MPN domain DUBs can act in isolation as

MPN+ domain DUBs can act in isolation, as exemplified by AMSH-LP, or in macromolecular assemblies to cleave poly ubiquitin chains of varied linkage types. In protein complexes, an MPN+ domain can associate with pseudo MPN domains (MPN–) for purposes that are not well understood. MPN– domains are recognizable by the absence of essential Zn2+-coordinating residues that are required for catalytic function. Established MPN+–MPN– domain pairings include CSN5 and CSN6 in the COP9 signalosome and RPN11 and RPN8 in the proteasome. While the mode of MPN+–MPN– interaction is conserved between these two systems (Deshaies, 2014, Lingaraju et al., 2014, Pathare et al., 2014, Worden et al., 2014), no precise function has been ascribed to either MPN– domain, and neither structure has been probed deeply for insights into how the MPN– domain might support MPN+ domain function. The importance of the MPN– subunit in BRISC and ARISC minimally serves a 2-fold purpose, first to support DUB activity and second to mediate protein–protein interactions. KIAA0157 and Abraxas are required for the catalytic function of BRCC36 in both complexes and enable association of two additional shared subunits (BRCC45 and MERIT40) to each holoenzyme. Moreover, BRCC36 interactions with KIAA0157 or Abraxas are necessary for association with the holoenzyme specific adaptor subunits, SHMT2 and RAP80, which target their respective DUB complex to sites of K63-Ub synthesis.
Results
Discussion Our protein NG25 mg and co-purification studies on ARISC and BRISC in insect cells, native mass spectrometry analysis of HsBRISC, crystallographic and SAXS analyses on the CfBRCC36–KIAA0157 complex, mutational and (in vitro and cellular) functional studies on ARISC and BRISC support the minimal architecture model for BRISC shown in Figure S2 (boxed). This model is applicable to ARISC with only the substitution of the paralogous subunit of KIAA0157 with Abraxas (40% identity, 65% similarity). Two BRCC36–KIAA0157 heterodimers assemble as a higher-order super dimer at the center of the holoenzyme. Each KIAA0157 subunit recruits a BRCC45 subunit (as inferred by mass spectrometry Figure S2B, red oval), which in turn recruits a MERIT40 subunit. The pseudo DUBs KIAA0157 and Abraxas are required for the catalytic function of BRCC36. Comparison of the BRCC36–KIAA0157 heterodimer structure with an inactive BRCC36 homodimer structure provides a model for understanding how this functional interplay is achieved. As shown in schematic form in Figure 5B, the binding of KIAA0157 to BRCC36 is required for stabilization of (1) the CCHB, (2) the E-loop of BRCC36 and optimal positioning of the catalytic glutamate side chain for catalysis, and (3) the Ins-1 loop of BRCC36 and its proper positioning for substrate binding. Stabilization of all three elements is supported through direct interactions between KIAA0157 and BRCC36 within a single heterodimer as well as through interactions across BRCC36–KIAA0157 heterodimers within a super dimer. Despite the modeled vicinity of the proximal ubiquitin moiety to the opposing BRCC36 protomer within the super dimer (Figure 7A), our data demonstrate that super-dimerization does not contribute positively to substrate recognition (Figure 7C).