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The ESCRT pathway’s AAA+ ATPase, Vps4p, remodels ESCRT-III complexes to drive membrane fission. Here, we use peptide binding assays to further the understanding of substrate specificity and the mechanism of autoinhibition. Our results revealed unexpected sequence preference to the substrate binding groove and an elegant mechanism of regulation that couples localization to substrate with release from autoinhibition.

The p97 AAA+ATPase is an essential and abundant regulator of protein homeostasis that plays a central role in unfolding ubiquitylated substrates. Here we report two cryo-EM structures of human p97 in complex with its p47 adaptor. One of the conformations is six-fold symmetric, corresponds to previously reported structures of p97, and lacks bound substrate. The other structure adopts a helical conformation, displays substrate running in an extended conformation through the pore of the p97 hexamer, and resembles structures reported for other AAA unfoldases. These findings support the model that p97 utilizes a “hand-over-hand” mechanism in which two residues of the substrate are translocated for hydrolysis of two ATPs, one in each of the two p97 AAA ATPase rings. Proteomics analysis supports the model that one p97 complex can bind multiple substrate adaptors or binding partners, and can process substrates with multiple types of ubiquitin modification. The p97 unfoldase is an essential and abundant enzyme that segregates its substrates from macromolecular complexes and organelle membranes. Here, authors determined the structure of human p97 in the act of unfolding an authentic substrate.

Many important cellular membrane fission reactions are driven by ESCRT pathways, which culminate in disassembly of ESCRT-III polymers by the AAA ATPase Vps4. We report a 4.3 Å resolution cryo-EM structure of the active Vps4 hexamer with its cofactor Vta1, ADP·BeFx, and an ESCRT-III substrate peptide. Four Vps4 subunits form a helix whose interfaces are consistent with ATP binding, is stabilized by Vta1, and binds the substrate peptide. The fifth subunit approximately continues this helix but appears to be dissociating. The final Vps4 subunit completes a notched-washer configuration as if transitioning between the ends of the helix. We propose that ATP binding propagates growth at one end of the helix while hydrolysis promotes disassembly at the other end, so that Vps4 ‘walks’ along ESCRT-III until it encounters the ordered N-terminal domain to destabilize the ESCRT-III lattice. This model may be generally applicable to other protein-translocating AAA ATPases. Membranes surround multiple compartments within cells as well as the cell itself. In living cells, these membranes are remodeled continuously. This allows cells to divide, move molecules between different compartments and perform other essential activities. One important remodeling event is known as fission, which splits a membrane into separate parts. Large repeating structures (or polymers) of ESCRT-III proteins play a crucial role in membrane fission. Breaking apart ESCRT-III polymers triggers membrane fission and also recycles the ESCRT-III proteins so that they can be used again. An enzyme called Vps4 converts chemical energy (stored in the form of a molecule called ATP) into the mechanical force that breaks apart the ESCRT-III polymers. The active form of Vps4 consists of six Vps4 subunits working together to form a complex that includes a cofactor protein called Vta1. Monroe et al. have now used a technique called cryo-electron microscopy to determine the structure of an active yeast Vps4-Vta1 complex while it is bound to a segment of an ESCRT-III protein. This revealed that four of the six Vps4 subunits form a helix (which resembles a spiral staircase) that binds ESCRT-III in its central pore. The structure implies that binding of ATP causes the Vps4 helix to grow at one end and that converting ATP into a molecule called ADP (to release energy) causes disassembly at the other end. The two additional Vps4 subunits move from the disassembling end to the growing end of the helix. In this manner, Vps4 ‘walks’ along ESCRT-III, thereby pulling it through the pore at the center of the Vps4 complex and triggering breakdown of the ESCRT-III polymer. Further work is now needed to understand exactly how this activity leads to membrane fission.

Eukaryotic cells employ the ribosome-associated quality control complex (RQC) to maintain homeostasis despite defects that cause ribosomes to stall. The RQC comprises the E3 ubiquitin ligase Ltn1p, the ATPase Cdc48p, Rqc1p, and Rqc2p. Upon ribosome stalling and splitting, the RQC assembles on the 60S species containing unreleased peptidyl-tRNA (60S:peptidyl–tRNA). Ltn1p and Rqc1p facilitate ubiquitination of the incomplete nascent chain, marking it for degradation. Rqc2p stabilizes Ltn1p on the 60S and recruits charged tRNAs to the 60S to catalyze elongation of the nascent protein with carboxy-terminal alanine and threonine extensions (CAT tails). By mobilizing the nascent chain, CAT tailing can expose lysine residues that are hidden in the exit tunnel, thereby supporting efficient ubiquitination. If the ubiquitin–proteasome system is overwhelmed or unavailable, CAT-tailed nascent chains can aggregate in the cytosol or within organelles like mitochondria. Here we identify Vms1p as a tRNA hydrolase that releases stalled polypeptides engaged by the RQC. The ribosome-associated quality control complex (RQC) functions to disassemble stalled ribosomes. Here the authors find that the tRNA hydrolase Vms1 is involved in the release of nascent peptide from stalled ribosomes.

