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In response to complement activation, the membrane attack complex (MAC) assembles from fluid-phase proteins to form pores in lipid bilayers. MAC directly lyses pathogens by a ‘multi-hit’ mechanism; however, sublytic MAC pores on host cells activate signalling pathways. Previous studies have described the structures of individual MAC components and subcomplexes; however, the molecular details of its assembly and mechanism of action remain unresolved. Here we report the electron cryo-microscopy structure of human MAC at subnanometre resolution. Structural analyses define the stoichiometry of the complete pore and identify a network of interaction interfaces that determine its assembly mechanism. MAC adopts a ‘split-washer’ configuration, in contrast to the predicted closed ring observed for perforin and cholesterol-dependent cytolysins. Assembly precursors partially penetrate the lipid bilayer, resulting in an irregular β-barrel pore. Our results demonstrate how differences in symmetric and asymmetric components of the MAC underpin a molecular basis for pore formation and suggest a mechanism of action that extends beyond membrane penetration. The membrane attack complex (MAC) is an immune effector that kills pathogens by forming pores in their membrane. Here the authors use cryo-electron microscopy to reveal that the full MAC is an asymmetric pore with a split-washer configuration and identify a network of interactions that provide a basis for sequential assembly.

The membrane attack complex (MAC) is one of the immune system’s first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant β-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how β-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions. The complement membrane attack complex (MAC) is a lytic immune pore that kills pathogens. Here the authors use cryoEM to provide a structural and biophysical mechanism for how β-pore forming proteins breach the lipid bilayer, providing pathways to explore pore-formation in molecular detail.

The activity of dynein is regulated by a number of adaptors that mediate its interaction with dynactin, effectively activating the motor complex while also connecting it to different cargos. The regulation of adaptors is consequently central to dynein physiology but remains largely unexplored. We now describe that one of the best-known dynein adaptors, BICD2, is effectively activated through phosphorylation. In G2, phosphorylation of BICD2 by CDK1 promotes its interaction with PLK1. In turn, PLK1 phosphorylation of a single residue in the N-terminus of BICD2 results in a structural change that facilitates the interaction with dynein and dynactin, allowing the formation of active motor complexes. Moreover, modified BICD2 preferentially interacts with the nucleoporin RanBP2 once RanBP2 has been phosphorylated by CDK1. BICD2 phosphorylation is central for dynein recruitment to the nuclear envelope, centrosome tethering to the nucleus and centrosome separation in the G2 and M phases of the cell cycle. This work reveals adaptor activation through phosphorylation as crucial for the spatiotemporal regulation of dynein activity. The dynein motor complex has a variety of important functions in both dividing and non-dividing cells. Here, Gallisà et al. describe a mode of regulation of dynein based on the phosphorylation of its adaptor BICD2 by the kinase PLK1, and how this is central for the regulation of centrosome position in G2 and M.

Colchicine has been used to treat gout and, more recently, to effectively prevent autoinflammatory diseases and both primary and recurrent episodes of pericarditis. The anti-inflammatory action of colchicine seems to result from irreversible inhibition of tubulin polymerization and microtubule (MT) assembly by binding to the tubulin heterodimer, avoiding the signal transduction required to the activation of the entire NLRP3 inflammasome. Emerging results show that the MT network is a potential regulator of cardiac mechanics. Here, we investigated how colchicine impacts in tubulin folding cofactors TBCA, TBCB, and TBCE activities. We show that TBCA is abundant in mouse heart insoluble protein extracts. Also, a decrease of the TBCA/β-tubulin complex followed by an increase of free TBCA is observed in human cells treated with colchicine. The presence of free TBCA is not observed in cells treated with other antimitotic agents such as nocodazole or cold shock, neither after translation inhibition by cycloheximide. In vitro assays show that colchicine inhibits tubulin heterodimer dissociation by TBCE/TBCB, probably by interfering with interactions of TBCE with tubulin dimers, leading to free TBCA. Manipulation of TBCA levels, either by RNAi or overexpression results in decreased levels of tubulin heterodimers. Together, these data strongly suggest that TBCA is mainly receiving β-tubulin from the dissociation of preexisting heterodimers instead of newly synthesized tubulins. The TBCE/TBCB+TBCA system is crucial for controlling the critical concentration of free tubulin heterodimers and MT dynamics in the cells by recycling the tubulin heterodimers. It is conceivable that colchicine affects tubulin heterodimer recycling through the TBCE/TBCB+TBCA system producing the known benefits in the treatment of pericardium inflammation.

SETD8 is a methyltransferase that is overexpressed in several cancers, which monomethylates H4K20 as well as other non-histone targets such as PCNA or p53. We here report novel SETD8 inhibitors, which were discovered while trying to identify chemicals that prevent 53BP1 foci formation, an event mediated by H4K20 methylation. Consistent with previous reports, SETD8 inhibitors induce p53 expression, although they are equally toxic for p53 proficient or deficient cells. Thermal stability proteomics revealed that the compounds had a particular impact on nucleoli, which was confirmed by fluorescent and electron microscopy. Similarly, Setd8 deletion generated nucleolar stress and impaired ribosome biogenesis, supporting that this was an on-target effect of SETD8 inhibitors. Furthermore, a genome-wide CRISPR screen identified an enrichment of nucleolar factors among those modulating the toxicity of SETD8 inhibitors. Accordingly, the toxicity of SETD8 inhibition correlated with MYC or mTOR activity, key regulators of ribosome biogenesis. Together, our study provides a new class of SETD8 inhibitors and a novel biomarker to identify tumors most likely to respond to this therapy.

