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Apoptosis, the intrinsic programmed cell death process, is mediated by the Bcl-2 family members Bak and Bax. Activation via formation of symmetric core dimers and oligomerization on the mitochondrial outer membrane (MOM) leads to permeabilization and cell death. Although this process is linked to the MOM, the role of the membrane in facilitating such pores is poorly understood. We recently described Bak core domain dimers, revealing lipid binding sites and an initial role of lipids in oligomerization. Here we describe simulations that identified localized clustering and interaction of triacylglycerides (TAGs) with a minimized Bak dimer construct. Coalescence of TAGs occurred beneath this Bak dimer, mitigating dimer-induced local membrane thinning and curvature in representative coarse-grain MOM and model membrane systems. Furthermore, the effects observed as a result of coarse-grain TAG cluster formation was concentration dependent, scaling from low physiological MOM concentrations to those found in other organelles. We find that increasing the TAG concentration in liposomes mimicking the MOM decreased the ability of activated Bak to permeabilize these liposomes. These results suggest that the presence of TAGs within a Bak-lipid membrane preserves membrane integrity and is associated with reduced membrane stress, suggesting a possible role of TAGs in Bak-mediated apoptosis.

Neutrophils help to clear pathogens and cellular debris, but can also cause collateral damage within inflamed tissues. Prolonged neutrophil residency within an inflammatory niche can exacerbate tissue pathology. Using both genetic and pharmacological approaches, we show that BCL-XL is required for the persistence of neutrophils within inflammatory sites in mice. We demonstrate that a selective BCL-XL inhibitor (A-1331852) has therapeutic potential by causing apoptosis in inflammatory human neutrophils ex vivo. Moreover, in murine models of acute and chronic inflammatory disease, it reduced inflammatory neutrophil numbers and ameliorated tissue pathology. In contrast, there was minimal effect on circulating neutrophils. Thus, we show a differential survival requirement in activated neutrophils for BCL-XL and reveal a new therapeutic approach to neutrophil-mediated diseases.

The BCL-2 family of proteins (including the prosurvival proteins BCL-2, BCL-XL, and MCL-1) is an important target for the development of novel anticancer therapeutics. Despite the challenges of targeting protein–protein interaction (PPI) interfaces with small molecules, a number of inhibitors (called BH3 mimetics) have entered the clinic and the BCL-2 inhibitor, ABT-199/venetoclax, is already proving transformative. For BCL-XL, new validated chemical series are desirable. Here, we outline the crystallography-guided development of a structurally distinct series of BCL-XL/BCL-2 inhibitors based on a benzoylurea scaffold, originally proposed as α-helix mimetics. We describe structure-guided exploration of a cryptic “p5” pocket identified in BCL-XL. This work yields novel inhibitors with submicromolar binding, with marked selectivity toward BCL-XL. Extension into the hydrophobic p2 pocket yielded the most potent inhibitor in the series, binding strongly to BCL-XLand BCL-2 (nanomolar-range half-maximal inhibitory concentration (IC50)) and displaying mechanism-based killing in cells engineered to depend on BCL-XLfor survival.

Apoptosis is a form of programmed cell death that is regulated by the balance between prosurvival and proapoptotic BCL-2 protein family members. Evasion of apoptosis is a hallmark of cancer that arises when this balance is tipped in favour of survival. One form of anticancer therapeutic, termed ‘BH3-mimetic drugs’, has been developed to directly activate the apoptosis machinery in malignant cells. These drugs bind to and inhibit specific prosurvival BCL-2 family proteins, thereby mimicking their interaction with the BH3 domains of proapoptotic BCL-2 family proteins. The BCL-2-specific inhibitor venetoclax is approved by the US Food and Drug Administration and many regulatory authorities worldwide for the treatment of chronic lymphocytic leukaemia and acute myeloid leukaemia. BH3-mimetic drugs targeting other BCL-2 prosurvival proteins have been tested in preclinical models of cancer, and drugs targeting MCL-1 or BCL-XLhave advanced into phase I clinical trials for certain cancers. As with all therapeutics, efficacy and tolerability need to be carefully balanced to achieve a therapeutic window whereby there is significant anticancer activity with an acceptable safety profile. In this Review, we outline the current state of BH3-mimetic drugs targeting various prosurvival BCL-2 family proteins and discuss emerging data regarding primary and acquired resistance to these agents and approaches that may overcome this. We highlight issues that need to be addressed to further advance the clinical application of BH3-mimetic drugs, both alone and in combination with additional anticancer agents (for example, standard chemotherapeutic drugs or inhibitors of oncogenic kinases), for improved responses in patients with cancer.

Plasmodium falciparumcauses the severe form of malaria that has high levels of mortality in humans. Blood-stage merozoites of P. falciparuminvade erythrocytes, and this requires interactions between multiple ligands from the parasite and receptors in hosts. These interactions include the binding of the Rh5–CyRPA–Ripr complex with the erythrocyte receptor basigin1,2, which is an essential step for entry into human erythrocytes. Here we show that the Rh5–CyRPA–Ripr complex binds the erythrocyte cell line JK-1 significantly better than does Rh5 alone, and that this binding occurs through the insertion of Rh5 and Ripr into host membranes as a complex with high molecular weight. We report a cryo-electron microscopy structure of the Rh5–CyRPA–Ripr complex at subnanometre resolution, which reveals the organization of this essential invasion complex and the mode of interactions between members of the complex, and shows that CyRPA is a critical mediator of complex assembly. Our structure identifies blades 4–6 of the β-propeller of CyRPA as contact sites for Rh5 and Ripr. The limited contacts between Rh5–CyRPA and CyRPA–Ripr are consistent with the dissociation of Rh5 and Ripr from CyRPA for membrane insertion. A comparision of the crystal structure of Rh5–basigin with the cryo-electron microscopy structure of Rh5–CyRPA–Ripr suggests that Rh5 and Ripr are positioned parallel to the erythrocyte membrane before membrane insertion. This provides information on the function of this complex, and thereby provides insights into invasion by P. falciparum.

