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Cryo-EM structures of an insecticidal Bt toxin reveal its mechanism of action on the membrane.

ABSTRACT: Insect pests are a major cause of crop losses worldwide, with an estimated economic cost of $470 billion annually. Biotechnological tools have been introduced to control such insects without the need for chemical pesticides; for instance, the development of transgenic plants harbouring genes encoding insecticidal proteins. The Vip3 (vegetative insecticidal protein 3) family proteins from Bacillus thuringiensis convey toxicity to species within the Lepidoptera, and have wide potential applications in commercial agriculture. Vip3 proteins are proposed to exert their insecticidal activity through pore formation, though to date there is no mechanistic description of how this occurs on the membrane. Here we present cryo-EM structures of a Vip3 family toxin in both inactive and activated forms in conjunction with structural and functional data on toxin-membrane interactions. Together these data demonstrate that activated Vip3Bc1 complex is able to insert into membranes in a highly efficient manner, indicating that receptor binding is the likely driver of Vip3 specificity.

SUBMITTER: Byrne MJ

PROVIDER: S-EPMC8121907 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Cryo-EM structures of PAC1 receptor reveal ligand binding mechanism.

Project description:The pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1R) belongs to the secretin receptor family and is widely distributed in the central neural system and peripheral organs. Abnormal activation of the receptor mediates trigeminovascular activation and sensitization, which is highly related to migraine, making PAC1R a potential therapeutic target. Elucidation of PAC1R activation mechanism would benefit discovery of therapeutic drugs for neuronal disorders. PAC1R activity is governed by pituitary adenylate cyclase-activating polypeptide (PACAP), known as a major vasodilator neuropeptide, and maxadilan, a native peptide from the sand fly, which is also capable of activating the receptor with similar potency. These peptide ligands have divergent sequences yet initiate convergent PAC1R activity. It is of interest to understand the mechanism of PAC1R ligand recognition and receptor activity regulation through structural biology. Here we report two near-atomic resolution cryo-EM structures of PAC1R activated by PACAP38 or maxadilan, providing structural insights into two distinct ligand binding modes. The structures illustrate flexibility of the extracellular domain (ECD) for ligands with distinct conformations, where ECD accommodates ligands in different orientations while extracellular loop 1 (ECL1) protrudes to further anchor the ligand bound in the orthosteric site. By structure-guided molecular modeling and mutagenesis, we tested residues in the ligand-binding pockets and identified clusters of residues that are critical for receptor activity. The structures reported here for the first time elucidate the mechanism of specificity and flexibility of ligand recognition and binding for PAC1R, and provide insights toward the design of therapeutic molecules targeting PAC1R.

| S-EPMC7196072 | biostudies-literature

Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding.

Project description:Transmembrane β barrel proteins are folded into the outer membrane (OM) of Gram-negative bacteria by the β barrel assembly machinery (BAM) via a poorly understood process that occurs without known external energy sources. Here, we used single-particle cryo-EM to visualize the folding dynamics of a model β barrel protein (EspP) by BAM. We found that BAM binds the highly conserved "β signal" motif of EspP to correctly orient β strands in the OM during folding. We also found that the folding of EspP proceeds via "hybrid-barrel" intermediates in which membrane integrated β sheets are attached to the essential BAM subunit, BamA. The structures show an unprecedented deflection of the membrane surrounding the EspP intermediates and suggest that β sheets progressively fold toward BamA to form a β barrel. Along with in vivo experiments that tracked β barrel folding while the OM tension was modified, our results support a model in which BAM harnesses OM elasticity to accelerate β barrel folding.

| S-EPMC8985213 | biostudies-literature

Cryo-EM structures of human STEAP4 reveal mechanism of iron(III) reduction.

Project description:Enzymes of the six-transmembrane epithelial antigen of the prostate (STEAP) family reduce Fe3+ and Cu2+ ions to facilitate metal-ion uptake by mammalian cells. STEAPs are highly upregulated in several types of cancer, making them potential therapeutic targets. However, the structural basis for STEAP-catalyzed electron transfer through an array of cofactors to metals at the membrane luminal side remains elusive. Here, we report cryo-electron microscopy structures of human STEAP4 in absence and presence of Fe3+-NTA. Domain-swapped, trimeric STEAP4 orients NADPH bound to a cytosolic domain onto axially aligned flavin-adenine dinucleotide (FAD) and a single b-type heme that cross the transmembrane-domain to enable electron transfer. Substrate binding within a positively charged ring indicates that iron gets reduced while in complex with its chelator. These molecular principles of iron reduction provide a basis for exploring STEAPs as therapeutic targets.

| S-EPMC6194020 | biostudies-literature

Cryo-EM structures of holo condensin reveal a subunit flip-flop mechanism.

Project description:Complexes containing a pair of structural maintenance of chromosomes (SMC) family proteins are fundamental for the three-dimensional (3D) organization of genomes in all domains of life. The eukaryotic SMC complexes cohesin and condensin are thought to fold interphase and mitotic chromosomes, respectively, into large loop domains, although the underlying molecular mechanisms have remained unknown. We used cryo-EM to investigate the nucleotide-driven reaction cycle of condensin from the budding yeast Saccharomyces cerevisiae. Our structures of the five-subunit condensin holo complex at different functional stages suggest that ATP binding induces the transition of the SMC coiled coils from a folded-rod conformation into a more open architecture. ATP binding simultaneously triggers the exchange of the two HEAT-repeat subunits bound to the SMC ATPase head domains. We propose that these steps result in the interconversion of DNA-binding sites in the catalytic core of condensin, forming the basis of the DNA translocation and loop-extrusion activities.

| S-EPMC7610691 | biostudies-literature

Author Correction: Cryo-EM structures of PAC1 receptor reveal ligand binding mechanism.

