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Gasdermins mediate inflammatory cell death after cleavage by caspases or other, unknown enzymes. The cleaved N-terminal fragments bind to acidic membrane lipids to form pores, but the mechanism of pore formation remains unresolved. Here we present the cryo-electron microscopy structures of the 27-fold and 28-fold single-ring pores formed by the N-terminal fragment of mouse GSDMA3 (GSDMA3-NT) at 3.8 and 4.2 Å resolutions, and of a double-ring pore at 4.6 Å resolution. In the 27-fold pore, a 108-stranded anti-parallel β-barrel is formed by two β-hairpins from each subunit capped by a globular domain. We identify a positively charged helix that interacts with the acidic lipid cardiolipin. GSDMA3-NT undergoes radical conformational changes upon membrane insertion to form long, membrane-spanning β-strands. We also observe an unexpected additional symmetric ring of GSDMA3-NT subunits that does not insert into the membrane in the double-ring pore, which may represent a pre-pore state of GSDMA3-NT. These structures provide a basis that explains the activities of several mutant gasdermins, including defective mutants that are associated with cancer. High-resolution cryo-electron microscopy structures of the membrane-pore-forming domain of the mouse gasdermin GSDMA3 show that it forms pores with 26-, 27- or 28-fold symmetry and indicate that it may also form a parallel, soluble, pre-pore ring structure.

ABCG2 is a transporter protein of the ATP-binding-cassette (ABC) family that is expressed in the plasma membrane in cells of various tissues and tissue barriers, including the blood–brain, blood–testis and maternal–fetal barriers 1 – 4 . Powered by ATP, it translocates endogenous substrates, affects the pharmacokinetics of many drugs and protects against a wide array of xenobiotics, including anti-cancer drugs 5 – 12 . Previous studies have revealed the architecture of ABCG2 and the structural basis of its inhibition by small molecules and antibodies 13 , 14 . However, the mechanisms of substrate recognition and ATP-driven transport are unknown. Here we present high-resolution cryo-electron microscopy (cryo-EM) structures of human ABCG2 in a substrate-bound pre-translocation state and an ATP-bound post-translocation state. For both structures, we used a mutant containing a glutamine replacing the catalytic glutamate (ABCG2 EQ ), which resulted in reduced ATPase and transport rates and facilitated conformational trapping for structural studies. In the substrate-bound state, a single molecule of estrone-3-sulfate (E 1 S) is bound in a central, hydrophobic and cytoplasm-facing cavity about halfway across the membrane. Only one molecule of E 1 S can bind in the observed binding mode. In the ATP-bound state, the substrate-binding cavity has collapsed while an external cavity has opened to the extracellular side of the membrane. The ATP-induced conformational changes include rigid-body shifts of the transmembrane domains, pivoting of the nucleotide-binding domains (NBDs), and a change in the relative orientation of the NBD subdomains. Mutagenesis and in vitro characterization of transport and ATPase activities demonstrate the roles of specific residues in substrate recognition, including a leucine residue that forms a ‘plug’ between the two cavities. Our results show how ABCG2 harnesses the energy of ATP binding to extrude E 1 S and other substrates, and suggest that the size and binding affinity of compounds are important for distinguishing substrates from inhibitors. Cryo-electron microscopy structures of the ABCG2 protein in ATP- and substrate-bound states reveal the location of substrate binding, conformational changes required for substrate translocation and how inhibitors might be distinguished from substrates.

