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  • br Results br Discussion LNX

    2020-08-03


    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 Myosmine sale 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. The RING domain was previously considered sufficient to confer ubiquitination by RING-type E3 ligases. However, recent reports have shown that sequences closest to the RING domain are also important in E3 ligase function. Residues like Tyr193 in RNF4 [40], Phe296 and Arg294 in BIRC7 [31], and Lys65 and Asn71 in TRIM25 [34], [37], which lie immediately after the RING domain, make extensive contacts with the Ub moiety to maintain a “closed” conformation. Moreover, sequence alignment shows that these amino acids lie at a similar position and can be clustered together (Fig. 4b). However, in the case of LNX1, additional amino acid residues are needed for such contacts, and these residues are found in the C-terminus, away from the RING domain (Fig. 4b). Interestingly, the buried surface area between the LNX1A and UbB was more than the area shared between LNX1A and UbA. This is contrary to the other reported structures for E3–E2~Ub. This may be due to the longer (128 aa) sequence of the LNX1 construct required for ubiquitination activity in our study, and this may explain the additional contacts shared between LNX1A and UbB. Moreover, we identified the interaction of Lys33 on UbB with the C-terminus of LNX1A, which is critical for the function of LNX1.