Skip to main content
. Author manuscript; available in PMC: 2017 Aug 25.
Published in final edited form as: Cell. 2016 Aug 25;166(5):1198–1214.e24. doi: 10.1016/j.cell.2016.07.027

Figure 5. Activity Profiling of ARIH1 Mutants: “Signatures” Identifying Functions in UB Ligation to Neddylated CRL Substrates.

Figure 5

(A) Hierarchical assay scheme to profile selected ARIH1 mutants in neddylated CRL pathway. The sum of results from all assays form a signature, or “fingerprint” identifying normal function(s) of a mutated surface. Color coding of functions is maintained in structures and signatures in (B)–(I), except signatures VII and VIII that signify dual functions (orange/purple and orange/red). Details are in Figure S5.

(B) Close up showing ARIH1 catalytic Cys that is normally sequestered by autoinhibitory intramolecular RING2-Ariadne (ARI) domain interface (4KBL.PDB). Hyperactive mutant sites are spheres: magenta, relieve autoinhibition even without neddylated CRL, thus termed “OPEN” due to mutationally opening the RING2-ARI interface; cyan, facilitate “opening” by neddylated CRL, monitored by reactivity with *UB-VME. OPEN mutant B was combined with other mutations for assays 3, 4, and 5 in subsequent profiles.

(C) Activity profile signatures of hyperactive mutants, normalized to WT ARIH1, defined by assays 1–3.

(D) Model of ARIH1 RING2~UB intermediate based on 4KBL.PDB and 4LJO.PDB. Sites of activity profile signatures indicating roles in catalytic UB binding or intrinsic ligation activity are in blue and purple spheres, respectively.

(E) Activity profile signatures for mutants in (D).

(F) ARIH1's RING1 and UBA domains modeled with docked E2 or UBL (NEDD8/UB), respectively (4KBL.PDB, 5EDV.PDB, 4UN2.PDB). Sites of mutants with activity profile signatures indicating roles in UB transfer from E2 (UBCH7) to ARIH1 and neddylated CRL-dependent UB transfer from UBCH7 to ARIH1 are in gray and green spheres, respectively.

(G) Activity profile signatures for mutants in (F).

(H) ARIH1 structure as in (B), rotated ≈60° about x, highlighting residues in RING2 and ARI domains with dual roles in two colors: neddylated CUL-dependent relief of ARIH1 autoinhibition, orange; role in intrinsic RING2-mediated UB ligation to a Lys nucleophile, purple; role in specifically targeting CRL substrate, red.

(I) Activity profile signatures for mutants in (H).