Christopher D. Lima

RNA surveillance and decay is carried out in part by helicase containing complexes that identify, capture and sometimes modify RNA before delivering it to the RNA exosome complex for processing or degradation. The MTREC core complex includes a Mtr4-like protein (Mtl1) helicase and Red1 that works with other cofactors and the RNA exosome in Schizosaccharomyces pombe to degrade nuclear transcripts in processes that can result in formation of facultative heterochromatin. The activities of Mtl1 remain uncharacterized as do contributions of Red1 to Mtl1 within MTREC. Here, we reconstitute the MTREC core complex, resolve structures by cryo-electron microscopy, and compare MTREC activities to S. pombe Mtr4 and Mtl1. We show that Mtl1 is more active relative to MTREC and Mtr4, that MTREC binds RNA better than Mtl1, and that Red1 includes an autoinhibitory coiled-coil domain that dimerizes MTREC and contacts the Mtl1 RecA domains to disrupt its ATPase active site. Together, these data suggest that Red1 may endow MTREC to bind RNA while slowing translocation so that it remains associated with RNA long enough to chaperone it to the RNA exosome for processing or degradation.

The AAA+ protein p97/VCP and its cofactor UFD1/NPL4 interact with and unfold ubiquitinated proteins to promote disaggregation and unfolding for recycling or to prepare substrates for proteasomal degradation. The cofactor Fas-associated factor 1 (FAF1) is suggested to reduce the length of ubiquitin chain required for substrate unfolding by UFD1/NPL4-p97 and to interact with SUMO. Here, we employ in vitro reconstitution of UFD1/NPL4-p97 and FAF1/UFD1/NPL4-p97 complexes and fluorescent substrates modified with SUMO2-polyubiquitin hybrid or polyubiquitin-only chains of varying lengths to assess initial rates of unfolding. These assays reveal that FAF1 enhances initial rates of unfolding relative to UFD1/NPL4-p97 in a manner that is independent of SUMO2 and semi-dependent on ubiquitin chain length. Unlike preferences observed for yeast Ufd1/Npl4-Cdc48, these data suggest that the FAF1 cofactor does not contribute to preferential unfolding of the SUMO2-polyubiquitin substrates tested. Further dissection of FAF1 reveals that it significantly increases the rate of unfolding for all ubiquitin chain lengths tested with its greatest differential impact observed when unfolding chains with four to ten ubiquitin molecules that are considered physiologically relevant. Using cryoEM we resolve a series of reconstructions that reveal FAF1/UFD1/NPL4-p97 bound to substrate in non-translocating and translocating states. Observed interactions between a helix of FAF1 and UFD1 throughout the unfolding process are consistent with AlphaFold models and recent reports suggesting that FAF1 may stabilize interactions between UFD1, NPL4, and p97 to promote substrate engagement and unfolding.

The human nucleoporin RanBP2/Nup358 interacts with SUMO1-modified RanGAP1 and the SUMO E2 Ubc9 at the nuclear pore complex (NPC) to promote export and disassembly of exportin Crm1/Ran(GTP)/cargo complexes. In mitosis, RanBP2/SUMO1-RanGAP1/Ubc9 remains intact after NPC disassembly and is recruited to kinetochores and mitotic spindles by Crm1 where it contributes to mitotic progression. RanBP2 binds SUMO1-RanGAP1/Ubc9 via motifs that also catalyze SUMO E3 ligase activity. Here, we resolve cryo-EM structures of a RanBP2 C-terminal fragment in complex with Crm1, SUMO1-RanGAP1/Ubc9, and two molecules of Ran(GTP). These structures reveal several interactions with Crm1 including a nuclear export signal (NES) for RanGAP1, the deletion of which mislocalizes RanGAP1 and the Ran GTPase in cells. Our structural and biochemical results support models in which RanBP2 E3 ligase activity is dependent on Crm1, the RanGAP1 NES and Ran GTPase cycling.

Conjugation of ubiquitin (Ub) and structurally related ubiquitin‐like proteins (Ubls), essential for many cellular processes, employs multi‐step reactions orchestrated by specific E1, E2 and E3 enzymes. The E1 enzyme activates the Ub/Ubl C‐terminus in an ATP‐dependent process that results in the formation of a thioester linkage with the E1 active site cysteine. The thioester‐activated Ub/Ubl is transferred to the active site of an E2 enzyme which then interacts with an E3 enzyme to promote conjugation to the target substrate. The E1‐E2‐E3 enzymatic cascades utilize labile intermediates, extensive conformational changes, and vast combinatorial diversity of short‐lived protein‐protein complexes to conjugate Ub/Ubl to various substrates in a regulated manner. In this review, we discuss various chemical tools and methods used to study the consecutive steps of Ub/Ubl activation and conjugation, which are often too elusive for direct studies. We focus on methods developed to probe enzymatic activities and capture and characterize stable mimics of the transient intermediates and transition states, thereby providing insights into fundamental mechanisms in the Ub/Ubl conjugation pathways.

