Open architecture of archaea MCM and dsDNA complexes resolved using monodispersed streptavidin affinity CryoEM.

The cryo-electron microscopy (cryoEM) method has enabled high-resolution structure determination of numerous biomolecules and complexes. Nevertheless, cryoEM sample preparation of challenging proteins and complexes, especially those with low abundance or with preferential orientation, remains a major hurdle. We developed an affinity-grid method employing monodispersed single particle streptavidin on a lipid monolayer to enhance particle absorption on the grid surface and alleviate sample exposure to the air-water interface.

In vitro evolution driven by epistasis reveals alternative cholesterol-specific binding motifs of perfringolysin O.

The crucial molecular factors that shape the interfaces of lipid-binding proteins with their target ligands and surfaces remain unknown due to the complex makeup of biological membranes. Cholesterol, the major modulator of bilayer structure in mammalian cell membranes, is recognized by various proteins, including the well-studied cholesterol-dependent cytolysins. Here, we use in vitro evolution to investigate the molecular adaptations that preserve the cholesterol specificity of perfringolysin O, the prototypical cholesterol-dependent cytolysin from Clostridium perfringens.

Structural basis for activity switching in polymerases determining the fate of let-7 pre-miRNAs.

Tumor-suppressor let-7 pre-microRNAs (miRNAs) are regulated by terminal uridylyltransferases TUT7 and TUT4 that either promote let-7 maturation by adding a single uridine nucleotide to the pre-miRNA 3' end or mark them for degradation by the addition of multiple uridines. Oligo-uridylation is increased in cells by enhanced TUT7/4 expression and especially by the RNA-binding pluripotency factor LIN28A. Using cryogenic electron microscopy, we captured high-resolution structures of active forms of TUT7 alone, of TUT7 plus pre-miRNA and of both TUT7 and TUT4 bound with pre-miRNA and LIN28A.

Peptide-based pore formation and cell membrane deformation: European Biophysics Journal Prizes at EBSA 2023.

The European Biophysics Journal Prizes awarded at the European Biophysical Societies Association (EBSA) Congress in Stockholm in the Summer of 2023 recognised papers published in 2020 and 2021 which made use of multiple complementing experimental, theoretical and computational approaches. One of the winning papers addressed the specific role of arginine residues within antimicrobial and cell-penetrating peptides, in promoting membrane defect stabilisation and pore formation. The other winning paper described the influence of atomic force microscopy probe geometry on the measurement of surface deformability, assessed for investigation of the differing viscoelastic properties of non-malignant and cancerous cells.

Cryo-EM structures of perforin-2 in isolation and assembled on a membrane suggest a mechanism for pore formation.

Perforin-2 (PFN2, MPEG1) is a key pore-forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane-bound pre-pore complex that converts to a pore-forming structure upon acidification; but its mechanism of conformational transition has been debated. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures of PFN2 in pre-pore and pore conformations in isolation and bound to liposomes.

Structural, Functional and Computational Studies of Membrane Recognition by Plasmodium Perforin-Like Proteins 1 and 2.

Perforin-like proteins (PLPs) play key roles in mechanisms associated with parasitic disease caused by the apicomplexan parasites Plasmodium and Toxoplasma. The T. gondii PLP1 (TgPLP1) mediates tachyzoite egress from cells, while the five Plasmodium PLPs carry out various roles in the life cycle of the parasite and with respect to the molecular basis of disease.

Hedgehog-Interacting Protein is a multimodal antagonist of Hedgehog signalling.

Hedgehog (HH) morphogen signalling, crucial for cell growth and tissue patterning in animals, is initiated by the binding of dually lipidated HH ligands to cell surface receptors. Hedgehog-Interacting Protein (HHIP), the only reported secreted inhibitor of Sonic Hedgehog (SHH) signalling, binds directly to SHH with high nanomolar affinity, sequestering SHH. Here, we report the structure of the HHIP N-terminal domain (HHIP-N) in complex with a glycosaminoglycan (GAG).

Electron microscopy as a critical tool in the determination of pore forming mechanisms in proteins.

Electron microscopy has consistently played an important role in the description of pore-forming protein systems. The discovery of pore-forming proteins has depended on visualization of the structural pores formed by their oligomeric protein complexes, and as electron microscopy has advanced technologically so has the degree of insight it has been able to give. This review considers a large number of published studies of pore-forming complexes in prepore and pore states determined using single-particle cryo-electron microscopy.

Structure and mechanism of bactericidal mammalian perforin-2, an ancient agent of innate immunity.

Perforin-2 (MPEG1) is thought to enable the killing of invading microbes engulfed by macrophages and other phagocytes, forming pores in their membranes. Loss of perforin-2 renders individual phagocytes and whole organisms significantly more susceptible to bacterial pathogens. Here, we reveal the mechanism of perforin-2 activation and activity using atomic structures of pre-pore and pore assemblies, high-speed atomic force microscopy, and functional assays.