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1, and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.

TELO2‐TTI1‐TTI2 (TTT) and R2TP are multi‐subunit chaperones that cooperate with HSP90 to assemble matured complexes of the PIKK family of kinases, including mTOR complex 1 (mTORC1). WAC, a protein previously implicated in transcription, H2B ubiquitination, and autophagy, was recently identified as a regulator of mTORC1 in response to glucose and glutamine availability, acting in concert with R2TP and TTT. However, the molecular basis of the interactions of WAC with R2TP and TTT and their role in mTORC1 regulation remains poorly defined. Here, we characterized the interactions of WAC with mTOR, R2TP, and TTT and how these are affected by nutrient conditions. Using purified proteins, we establish that WAC directly binds to mTOR‐mLST8, R2TP, and TELO2, but not TTI1 and TTI2. In cells, WAC is part of complexes containing components of mTORC1, R2TP, and TTT, and these associations are modulated by nutrient availability. Notably, WAC and TELO2 strongly associate with mTOR under glucose and glutamine deprivation, and these interactions are weakened minutes after nutrient refeeding. These dynamics correlate with changes in mTORC1 activity. Transcriptomic and proteomic analysis shows that WAC, mTOR, R2TP, and TTT are co‐expressed across several human cancers, supporting that WAC is part of a functional pathway with mTOR, R2TP, and TTT. Together, our findings reveal the formation and disassembly of a WAC complex with mTOR and TELO2 that contributes to regulate mTORC1 in response to glucose and glutamine availability. TTT and R2TP chaperone complexes are required for the assembly and activation of mTORC1. WAC directly interacts with components of TTT, R2TP, and mTORC1, and these interactions are affected by the availability of glucose and glutamine, correlating with changes in mTORC1 activity. Thus, the interaction of WAC with TTT and R2TP could contribute to regulating mTORC1 in response to nutrients.

Microtubule nucleation by the γ-tubulin ring complex (γTuRC) is spatiotemporally regulated and in higher eukaryotes is thought to involve a transition from an inactive open to an active closed conformation that matches the microtubule geometry. However, γTuRC activators only promote a partially closed conformation, raising the question of whether complete closure is required for activation. Combining in vitro nucleation assays and cryo-EM, we find that centrosomin motif 1 (CM1), a conserved element of several γTuRC regulators, potently accelerates human γTuRC-mediated microtubule nucleation by facilitating complete closure of γTuRC as the nascent microtubule assembles. A 3.7 Å cryo-EM structure identifies the γTuRC latch and several interactions involved in conformational closure. Notably, the distinct subunits that keep γTuRC open and inactive in higher eukaryotes also participate in its closure and activation. This work provides additional insight into the logic of the human γTuRC architecture and its activation by CM1.

Eukaryotic ectotherms of the Southern Ocean face energetic challenges to protein folding assisted by the cytosolic chaperonin CCT. We hypothesize that CCT and its client proteins (CPs) have co-evolved molecular adaptations that facilitate CCT–CP interaction and the ATP-driven folding cycle at low temperature. To test this hypothesis, we compared the functional and structural properties of CCT–CP systems from testis tissues of an Antarctic fish, Gobionotothen gibberifrons (Lönnberg) (habitat/body T = −1.9 to +2°C), and of the cow (body T = 37°C). We examined the temperature dependence of the binding of denatured CPs (β-actin, β-tubulin) by fish and bovine CCTs, both in homologous and heterologous combinations and at temperatures between −4°C and 20°C, in a buffer conducive to binding of the denatured CP to the open conformation of CCT. In homologous combination, the percentage of G. gibberifrons CCT bound to CP declined linearly with increasing temperature, whereas the converse was true for bovine CCT. Binding of CCT to heterologous CPs was low, irrespective of temperature. When reactions were supplemented with ATP, G. gibberifrons CCT catalyzed the folding and release of actin at 2°C. The ATPase activity of apo-CCT from G. gibberifrons at 4°C was ∼2.5-fold greater than that of apo-bovine CCT, whereas equivalent activities were observed at 20°C. Based on these results, we conclude that the catalytic folding cycle of CCT from Antarctic fishes is partially compensated at their habitat temperature, probably by means of enhanced CP-binding affinity and increased flexibility of the CCT subunits.

CCT/TRiC is a eukaryotic multi-subunit chaperonin that promotes the correct folding of many cytosolic proteins, including tubulin, within its cavity. Now the crystal structure of CCT in its open state is solved to 5.5-Å resolution and, together with EM and biochemical analysis, allows the observation of a bound tubulin molecule interacting with CCT loops in the apical and equatorial domains. Protein folding is assisted by molecular chaperones. CCT (chaperonin containing TCP-1, or TRiC) is a 1-MDa oligomer that is built by two rings comprising eight different 60-kDa subunits. This chaperonin regulates the folding of important proteins including actin, α-tubulin and β-tubulin. We used an electron density map at 5.5 Å resolution to reconstruct CCT, which showed a substrate in the inner cavities of both rings. Here we present the crystal structure of the open conformation of this nanomachine in complex with tubulin, providing information about the mechanism by which it aids tubulin folding. The structure showed that the substrate interacts with loops in the apical and equatorial domains of CCT. The organization of the ATP-binding pockets suggests that the substrate is stretched inside the cavity. Our data provide the basis for understanding the function of this chaperonin.