub in a “closed” configuration. In other complex structures, like RNF4–UbcH5a~Ub [32] and BIRC7–UbcH5b~Ub [31], the C-terminal region is responsible for dimerization and stabilization of the E2~Ub complex.