Exploring the genetic basis of congenital cataracts in two families identifies a molecule, lanosterol, which prevents intracellular protein aggregation of various cataract-causing mutant crystallins, and which can reduce cataract severity and increase lens transparency in vivo in dogs. Lanosterol counters cataract formation In a study of the genetic basis of congenital cataract formation in two families, Kang Zhang and colleagues demonstrate that lanosterol, a sterol present naturally in the lens, can prevent intracellular aggregation of various cataract-causing mutant crystallin proteins. The mutations identified in the genetic study impair the function of lanosterol synthase, an enzyme for synthesizing lanosterol. In dogs with naturally occurring cataracts, the application of eye drops containing lanosterol for six weeks reduced cataract severity and increased lens transparency, suggesting that lanosterol or molecules with similar activity might provide an alternative to surgery for the management of cataracts. The human lens is comprised largely of crystallin proteins assembled into a highly ordered, interactive macro-structure essential for lens transparency and refractive index. Any disruption of intra- or inter-protein interactions will alter this delicate structure, exposing hydrophobic surfaces, with consequent protein aggregation and cataract formation. Cataracts are the most common cause of blindness worldwide, affecting tens of millions of people 1 , and currently the only treatment is surgical removal of cataractous lenses. The precise mechanisms by which lens proteins both prevent aggregation and maintain lens transparency are largely unknown. Lanosterol is an amphipathic molecule enriched in the lens. It is synthesized by lanosterol synthase (LSS) in a key cyclization reaction of a cholesterol synthesis pathway. Here we identify two distinct homozygous LSS missense mutations (W581R and G588S) in two families with extensive congenital cataracts. Both of these mutations affect highly conserved amino acid residues and impair key catalytic functions of LSS. Engineered expression of wild-type, but not mutant, LSS prevents intracellular protein aggregation of various cataract-causing mutant crystallins. Treatment by lanosterol, but not cholesterol, significantly decreased preformed protein aggregates both in vitro and in cell-transfection experiments. We further show that lanosterol treatment could reduce cataract severity and increase transparency in dissected rabbit cataractous lenses in vitro and cataract severity in vivo in dogs. Our study identifies lanosterol as a key molecule in the prevention of lens protein aggregation and points to a novel strategy for cataract prevention and treatment.

Amyloid aggregation of α-synuclein (α-syn) is closely associated with Parkinson’s disease (PD) and other synucleinopathies. Several single amino-acid mutations (e.g. E46K) of α-syn have been identified causative to the early onset of familial PD. Here, we report the cryo-EM structure of an α-syn fibril formed by N-terminally acetylated E46K mutant α-syn (Ac-E46K). The fibril structure represents a distinct fold of α-syn, which demonstrates that the E46K mutation breaks the electrostatic interactions in the wild type (WT) α-syn fibril and thus triggers the rearrangement of the overall structure. Furthermore, we show that the Ac-E46K fibril is less resistant to harsh conditions and protease cleavage, and more prone to be fragmented with an enhanced seeding capability than that of the WT fibril. Our work provides a structural view to the severe pathology of the PD familial mutation E46K of α-syn and highlights the importance of electrostatic interactions in defining the fibril polymorphs. The E46K α-synuclein mutation causes familial Parkinson’s disease. Here, the authors present the cryo-EM structure of N-terminally acetylated E46K α-synuclein fibrils and find that it is distinct from other known α-synuclein fibril structures.

Mutants of the C 4 -symmetric protein RhuA were designed to self-assemble into two-dimensional crystalline lattices with precise spatial arrangements and patterns; the lattices of one of the variants are auxetic and deform perpendicularly to an applied force in a way that is contrary to what is generally expected in typical materials. Protein assemblies designed to surprise Auxetic materials are those that, because of their internal structure, deform perpendicularly to an applied force in a manner opposite to what is generally expected. So, when stretched, they get thicker across their width, and when compressed they get thinner. Akif Tezcan and colleagues have created a crystalline protein lattice that demonstrates such behaviour, dependent on the positioning and type of linkages between each individual protein unit. They designed mutants of the C 4 -symmetric protein RhuA to self-assemble into two-dimensional crystalline lattices with precise spatial arrangements and patterns. Disulfide bonds and metal-mediated coordination between units provide a balance between robustness and flexibility, such that large, low-defect lattices are formed that exhibit coherent rotational motion in response to an applied stress. Two-dimensional (2D) crystalline materials possess unique structural, mechanical and electronic properties 1 , 2 that make them highly attractive in many applications 3 , 4 , 5 . Although there have been advances in preparing 2D materials that consist of one or a few atomic or molecular layers 6 , 7 , bottom-up assembly of 2D crystalline materials remains a challenge and an active area of development 8 , 9 , 10 . More challenging is the design of dynamic 2D lattices that can undergo large-scale motions without loss of crystallinity. Dynamic behaviour in porous three-dimensional (3D) crystalline solids has been exploited for stimuli-responsive functions and adaptive behaviour 11 , 12 , 13 . As in such 3D materials, integrating flexibility and adaptiveness into crystalline 2D lattices would greatly broaden the functional scope of 2D materials. Here we report the self-assembly of unsupported, 2D protein lattices with precise spatial arrangements and patterns using a readily accessible design strategy. Three single- or double-point mutants of the C 4 -symmetric protein RhuA were designed to assemble via different modes of intermolecular interactions (single-disulfide, double-disulfide and metal-coordination) into crystalline 2D arrays. Owing to the flexibility of the single-disulfide interactions, the lattices of one of the variants ( C98 RhuA) are essentially defect-free and undergo substantial, but fully correlated, changes in molecular arrangement, yielding coherently dynamic 2D molecular lattices. C98 RhuA lattices display a Poisson’s ratio of −1—the lowest thermodynamically possible value for an isotropic material—making them auxetic.

