Molecular recognition of M1-linked ubiquitin chains by native and phosphorylated UBAN domains

Abstract

Although the Ub-binding domain in ABIN proteins and NEMO (UBAN) is highly conserved, UBAN-containing proteins exhibit different Ub-binding properties, resulting in their diverse biological roles. Post-translational modifications further control UBAN domain specificity for poly-Ub chains. However, precisely, how the UBAN domain structurally confers such functional diversity remains poorly understood. Here we report crystal structures of ABIN-1 alone and in complex with one or two M1-linked di-Ub chains. ABIN-1 UBAN forms a homo-dimer that provides two symmetrical Ub-binding sites on either side of the coiled-coil structure. Moreover, crystal structures of ABIN1 UBAN in complex with di-Ub chains reveal a concentration-dependency of UBAN/di-Ub binding stoichiometry. Analysis of UBAN/M1-linked di-Ub binding characteristics indicates that phosphorylated S473 in OPTN and its corresponding phospho-mimetic residue in ABIN-1 (E484) are essential for high affinity interactions with M1-linked Ub chains. Also, a phospho-mimetic mutation of A303 in NEMO, corresponding to S473 of OPTN, increases binding affinity for M1-linked Ub chains. These findings are in line with the diverse physiological roles of UBAN domains, as phosphorylation of OPTN UBAN is required to enhance its binding to Ub during mitophagy.

The Anti-Inflammatory Protein TNIP1 Is Intrinsically Disordered with Structural Flexibility Contributed by Its AHD1-UBAN Domain

“…This was supported by compatible data from analytical gel filtration and DLS ( Figures 6 and 7, respectively) experiments which determined the molecular radius of TNIP1 417-509 at 28.1 and 29.6 Å,~1.8× greater than would be expected for a globular form of a protein with similar predicted molecular weight. The analytical gel-filtration and DLS experiments revealed initial data with a potential dimer population of TNIP1 417-509 , which is not unexpected based on reports of the UBAN-only region of murine TNIP1 [21] presenting the UBAN entirely as a coiled-coil. In contrast, our data suggest a mixed population with a secondary peak ( Figure 6-double asterisk) from analytical gel-filtration and PDI of 0.291, indicating only moderate polydispersity within our TNIP1 417-509 samples.…”

“…Furthermore, TNIP1 features a phospho-mimetic residue (E470) that corresponds to a critical phosphorylation site in the UBAN of optineurin. Mutation of the E470 residue of TNIP1 promotes a decrease in affinity for polyubiquitin [21]. The expected intrinsically disordered nature of the full-length TNIP1 was found conserved across 23 species (all species had average PONDR-FIT residue scores greater than 0.6 ranging from 0.62-0.71) despite Clustal Omega alignment showing less than 50% sequence identity (Supplemental Table S2B).…”

“…Considering peptide regions expressed in structural studies for the analogous regions of human TNIP2 and mouse TNIP1 [21,22], we generated a human TNIP1 AA417-509 expression construct (TNIP1 417-509 ) with a C-terminus HIS6 tag (12.7 kDa total predicted molecular weight (MW)). As with the full-length protein work above, solubilization and purification of the TNIP1 417-509 protein was accomplished with heat lysis of bacterial pellets (Figure 4, TNIP1-lane five) which in multiple reports of recombinant expression of IDPs has proven to promote enrichment of the intact disordered protein and preferential reduction in other heat-denatured proteins (e.g., proteases).…”

LUBAC-mediated linear ubiquitination: a crucial regulator of immune signaling

“…79) The ABIN1 protein has a UBAN domain that binds to linear ubiquitin chains with high affinity. 80) Loss of ABIN1 or the ubiquitin-binding activity of ABIN1 (ABIN1 D485N) in mice causes glomerulonephritis, which is characteristic of lupus nephritis with a high titer of pathogenic autoantibodies, including anti- nuclear antibody and anti-double strand DNA antibody. 81), 82) The involvement of TLR7-MyD88-IRAK4 signaling pathways in lupus nephritis has been suggested, 83) and lupus nephritis in ABIN1 D485N mice is ameliorated by IRAK4 inhibition.…”

Receptor-interacting protein kinase 1 (RIPK1) as a therapeutic target

“…TNIP1 , encoding the ABIN1 protein, is one of the highest scoring non-MHC genes associated with various autoimmune and autoinflammatory diseases, including psoriasis, psoriatic arthritis, systemic sclerosis and systemic lupus erythematosus (SLE) across multiple GWAS 54 . ABIN1, a ubiquitin binding protein with a UBAN domain that binds preferentially to linear ubiquitin chains, interacts with A20 to negatively regulate the activation of MAPK and NF-κB-mediated gene transcription downstream of TNFR1 and TLRs 89 – 91 . ABIN1, which is recruited into the TNFR1 signalling complex by binding linear ubiquitin chains, in turn promotes the recruitment of A20 (ref.…”