Structural And Functional Insights Into Human MEX-3C Ring Finger Domain As An E3 Ubiquitin Ligase

E3 ubiquitin ligases(E3s)are the most attractive targets for potential drug discovery owing to substrate specificity.The E3 ubiquitin ligases are large proteins,working as multi subunit complexes.E3s control the half-life of different proteins and are crucial to ensure the appropriate destruction of transcription factors that orchestrate worldwide cellular processes such as cell division,differentiation,and apoptosis.All of them belongs to two families,which are Ring(Really Interesting New Gene)and HECT(Homologous to the E6AP Carboxyl Terminus).The RING-in-between-RING(RBR)E3s are a curious family of E3 ubiquitin ligases.RBR E3 ligases are unusual E3 ligases,containing multiple RING domains and facilitated ubiquitination in a similar manner to the RING E3 enzymes.The process of ubiquitination modification consists of three main enzymatic steps with the sequential process of a ubiquitin-activating enzyme(E1),a ubiquitin-conjugating enzyme(E2)and a ubiquitin ligase(E3).A family of protein homologous to the Caenorhabditis elegans(C.elegans)MEX-3(MEX-3A-3D)was identified in human and also mice,involved with the post-transcriptional regulation.All of them have two K homology(KH)RNA-binding domains at the N-terminal and a Ring finger domain at the C-terminal.Among these four homologous,MEX-3C has been reported to be involved in several diseases,including cancer,genetic hypertension,postnatal growth,and immune responses.Recent study has illustrated that the human MEX-3C(hMEX-3C)-mediated RIG-I ubiquitination is necessary to the antiviral immune responses.Furthermore,MEX-3C has been demonstrated to be associated with the degradation of mRNA,including the post-transcriptional regulation of ordinary Human Leukocyte Antigen Serotype A2(HLA-A2)allotypes.MEX-3C can bind to the 3’UTR of HLA-A2,and induce Ring finger domain dependent mRNA degradation.However,the structural basis on the ubiquitination catalyzed by the Ring finger domain of hMEX-3C remains to be elucidated.In our study,we determined the crystal structure the Ring finger domain of hMEX-3C and superimposed the structure of the Ring finger domain with the complex structure of MDM2-MDMX-UbcH5b-Ub.Our autoubiquitination assays revealed that the Ring finger domain of hMEX-3C acts as an E3 ubiquitin ligase in vitro and interacts with specific E2 to mediate ubiquitination.Furthermore,we figured out several principal residues in the Ring finger domain of hMEX-3C probably associated with the interaction with the E2-Ub conjugate and analyzed the activities of the E3 ubiquitin ligase of wild type(WT)and mutants at the key sites.In addition,the Zinc(Zn)chelation experiment suggested that the intact structural stability is important for the activity of self-ubiquitination of the Ring finger domain of hMEX-3C.Based on these results,our studies provide the new observation into the mechanism of the Ring finger domain of hMEX-3C that may play an essential role to elicit the antiviral immune responses and therapeutic interventions.