A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell 1 . For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP4 2 , 3 . Here we describe the use of electron cryomicroscopy to determine how VP4 performs this function and show that when activated by cleavage to VP8* and VP5*, VP4 can rearrange on the virion surface from an ‘upright’ to a ‘reversed’ conformation. The reversed structure projects a previously buried ‘foot’ domain outwards into the membrane of the host cell to which the virion has attached. Electron cryotomograms of virus particles entering cells are consistent with this picture. Using a disulfide mutant of VP4, we have also stabilized a probable intermediate in the transition between the two conformations. Our results define molecular mechanisms for the first steps of the penetration of rotaviruses into the membranes of target cells and suggest similarities with mechanisms postulated for other viruses. Electron cryomicroscopy and cryotomography studies reveal that rotaviruses attach to a target cell through the outer-layer protein VP4, which—following cleavage—rearranges to enable perforation of the membrane and delivery of the viral genome into the host cell.

Membrane fission is a fundamental process in the regulation and remodelling of cell membranes. Dynamin, a large GTPase, mediates membrane fission by assembling around, constricting and cleaving the necks of budding vesicles 1 . Here we report a 3.75 Å resolution cryo-electron microscopy structure of the membrane-associated helical polymer of human dynamin-1 in the GMPPCP-bound state. The structure defines the helical symmetry of the dynamin polymer and the positions of its oligomeric interfaces, which were validated by cell-based endocytosis assays. Compared to the lipid-free tetramer form 2 , membrane-associated dynamin binds to the lipid bilayer with its pleckstrin homology domain (PHD) and self-assembles across the helical rungs via its guanine nucleotide-binding (GTPase) domain 3 . Notably, interaction with the membrane and helical assembly are accommodated by a severely bent bundle signalling element (BSE), which connects the GTPase domain to the rest of the protein. The BSE conformation is asymmetric across the inter-rung GTPase interface, and is unique compared to all known nucleotide-bound states of dynamin. The structure suggests that the BSE bends as a result of forces generated from the GTPase dimer interaction that are transferred across the stalk to the PHD and lipid membrane. Mutations that disrupted the BSE kink impaired endocytosis. We also report a 10.1 Å resolution cryo-electron microscopy map of a super-constricted dynamin polymer showing localized conformational changes at the BSE and GTPase domains, induced by GTP hydrolysis, that drive membrane constriction. Together, our results provide a structural basis for the mechanism of action of dynamin on the lipid membrane. A cryo-electron microscopy structure of human dynamin-1 demonstrates conformational changes and sheds light on the fission of membranes during endocytosis.

Twinkle is the mammalian helicase vital for replication and integrity of mitochondrial DNA. Over 90 Twinkle helicase disease variants have been linked to progressive external ophthalmoplegia and ataxia neuropathies among other mitochondrial diseases. Despite the biological and clinical importance, Twinkle represents the only remaining component of the human minimal mitochondrial replisome that has yet to be structurally characterized. Here, we present 3-dimensional structures of human Twinkle W315L. Employing cryo-electron microscopy (cryo-EM), we characterize the oligomeric assemblies of human full-length Twinkle W315L, define its multimeric interface, and map clinical variants associated with Twinkle in inherited mitochondrial disease. Cryo-EM, crosslinking-mass spectrometry, and molecular dynamics simulations provide insight into the dynamic movement and molecular consequences of the W315L clinical variant. Collectively, this ensemble of structures outlines a framework for studying Twinkle function in mitochondrial DNA replication and associated disease states.