The hexameric AAA ATPase Vps4 drives membrane fission by remodeling and disassembling ESCRT-III filaments. Building upon our earlier 4.3 Å resolution cryo-EM structure (Monroe et al., 2017 ), we now report a 3.2 Å structure of Vps4 bound to an ESCRT-III peptide substrate. The new structure reveals that the peptide approximates a β-strand conformation whose helical symmetry matches that of the five Vps4 subunits it contacts directly. Adjacent Vps4 subunits make equivalent interactions with successive substrate dipeptides through two distinct classes of side chain binding pockets formed primarily by Vps4 pore loop 1. These pockets accommodate a wide range of residues, while main chain hydrogen bonds may help dictate substrate-binding orientation. The structure supports a ‘conveyor belt’ model of translocation in which ATP binding allows a Vps4 subunit to join the growing end of the helix and engage the substrate, while hydrolysis and release promotes helix disassembly and substrate release at the lagging end.

The Cdc48 AAA+ ATPase is an abundant and essential enzyme that unfolds substrates in multiple protein quality control pathways. The enzyme includes two conserved AAA+ ATPase motor domains, D1 and D2, that assemble as hexameric rings with D1 stacked above D2. Here, we report an ensemble of native structures of Cdc48 affinity purified from budding yeast lysate in complex with the adaptor Shp1 in the act of unfolding substrate. Our analysis reveals a continuum of structural snapshots that spans the entire translocation cycle. These data uncover elements of Shp1-Cdc48 interactions and support a ‘hand-over-hand’ mechanism in which the sequential movement of individual subunits is closely coordinated. D1 hydrolyzes ATP and disengages from substrate prior to D2, while D2 rebinds ATP and re-engages with substrate prior to D1, thereby explaining the dominant role played by the D2 motor in substrate translocation/unfolding. The Cdc48 enzyme is an abundant and essential enzyme that functions in many cellular pathways as a protein unfoldase. Here, the authors determine an ensemble of Cdc48 structures that capture snapshots of its unfolding action using a ‘hand-over-hand’ mechanism.

The ESCRT pathway facilitates membrane fission events during enveloped virus budding, multivesicular body formation, and cytokinesis. To promote HIV budding and cytokinesis, the ALIX protein must bind and recruit CHMP4 subunits of the ESCRT-III complex, which in turn participate in essential membrane remodeling functions. Here, we report that the Bro1 domain of ALIX binds specifically to C-terminal residues of the human CHMP4 proteins (CHMP4A-C). Crystal structures of the complexes reveal that the CHMP4 C-terminal peptides form amphipathic helices that bind across the conserved concave surface of ALIXBro₁. ALIX-dependent HIV-1 budding is blocked by mutations in exposed ALIXBro₁ residues that help contribute to the binding sites for three essential hydrophobic residues that are displayed on one side of the CHMP4 recognition helix (M/L/IxxLxxW). The homologous CHMP1-3 classes of ESCRT-III proteins also have C-terminal amphipathic helices, but, in those cases, the three hydrophobic residues are arrayed with L/I/MxxxLxxL spacing. Thus, the distinct patterns of hydrophobic residues provide a \"code\" that allows the different ESCRT-III subunits to bind different ESCRT pathway partners, with CHMP1-3 proteins binding MIT domain-containing proteins, such as VPS4 and Vta1/LIP5, and CHMP4 proteins binding Bro1 domain-containing proteins, such as ALIX.

Bone morphogenetic protein4 (BMP4) plays numerous roles during embryogenesis and can signal either alone as a homodimer, or together with BMP7 as a more active heterodimer. BMPs are generated as inactive precursor proteins that dimerize and are cleaved to generate the bioactive ligand and inactive prodomain fragments. In humans, heterozygous mutations within the prodomain of BMP4 are associated with birth defects. We studied the effect of two of these mutations (p.S91C and p.E93G), which disrupt a conserved FAM20C phosphorylation motif, on ligand activity. We compared the activity of ligands generated from BMP4, BMP4 S91C , or BMP4 E93G in Xenopus embryos and found that these mutations reduce the activity of BMP4 homodimers but not BMP4/7 heterodimers. We generated Bmp4 S91C and Bmp4 E93G knock-in mice and found that Bmp4 S91C/S91C mice die by E11.5 and display reduced BMP activity in multiple tissues including the heart. Most Bmp4 E93G/E93G mice die before weaning and Bmp4 −/E93G mutants die prenatally with reduced or absent eyes, heart, and ventral body wall closure defects. Mouse embryonic fibroblasts (MEFs) isolated from Bmp4 S91C and Bmp4 E93G embryos show accumulation of BMP4 precursor protein, reduced levels of cleaved BMP ligand and reduced BMP activity relative to MEFs from wild type littermates. Because Bmp7 is not expressed in MEFs, the accumulation of unprocessed BMP4 precursor protein in mice carrying these mutations most likely reflects an inability to cleave BMP4 homodimers, leading to reduced levels of ligand and BMP activity in vivo. Our results suggest that phosphorylation of the BMP4 prodomain is required for proteolytic activation of BMP4 homodimers, but not heterodimers.

During HIV-1 entry, the highly conserved gp41 N-trimer pocket region becomes transiently exposed and vulnerable to inhibition. Using mirror-image phage display and structure-assisted design, we have discovered protease-resistant D-amino acid peptides (D-peptides) that bind the N-trimer pocket with high affinity and potently inhibit viral entry. We also report high-resolution crystal structures of two of these D-peptides in complex with a pocket mimic that suggest sources of their high potency. A trimeric version of one of these peptides is the most potent pocket-specific entry inhibitor yet reported by three orders of magnitude (IC₅₀ = 250 pM). These results are the first demonstration that D-peptides can form specific and high-affinity interactions with natural protein targets and strengthen their promise as therapeutic agents. The D-peptides described here address limitations associated with current L-peptide entry inhibitors and are promising leads for the prevention and treatment of HIV/AIDS.