Conformational Flexibility Is a Key Determinant for the Lytic Activity of the Pore-Forming Protein, Cytolysin A.

Several bacterial infections are mediated by pore-forming toxins (PFTs), a subclass of proteins that oligomerize on mammalian cell membranes forming lytic nanopores. Cytolysin A (ClyA), an α-PFT, undergoes a dramatic conformational change restructuring its two membrane-binding motifs (the ß-tongue and the N-terminus helix), during pore formation. A complete molecular picture for this key transition and the driving force behind the secondary structure change upon membrane binding remain elusive. Using all-atom molecular dynamics (MD) simulations of the ClyA monomer and string method based free energy computations with path collective variables, we illustrate that an unfolded ß-tongue motif is an on-pathway intermediate during the transition to the helix-turn-helix motif of the protomer. An aggregate of 28 µs of all-atom thermal unfolding MD simulations of wild-type ClyA and its single point mutants reveal that the membrane-binding motifs of the ClyA protein display high structural flexibility in water. However, point mutations in these motifs lead to a distinct reduction in the flexibility, especially in the ß-tongue, thereby stabilizing the pretransition secondary structure. Resistance to unfolding was further corroborated by MD simulations of the ß-tongue mutant motif in the membrane. Combined with the thermal unfolding simulations, we posit that the ß-tongue as well as N-terminal mutants that lower the tendency to unfold and disorder the ß-tongue are detrimental to pore formation by ClyA and its lytic activity. Erythrocyte turbidity and vesicle leakage assays indeed reveal a loss of activity for the ß-tongue mutant, and delayed kinetics for the N-terminus mutants. On the other hand, a point mutation in the extracellular domain that did not abrogate lytic activity displayed similar unfolding characteristics as the wild type. Thus, attenuation of conformational flexibility in membrane-binding motifs correlates with reduced lytic and leakage activity. Combined with secondary structure changes observed in the membrane bound states, our study shows that the tendency to unfold in the ß-tongue region is a critical step in the conformational transition and bistability of the ClyA protein and mutants that disrupt this tendency reduced pore formation. Overall, our finding suggests that inherent flexibility in the protein could play a wider and hitherto unrecognized role in membrane-mediated conformational transitions of PFTs and other membrane protein transformations.

Single-molecule Diffusion and Assembly on Polymer-crowded Lipid Membranes.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer (SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Evaluation of spike protein antigens for SARS-CoV-2 serology.

BACKGROUND: Spike protein domains are being used in various serology-based assays to detect prior exposure to SARS-CoV-2 virus. However, there has been limited comparison of antibody titers against various spike protein antigens among COVID-19 infected patients. METHODS: We compared four spike proteins (RBD, S1, S2 and a stabilized spike trimer (ST)) representing commonly used antigens for their reactivity to human IgG antibodies using indirect ELISA in serum from COVID-19 patients and pre-2020 samples. ST ELISA was also compared against the EUROIMMUN IgG ELISA test. Further, we estimated time appropriate IgG and IgA seropositivity rates in COVID-19 patients using a panel of sera samples collected longitudinally from the day of onset of symptoms (DOS). RESULTS: Among the four spike antigens tested, the ST demonstrated the highest sensitivity (86.2 %; 95 % CI: 77.8-91.7 %), while all four antigens showed high specificity to COVID-19 sera (94.7-96.8 %). 13.8 % (13/94) of the samples did not show seroconversion in any of the four antigen-based assays. In a double-blinded head-to-head comparison, ST based IgG ELISA displayed a better sensitivity (87.5 %, 95 % CI: 76.4-93.8 %) than the EUROIMMUN IgG ELISA (67.9 %, 95 % CI: 54.8-78.6 %). Further, in ST-based assays, we found 48 % and 50 % seroconversion in the first six days (from DOS) for IgG and IgA antibodies, respectively, which increased to 84 % (IgG) and 85 % (IgA) for samples collected ≥22 days from DOS. CONCLUSIONS: Comparison of spike antigens demonstrates that spike trimer protein is a superior option as an ELISA antigen for COVID-19 serology.

Cholesterol promotes Cytolysin A activity by stabilizing the intermediates during pore formation.

Pore-forming toxins (PFTs) form nanoscale pores across target membranes causing cell death. Cytolysin A (ClyA) from Escherichia coli is a prototypical α-helical toxin that contributes to cytolytic phenotype of several pathogenic strains. It is produced as a monomer and, upon membrane exposure, undergoes conformational changes and finally oligomerizes to form a dodecameric pore, thereby causing ion imbalance and finally cell death. However, our current understanding of this assembly process is limited to studies in detergents, which do not capture the physicochemical properties of biological membranes. Here, using single-molecule imaging and molecular dynamics simulations, we study the ClyA assembly pathway on phospholipid bilayers. We report that cholesterol stimulates pore formation, not by enhancing initial ClyA binding to the membrane but by selectively stabilizing a protomer-like conformation. This was mediated by specific interactions by cholesterol-interacting residues in the N-terminal helix. Additionally, cholesterol stabilized the oligomeric structure using bridging interactions in the protomer-protomer interfaces, thereby resulting in enhanced ClyA oligomerization. This dual stabilization of distinct intermediates by cholesterol suggests a possible molecular mechanism by which ClyA achieves selective membrane rupture of eukaryotic cell membranes. Topological similarity to eukaryotic membrane proteins suggests evolution of a bacterial α-toxin to adopt eukaryotic motifs for its activation. Broad mechanistic correspondence between pore-forming toxins hints at a wider prevalence of similar protein membrane insertion mechanisms.

Single-molecule fluorescence imaging: Generating insights into molecular interactions in virology.

Single-molecule fluorescence methods remain a challenging yet information-rich set of techniques that allow one to probe the dynamics, stoichiometry and conformation of biomolecules one molecule at a time. Viruses are small (nanometers) in size, can achieve cellular infections with a small number of virions and their lifecycle is inherently heterogeneous with a large number of structural and functional intermediates. Single-molecule measurements that reveal the complete distribution of properties rather than the average can hence reveal new insights into virus infections and biology that are inaccessible otherwise. This article highlights some of the methods and recent applications of single-molecule fluorescence in the field of virology. Here, we have focused on new findings in virus-cell interaction, virus cell entry and transport, viral membrane fusion, genome release, replication, translation, assembly, genome packaging, egress and interaction with host immune proteins that underline the advantage of single-molecule approach to the question at hand. Finally, we discuss the challenges, outlook and potential areas for improvement and future use of single-molecule fluorescence that could further aid our understanding of viruses.