The human nucleoporin RanBP2/Nup358 interacts with SUMO1-modified RanGAP1 and the SUMO E2 Ubc9 at the nuclear pore complex (NPC) to promote export and disassembly of exportin Crm1/Ran(GTP)/cargo complexes. In mitosis, RanBP2/SUMO1-RanGAP1/Ubc9 remains intact after NPC disassembly and is recruited to kinetochores and mitotic spindles by Crm1 where it contributes to mitotic progression. Interestingly, RanBP2 binds SUMO1-RanGAP1/Ubc9 via motifs that also catalyze SUMO E3 ligase activity. Here, we resolve cryo-EM structures of a RanBP2 C-terminal fragment in complex with Crm1, SUMO1-RanGAP1/Ubc9, and two molecules of Ran(GTP). These structures reveal several unanticipated interactions with Crm1 including a nuclear export signal (NES) for RanGAP1, the deletion of which mislocalizes RanGAP1 and the Ran GTPase in cells. Our structural and biochemical results support models in which RanBP2 E3 ligase activity is dependent on Crm1, the RanGAP1 NES and Ran GTPase cycling.

Transthiolation (also known as transthioesterification) reactions are used in the biosynthesis of acetyl coenzyme A, fatty acids and polyketides, and for post-translational modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins^1–3. For the Ub pathway, E1 enzymes catalyse transthiolation from an E1~Ub thioester to an E2~Ub thioester. Transthiolation is also required for transfer of Ub from an E2~Ub thioester to HECT (homologous to E6AP C terminus) and RBR (ring-between-ring) E3 ligases to form E3~Ub thioesters^4–6. How isoenergetic transfer of thioester bonds is driven forward by enzymes in the Ub pathway remains unclear. Here we isolate mimics of transient transthiolation intermediates for E1–Ub(T)–E2 and E2–Ub(T)–E3^HECT complexes (where T denotes Ub in a thioester or Ub undergoing transthiolation) using a chemical strategy with native enzymes and near-native Ub to capture and visualize a continuum of structures determined by single-particle cryo-electron microscopy. These structures and accompanying biochemical experiments illuminate conformational changes in Ub, E1, E2 and E3 that are coordinated with the chemical reactions to facilitate directional transfer of Ub from each enzyme to the next.

Ubiquitin (Ub) regulates a wide array of cellular processes through post-translational modification of protein substrates. Ub is conjugated at its C-terminus to target proteins via an enzymatic cascade in which covalently bound Ub thioesters are transferred from E1 activating enzymes to E2 conjugating enzymes, and then to certain E3 protein ligases. These transthioesterification reactions proceed via transient tetrahedral intermediates. A variety of chemical strategies have been used to capture E1-Ub-E2 and E2-Ub-E3 mimics, but these introduce modifications that disrupt atomic spacing at the linkage point relative to the native tetrahedral intermediate. We have developed a biselectrophilic PSAN warhead that can be installed in place of the conserved C-terminal glycine in Ub and used to form ternary protein complexes linked via cyanomethyldithioacetals that closely mimic the native tetrahedral intermediates. Investigation of the reactivity of the warhead and substituted analogues led to an effective semisynthetic route to Ub^-1-PSAN, which was used to form a ternary E1-Ub*-E2 complex as a mimic of the transthioesterification intermediate.

The Ufd1/Npl4/Cdc48 complex is a universal protein segregase that plays key roles in eukaryotic cellular processes. Its functions orchestrating the clearance or removal of polyubiquitylated targets are established; however, prior studies suggest that the complex also targets substrates modified by the ubiquitin-like protein SUMO. Here, we show that interactions between Ufd1 and SUMO enhance unfolding of substrates modified by SUMO–polyubiquitin hybrid chains by the budding yeast Ufd1/Npl4/Cdc48 complex compared to substrates modified by polyubiquitin chains, a difference that is accentuated when the complex has a choice between these substrates. Incubating Ufd1/Npl4/Cdc48 with a substrate modified by a SUMO–polyubiquitin hybrid chain produced a series of single-particle cryo-EM structures that reveal features of interactions between Ufd1/Npl4/Cdc48 and ubiquitin prior to and during unfolding of ubiquitin. These results are consistent with cellular functions for SUMO and ubiquitin modifications and support a physical model wherein Ufd1/Npl4/Cdc48, SUMO, and ubiquitin conjugation pathways converge to promote clearance of proteins modified with SUMO and polyubiquitin.