Necroptosis is a regulated caspase-independent form of necrotic cell death that results in an inflammatory phenotype. This process contributes profoundly to the pathophysiology of numerous neurodegenerative, cardiovascular, infectious, malignant, and inflammatory diseases. Receptor-interacting protein kinase 1 (RIPK1), RIPK3, and the mixed lineage kinase domain-like protein (MLKL) pseudokinase have been identified as the key components of necroptosis signaling and are the most promising targets for therapeutic intervention. Here, we review recent developments in the field of small-molecule inhibitors of necroptosis signaling, provide guidelines for their use as chemical probes to study necroptosis, and assess the therapeutic challenges and opportunities of such inhibitors in the treatment of a range of clinical indications.

The membrane-spanning α helices of single-pass receptors play crucial roles in stabilizing oligomeric structures and transducing biochemical signals across the membrane. Probing intermolecular transmembrane interactions in single-pass receptors presents unique challenges, reflected in a gross underrepresentation of their membrane-embedded domains in structural databases. Here, we present two high-resolution structures of transmembrane assemblies from a eukaryotic single-pass protein crystallized in a lipidic membrane environment. Trimeric and tetrameric structures of the immunoreceptor signaling module DAP12, determined to 1.77-Å and 2.14-Å resolution, respectively, are organized by the same polar surfaces that govern intramembrane assembly with client receptors. We demonstrate that, in addition to the well-studied dimeric form, these trimeric and tetrameric structures are made in cells, and their formation is competitive with receptor association in the ER. The polar transmembrane sequences therefore act as primary determinants of oligomerization specificity through interplay between charge shielding and sequestration of polar surfaces within helix interfaces.

An attractive approach for developing therapeutic peptides is to enhance binding to their targets by stabilizing their α-helical conformation, for example, stabilized BimBH3 peptides (BimSAHB) designed to induce apoptosis. Unexpectedly, we found that such modified peptides have reducedaffinity for their targets, the pro-survival Bcl-2 proteins. We attribute this loss in affinity to disruption of a network of stabilizing intramolecular interactions present in the bound state of the native peptide. Altering this network may compromise binding affinity, as in the case of the BimBH3 stapled peptide studied here. Moreover, cells exposed to these peptides do not readily undergo apoptosis, strongly indicating that BimSAHB is not inherently cell permeable.

A model for the cytoplasmic domain of the M2 proton channel of influenza A virus was formulated based primarily on the cytoplasmic domain of the Vpu protein of HIV-1 using sequence similarity and structure prediction techniques. The model consists of a pair of antiparallel helices followed by a strand parallel to the first helix. Structural analogies with other proteins contribute support for features of the model and suggest ways in which the M2 cytoplasmic domain can interact with other viral and cellular factors.

Previously, two binding sites for interleukin 5 (IL-5) were identified on the IL-5 receptor alpha chain (IL-5Rα). They are located within the CD loop of the first fibronectin type III (FnIII)-like domain and the EF loop of the second FnIII-like domain. The first binding site was identified by exploiting the different abilities of human IL-5Rα (hIL-5Rα) and mouse IL-5Rα (mIL-5Rα) to bind hIL-5. Here we show that ovine IL-5 (oIL-5) has the ability to activate the hIL-5Rα but not the mIL-5Rα. By using chimeras of the mIL-5Rα and hIL-5Rα we demonstrate that residues within the first and third FnIII-like domains of mIL-5Rα are responsible for this lack of activity. Furthermore, mutation of residues on hIL-5Rα to mIL-5Rα within the predicted DE and FG loop regions of the third FnIII domain reduces oIL-5 activity. These results show that regions of the third FnIII domain of IL-5Rα are involved in binding, in addition to the regions in domains one and two of the IL-5Rα that were identified in an earlier study.

In mice there are two forms of the beta chain used in the IL3 receptor system, βcomand βIL3. βcomis used by the IL3, IL5 and GM‐CSF receptors whereas βIL3is only used in the IL3 receptor. In this work an assay was developed to identify residues of βIL3that restrict IL5 activity. It was found that such residues reside within the 2nd CRM of the molecule. Furthermore, when residues in the βIL3B′‐C′ loop were replaced with βcomsequence a form of βIL3was produced that was able to respond to IL5. This region is also responsible for IL3 binding to βIL3in the absence of alpha chain. It is therefore an important structural motif of βcomand βIL3responsible for both ligand interaction and specificity.

Solving the structure of the hinge domain of an epigenetic regulator highlights residues involved in dimerization and function.

Blombery: Janssen: Honoraria; Invivoscribe: Honoraria; Novartis: Consultancy. Anderson:Walter and Eliza Hall Institute: Employment, Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.. Seymour:Acerta: Consultancy; Celgene: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy. Huang:Genentech: Patents & Royalties: DCSH is an employee of the Walter and Eliza Hall Institute which receives milestone and royalty payments related to venetoclax. Roberts:AbbVie: Other: Unremunerated speaker for AbbVie, Research Funding; Australasian Leukaemia and Lymphoma Group: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Walter and Eliza Hall Institute: Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.; BeiGene: Research Funding.