High-Throughput Assay for Characterizing Rpn11 Deubiquitinase Activity.

Rpn11 is an essential metalloprotease responsible for the en bloc removal of ubiquitin chains from protein substrates that are targeted for degradation by the 26S proteasome. A unique feature of Rpn11 is that its deubiquitinase (DUB) activity is greatly stimulated by the mechanical translocation of the substrate into the proteasomal AAA+ (ATPase Associated with diverse cellular Activities) motor, which delivers the scissile isopeptide bond between a substrate lysine and the proximal moiety of an attached ubiquitin chain to the DUB catalytic active site. As a consequence, Rpn11 cleaves at the base of ubiquitin chains and lacks selectivity towards specific ubiquitin-chain linkage types, which is in contrast to other DUBs, including the related AMSH that selectively cleaves Lys63-linked chains. Prevention of Rpn11's deubiquitinase activity leads to inhibition of proteasomal degradation by stalling substrate translocation. With the proteasome as an approved anticancer target, Rpn11 is therefore an attractive point of attack for the development of new inhibitors, which requires robust biochemical assays to measure DUB activity. Here we describe a method for the purification of the Rpn8/Rpn11 heterodimer and ubiquitin-GC-TAMRA, a model substrate that can be used to characterize the DUB activity of Rpn11 in isolation without the need of purifying 26S proteasomes. This assay thus enables a high-throughput screening platform for Rpn11-targeted small-molecule discovery.

Multistate structures of the MLL1-WRAD complex bound to H2B-ubiquitinated nucleosome.

The human Mixed Lineage Leukemia-1 (MLL1) complex methylates histone H3K4 to promote transcription and is stimulated by monoubiquitination of histone H2B. Recent structures of the MLL1-WRAD core complex, which comprises the MLL1 methyltransferase, WDR5, RbBp5, Ash2L, and DPY-30, have revealed variability in the docking of MLL1-WRAD on nucleosomes. In addition, portions of the Ash2L structure and the position of DPY30 remain ambiguous. We used an integrated approach combining cryoelectron microscopy (cryo-EM) and mass spectrometry cross-linking to determine a structure of the MLL1-WRAD complex bound to ubiquitinated nucleosomes. The resulting model contains the Ash2L intrinsically disordered region (IDR), SPRY insertion region, Sdc1-DPY30 interacting region (SDI-motif), and the DPY30 dimer. We also resolved three additional states of MLL1-WRAD lacking one or more subunits, which may reflect different steps in the assembly of MLL1-WRAD. The docking of subunits in all four states differs from structures of MLL1-WRAD bound to unmodified nucleosomes, suggesting that H2B-ubiquitin favors assembly of the active complex. Our results provide a more complete picture of MLL1-WRAD and the role of ubiquitin in promoting formation of the active methyltransferase complex.

Lethal factor unfolding is the most force-dependent step of anthrax toxin translocation.

Cellular compartmentalization requires machinery capable of translocating polypeptides across membranes. In many cases, transported proteins must first be unfolded by means of the proton motive force and/or ATP hydrolysis. Anthrax toxin, which is composed of a channel-forming protein and two substrate proteins, is an attractive model system to study translocation-coupled unfolding, because the applied driving force can be externally controlled and translocation can be monitored directly by using electrophysiology. By controlling the driving force and introducing destabilizing point mutations in the substrate, we identified the barriers in the transport pathway, determined which barrier corresponds to protein unfolding, and mapped how the substrate protein unfolds during translocation. In contrast to previous studies, we find that the protein's structure next to the signal tag is not rate-limiting to unfolding. Instead, a more extensive part of the structure, the amino-terminal beta-sheet subdomain, must disassemble to cross the unfolding barrier. We also find that unfolding is catalyzed by the channel's phenylalanine-clamp active site. We propose a broad molecular mechanism for translocation-coupled unfolding, which is applicable to both soluble and membrane-embedded unfolding machines.