Project description:An amendment to this paper has been published and can be accessed via a link at the top of the paper.

| S-EPMC7265381 | biostudies-literature

Cryo-EM Structures of CusA Reveal a Mechanism of Metal-Ion Export.

Project description:Gram-negative bacteria utilize the resistance-nodulation-cell division (RND) superfamily of efflux pumps to expel a variety of toxic compounds from the cell. The Escherichia coli CusA membrane protein, which recognizes and extrudes biocidal Cu(I) and Ag(I) ions, belongs to the heavy-metal efflux (HME) subfamily of RND efflux pumps. We here report four structures of the trimeric CusA heavy-metal efflux pump in the presence of Cu(I) using single-particle cryo-electron microscopy (cryo-EM). We discover that different CusA protomers within the trimer are able to bind Cu(I) ions simultaneously. Our structural data combined with molecular dynamics (MD) simulations allow us to propose a mechanism for ion transport where each CusA protomer functions independently within the trimer.IMPORTANCE The bacterial RND superfamily of efflux pumps mediate resistance to a variety of biocides, including Cu(I) and Ag(I) ions. Here we report four cryo-EM structures of the trimeric CusA pump in the presence of Cu(I). Combined with MD simulations, our data indicate that each CusA protomer within the trimer recognizes and extrudes Cu(I) independently.

| S-EPMC8092243 | biostudies-literature

Cryo-EM structures reveal the activation and substrate recognition mechanism of human enteropeptidase.

Project description:Enteropeptidase (EP) initiates intestinal digestion by proteolytically processing trypsinogen, generating catalytically active trypsin. EP dysfunction causes a series of pancreatic diseases including acute necrotizing pancreatitis. However, the molecular mechanisms of EP activation and substrate recognition remain elusive, due to the lack of structural information on the EP heavy chain. Here, we report cryo-EM structures of human EP in inactive, active, and substrate-bound states at resolutions from 2.7 to 4.9 Å. The EP heavy chain was observed to clamp the light chain with CUB2 domain for substrate recognition. The EP light chain N-terminus induced a rearrangement of surface-loops from inactive to active conformations, resulting in activated EP. The heavy chain then served as a hinge for light-chain conformational changes to recruit and subsequently cleave substrate. Our study provides structural insights into rearrangements of EP surface-loops and heavy chain dynamics in the EP catalytic cycle, advancing our understanding of EP-associated pancreatitis.

| S-EPMC9663175 | biostudies-literature

Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP-AMP.

Project description:Infections by pathogens that contain DNA trigger the production of type-I interferons and inflammatory cytokines through cyclic GMP-AMP synthase, which produces 2'3'-cyclic GMP-AMP (cGAMP) that binds to and activates stimulator of interferon genes (STING; also known as TMEM173, MITA, ERIS and MPYS)1-8. STING is an endoplasmic-reticulum membrane protein that contains four transmembrane helices followed by a cytoplasmic ligand-binding and signalling domain9-13. The cytoplasmic domain of STING forms a dimer, which undergoes a conformational change upon binding to cGAMP9,14. However, it remains unclear how this conformational change leads to STING activation. Here we present cryo-electron microscopy structures of full-length STING from human and chicken in the inactive dimeric state (about 80 kDa in size), as well as cGAMP-bound chicken STING in both the dimeric and tetrameric states. The structures show that the transmembrane and cytoplasmic regions interact to form an integrated, domain-swapped dimeric assembly. Closure of the ligand-binding domain, induced by cGAMP, leads to a 180° rotation of the ligand-binding domain relative to the transmembrane domain. This rotation is coupled to a conformational change in a loop on the side of the ligand-binding-domain dimer, which leads to the formation of the STING tetramer and higher-order oligomers through side-by-side packing. This model of STING oligomerization and activation is supported by our structure-based mutational analyses.

| S-EPMC6859894 | biostudies-literature

Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader.

Project description:Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the Saccharomyces cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale 'crab-claw' expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC's switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.

| S-EPMC8893722 | biostudies-literature

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Project description:The epithelial anion transporter SLC26A9 contributes to airway surface hydration and gastric acid production. Colocalizing with CFTR, SLC26A9 has been proposed as a target for the treatment of cystic fibrosis. To provide molecular details of its transport mechanism, we present cryo-EM structures and a functional characterization of murine Slc26a9. These structures define the general architecture of eukaryotic SLC26 family members and reveal an unusual mode of oligomerization which relies predominantly on the cytosolic STAS domain. Our data illustrates conformational transitions of Slc26a9, supporting a rapid alternate-access mechanism which mediates uncoupled chloride transport with negligible bicarbonate or sulfate permeability. The characterization of structure-guided mutants illuminates the properties of the ion transport path, including a selective anion binding site located in the center of a mobile module within the transmembrane domain. This study thus provides a structural foundation for the understanding of the entire SLC26 family and potentially facilitates their therapeutic exploitation.

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