Structures of monomeric and oligomeric forms of the perforin-like protein 1.

and are the parasitic agents of toxoplasmosis and malaria, respectively, and use perforin-like proteins (PLPs) to invade host organisms and complete their life cycles. The PLP1 (PLP1) is required for efficient exit from parasitophorous vacuoles in which proliferation occurs. We report structures of the membrane attack complex/perforin (MACPF) and Apicomplexan PLP C-terminal β-pleated sheet (APCβ) domains of PLP1.

Structural Transitions of the Conserved and Metastable Hantaviral Glycoprotein Envelope.

Hantaviruses are zoonotic pathogens that cause severe hemorrhagic fever and pulmonary syndrome. The outer membrane of the hantavirus envelope displays a lattice of two glycoproteins, Gn and Gc, which orchestrate host cell recognition and entry. Here, we describe the crystal structure of the Gn glycoprotein ectodomain from the Asiatic Hantaan virus (HTNV), the most prevalent pathogenic hantavirus.

Repurposing a pore: highly conserved perforin-like proteins with alternative mechanisms.

Pore-forming proteins play critical roles in pathogenic attack and immunological defence. The membrane attack complex/perforin (MACPF) group of homologues represents, with cholesterol-dependent cytolysins, the largest family of such proteins. In this review, we begin by describing briefly the structure of MACPF proteins, outlining their common mechanism of pore formation.

Structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain.

Kindlins co-activate integrins alongside talin. They possess, like talin, a FERM domain (4.1-erythrin-radixin-moiesin domain) comprising F0-F3 subdomains, but with a pleckstrin homology (PH) domain inserted in the F2 subdomain that enables membrane association.

Structural Basis for Plexin Activation and Regulation.

Class A plexins (PlxnAs) act as semaphorin receptors and control diverse aspects of nervous system development and plasticity, ranging from axon guidance and neuron migration to synaptic organization. PlxnA signaling requires cytoplasmic domain dimerization, but extracellular regulation and activation mechanisms remain unclear. Here we present crystal structures of PlxnA (PlxnA1, PlxnA2, and PlxnA4) full ectodomains.

Crystal structure of an invertebrate cytolysin pore reveals unique properties and mechanism of assembly.

The invertebrate cytolysin lysenin is a member of the aerolysin family of pore-forming toxins that includes many representatives from pathogenic bacteria. Here we report the crystal structure of the lysenin pore and provide insights into its assembly mechanism. The lysenin pore is assembled from nine monomers via dramatic reorganization of almost half of the monomeric subunit structure leading to a β-barrel pore ∼10 nm long and 1.

Initiation of T cell signaling by CD45 segregation at 'close contacts'.

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent.

Measuring kinetic drivers of pneumolysin pore structure.

Most membrane attack complex-perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins are thought to form pores in target membranes by assembling into pre-pore oligomers before undergoing a pre-pore to pore transition. Assembly during pore formation is into both full rings of subunits and incomplete rings (arcs). The balance between arcs and full rings is determined by a mechanism dependent on protein concentration in which arc pores arise due to kinetic trapping of the pre-pore forms by the depletion of free protein subunits during oligomerization.

Protein-lipid interactions and non-lamellar lipidic structures in membrane pore formation and membrane fusion.

Pore-forming proteins and peptides act on their targeted lipid bilayer membranes to increase permeability. This approach to the modulation of biological function is relevant to a great number of living processes, including; infection, parasitism, immunity, apoptosis, development and neurodegeneration. While some pore-forming proteins/peptides assemble into rings of subunits to generate discrete, well-defined pore-forming structures, an increasing number is recognised to form pores via mechanisms which co-opt membrane lipids themselves.

An optimized protocol for expression and purification of murine perforin in insect cells.

Perforin (PFN) is one of the most important protein effectors of the immune system. It is produced by cytotoxic T lymphocytes and natural killer cells and helps with the clearance of virus-infected and tumor cells. PFN is a pore-forming protein that readily binds to the lipid membranes of target cells, oligomerizes at the cell surface and forms transmembrane pores that allow passage of ions and other larger molecules.

Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins.

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles.

Improved crystallization and diffraction of caffeine-induced death suppressor protein 1 (Cid1).

The post-transcriptional addition of uridines to the 3'-end of RNAs is an important regulatory process that is critical for coding and noncoding RNA stability. In fission yeast and metazoans this untemplated 3'-uridylylation is catalysed by a single family of terminal uridylyltransferases (TUTs) whose members are adapted to specific RNA targets. In Schizosaccharomyces pombe the TUT Cid1 is responsible for the uridylylation of polyadenylated mRNAs, targeting them for destruction.