Cryo-electron microscopy reveals the structure of the Kaposi’s sarcoma-associated herpesvirus capsid, and experiments with polypeptides that mimic the smallest capsid protein demonstrate the potential for structure-derived insights to help to develop antiviral agents. KSHV structure gives clues to stop viral replication Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, a cancer that commonly affects patients with AIDS. KSHV is an enormous virus with nearly 3,000 proteins, which has made determining its structure challenging. Hong Zhou and colleagues used cryo-electron microscopy to solve the structure of KSHV's capsid to 4.2 Å resolution. Their atomic model, corroborated by mutagenesis analysis, reveals molecular interactions that are important for stabilizing the capsid. The authors show experimentally that these interactions can be exploited to inhibit virus replication. Kaposi’s sarcoma-associated herpesvirus (KSHV) causes Kaposi’s sarcoma 1 , 2 , a cancer that commonly affects patients with AIDS 3 and which is endemic in sub-Saharan Africa 4 . The KSHV capsid is highly pressurized by its double-stranded DNA genome, as are the capsids of the eight other human herpesviruses 5 . Capsid assembly and genome packaging of herpesviruses are prone to interruption 6 , 7 , 8 , 9 and can therefore be targeted for the structure-guided development of antiviral agents. However, herpesvirus capsids—comprising nearly 3,000 proteins and over 1,300 Å in diameter—present a formidable challenge to atomic structure determination 10 and functional mapping of molecular interactions. Here we report a 4.2 Å resolution structure of the KSHV capsid, determined by electron-counting cryo-electron microscopy, and its atomic model, which contains 46 unique conformers of the major capsid protein (MCP), the smallest capsid protein (SCP) and the triplex proteins Tri1 and Tri2. Our structure and mutagenesis results reveal a groove in the upper domain of the MCP that contains hydrophobic residues that interact with the SCP, which in turn crosslinks with neighbouring MCPs in the same hexon to stabilize the capsid. Multiple levels of MCP–MCP interaction—including six sets of stacked hairpins lining the hexon channel, disulfide bonds across channel and buttress domains in neighbouring MCPs, and an interaction network forged by the N-lasso domain and secured by the dimerization domain—define a robust capsid that is resistant to the pressure exerted by the enclosed genome. The triplexes, each composed of two Tri2 molecules and a Tri1 molecule, anchor to the capsid floor via a Tri1 N-anchor to plug holes in the MCP network and rivet the capsid floor. These essential roles of the MCP N-lasso and Tri1 N-anchor are verified by serial-truncation mutageneses. Our proof-of-concept demonstration of the use of polypeptides that mimic the smallest capsid protein to inhibit KSHV lytic replication highlights the potential for exploiting the interaction hotspots revealed in our atomic structure to develop antiviral agents.

Naturally occurring point mutations in apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein (HDL), may affect plasma HDL-cholesterol levels and cardiovascular risk. Here, we evaluated the effect of human apoA-I mutations L144R (associated with low HDL-cholesterol), L178P (associated with low HDL-cholesterol and increased cardiovascular risk) and A164S (associated with increased cardiovascular risk and mortality without low HDL-cholesterol) on the structural integrity and functions of lipid-free and lipoprotein-associated apoA-I in an effort to explain the phenotypes of subjects carrying these mutations. All three mutants, in lipid-free form, presented structural and thermodynamic aberrations, with apoA-I[L178P] presenting the greatest thermodynamic destabilization. Additionally, apoA-I[L178P] displayed reduced ABCA1-mediated cholesterol efflux capacity. When in reconstituted HDL (rHDL), apoA-I[L144R] and apoA-I[L178P] were more thermodynamically destabilized compared to wild-type apoA-I, both displayed reduced SR-BI-mediated cholesterol efflux capacity and apoA-I[L144R] showed severe LCAT activation defect. ApoA-I[A164S] was thermodynamically unaffected when in rHDL, but exhibited a series of functional defects. Specifically, it had reduced ABCG1-mediated cholesterol and 7-ketocholesterol efflux capacity, failed to reduce ROS formation in endothelial cells and had reduced capacity to induce endothelial cell migration. Mechanistically, the latter was due to decreased capacity of rHDL-apoA-I[A164S] to activate Akt kinase possibly by interacting with endothelial LOX-1 receptor. The impaired capacity of rHDL-apoA-I[A164S] to preserve endothelial function may be related to the increased cardiovascular risk for this mutation. Overall, our structure–function analysis of L144R, A164S and L178P apoA-I mutants provides insights on how HDL-cholesterol levels and/or atheroprotective properties of apoA-I/HDL are impaired in carriers of these mutations.