Diego Carlero

Pore-forming proteins exemplify the transformative potential of biological molecules. Produced as soluble monomers, they assemble into multimeric membrane-inserted complexes in response to specific membrane environments. Actinoporins, a class of pore-forming proteins from sea anemones, target membranes to kill cells. Here, we report cryogenic electron microscopy structures of two actinoporins, fragaceatoxin C and sticholysin II, reconstituted in lipid membranes. The structures reveal an ordered arrangement of dozens of lipid molecules that form an integral part of the pore architecture. We also captured distinct oligomeric intermediates, arc-shaped assemblies with monomers in transitional conformations, representing key snapshots along the pore formation pathway. These data provide direct structural evidence for a stepwise mechanism in which monomers sequentially bind the membrane and undergo conformational changes that drive pore assembly and membrane disruption. Our findings reveal how these proteins reshape membranes and offer mechanistic insights into their cytolytic activity. This work broadens our understanding of pore-forming proteins, which are gaining increasing relevance in diverse biotechnological applications.

Pore-forming proteins exemplify the transformative potential of biological molecules. Initially produced in a monomeric, water-soluble form, they spontaneously assemble into multimeric integral membrane proteins in the presence of suitable target lipids. Their functions include roles in apoptosis, cell signaling, immunity, as well as attack and defense systems between different organisms. This latter group encompasses actinoporins, a family of pore-forming toxins from sea anemones that kill target cells by perforating their plasma membrane. Here, we have determined the structures of two such toxins, fragaceatoxin C and sticholysin II, in a membrane environment using cryogenic electron microscopy. The structures reveal how dozens of lipid molecules interact in an orderly manner, forming an intrinsic part of the pore. We have also isolated different pore-forming intermediates, where only a fraction of the constituent monomers is incorporated, exhibiting non-closed, arc-shaped structures. Based on these structures we propose a mechanism of action where the sequential assembly of toxin monomers onto the membrane, accompanied by conformational changes, triggers pore formation and membrane perforation. Our results contribute to a better understanding of the transforming capacity of these pore-forming proteins, which are becoming increasingly important for their diverse biotechnological applications.

Single-stranded RNA viruses (ssRNAv) are characterized by their biological diversity and great adaptability to different hosts; traits which make them a major threat to human health due to their potential to cause zoonotic outbreaks. A detailed understanding of the mechanisms involved in viral proliferation is essential to address the challenges posed by these pathogens. Key to these processes are ribonucleoproteins (RNPs), the genome-containing RNA-protein complexes whose function is to carry out viral transcription and replication. Structural determination of RNPs can provide crucial information on the molecular mechanisms of these processes, paving the way for the development of new, more effective strategies to control and prevent the spread of ssRNAv diseases. In this scenario, cryogenic electron microscopy (cryoEM), relying on the technical and methodological revolution it has undergone in recent years, can provide invaluable help in elucidating how these macromolecular complexes are organized, packaged within the virion, or the functional implications of these structures. In this review, we summarize some of the most prominent achievements by cryoEM in the study of RNP and nucleocapsid structures in lipid-enveloped ssRNAv.

Summary: In the present study we report the functional and structural characterization of 17T2, a new highly potent pan-neutralizing SARS-CoV-2 human monoclonal antibody (mAb) isolated from a convalescent COVID-19 individual infected during the first wave of the COVID-19 pandemic. 17T2 is a class 1 VH1-58/κ3-20 antibody, derived from a receptor binding domain (RBD)-specific IgA memory B cell and developed as a human recombinant IgG1. Functional characterization revealed that 17T2 mAb has a high and exceptionally broad neutralizing activity against all SARS-CoV-2 spike variants tested, including BQ.1.1. Moreover, 17T2 mAb has in vivo prophylactic activity against Omicron BA.1.1 infection in K18-hACE2 transgenic mice. 3D reconstruction from cryogenic-electron microscopy (cryo-EM) showed that 17T2 binds the Omicron BA.1 spike protein with the RBD domains in “up” position and recognizes an epitope overlapping with the receptor binding motif, as it is the case for other structurally similar neutralizing mAbs, including S2E12. Yet, unlike S2E12, 17T2 retained its high neutralizing activity against all Omicron sublineages tested, probably due to a larger contact area with the RBD, which could confer a higher resilience to spike mutations. These results highlight the impact of small structural antibody changes on neutralizing performance and identify 17T2 mAb as a potential candidate for future therapeutic and prophylactic interventions.

Effective airborne transmission of coronaviruses via liquid microdroplets requires a virion structure that must withstand harsh environmental conditions. Due to the demanding biosafety requirements for the study of human respiratory viruses, it is important to develop surrogate models to facilitate their investigation. Here we explore the mechanical properties and nanostructure of transmissible gastroenteritis virus (TGEV) virions in liquid milieu and their response to different chemical agents commonly used as biocides. Our data provide two-fold results on virus stability: First, while particles with larger size and lower packing fraction kept their morphology intact after successive mechanical aggressions, smaller viruses with higher packing fraction showed conspicuous evidence of structural damage and content release. Second, monitoring the structure of single TGEV particles in the presence of detergent and alcohol in real time revealed the stages of gradual degradation of the virus structure in situ. These data suggest that detergent is three orders of magnitude more efficient than alcohol in destabilizing TGEV virus particles, paving the way for optimizing hygienic protocols for viruses with similar structure, such as SARS-CoV-2.