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Abstract: Cholesterol is an essential sterol in cell membranes that regulates organization and fluidity. This biomolecule has been identified as one of the critical factors in the internalization process of various viruses in human cells. Therefore, understanding these mechanisms is crucial for a deeper comprehension of viral pathogenicity in the search for practical therapeutic approaches against viral diseases. The biochemical and biophysical processes related to these diseases are highly complex. For this reason, studying model systems capable of mimicking the interaction of lipid membranes with cholesterol and proteins is fundamental. In this work, we propose to study the structural and elastic changes in mono-, bi-, and tridimensional lipid systems composed of dipalmitoylphosphatidylcholine (PC) with varying amounts of cholesterol in the presence and absence of the S protein (Spike) and its receptor-binding domain (RBD) from SARS-CoV-2. To characterize these systems, we used both experimental and theoretical approaches such as Langmuir trough, atomic force microscopy (AFM), small-angle X-ray scattering (SAXS), electrochemical methods, and molecular dynamics (MD) simulations. With the interpretation of all results obtained in this work, it was possible to propose a structural model of the membrane in the presence of cholesterol and the interaction with the Spike protein and RBD. The behavior of the adsorption isotherm and SAXS data, together with the results provided by MD simulations, led us to conclude that cholesterol in PC monolayers promotes local alterations, inducing the formation of more rigid membrane regions. More importantly, cholesterol plays a crucial role in facilitating the allocation of SARS-CoV-2 proteins in lipid systems. This is especially true for the Spike protein, which displayed a non-ACE2 mediated stable binding to the lipid membrane with high internalization.

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Abstract: Enterohaemorrhagic Escherichia coli causes sporadic, and sometimes large-scale, food poisoning outbreaks, for which antibiotic treatment in humans is contraindicated. As an alternative form of therapy, previous studies developed the family of salicylidene acylhydrazide (SA) anti-virulence compounds. One target of the SA compounds is AdhE, an enzyme that converts acetyl-CoA to ethanol and vice versa. AdhE oligomerizes, forming helicoidal filaments, heterogeneous in length, called spirosomes. We show it is possible to only partially fractionate AdhE spirosomes because in vitro they oligomerize in the absence of stimuli, and that spirosome formation is necessary to regulate the direction of AdhE enzymatic reactions. We also show that the SA compound ME0054 binds and perturbs AdhE spirosomes, enhancing the conversion of ethanol to acetyl-CoA. This mechanistic understanding of how ME0054 impacts AdhE function will help in the development of SA compounds as novel anti-virulence inhibitors.

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Abstract: The SARS-CoV-2 nucleocapsid (N) protein is essential for the viral life cycle, facilitating RNA packaging, replication, and host-cell interactions. Its ability to self-assemble and undergo phase separation is critical for these functions but remains poorly understood. Using an integrated approach combining small-angle X-ray scattering (SAXS), nuclear magnetic resonance spectroscopy, computational modeling, and biophysical assays, we uncover key mechanisms underpinning N-protein’s dynamic self-assembly. We show that the N-protein’s interdomain linker (IDL) contains a conserved coiled-coil (CC) motif that drives transient interactions between protein subunits, enabling the formation of progressively larger complexes at higher concentrations. SAXS analysis and ensemble modeling reveal that the IDL exists in a concentration-dependent equilibrium between monomeric, dimeric, and trimeric states. The CC motif facilitates parallel, head-to-head oligomerization of N-protein dimers, transitioning between compact (closed) and extended (open) configurations depending on the interaction network within the IDL. This linker-driven assembly modulates phase separation, impacting the size, stability, and dynamics of biomolecular condensates. Here, we present the most comprehensive conformational landscape analysis of the N-protein to date, providing a detailed model of its self-assembly and phase separation. Our findings highlight how the structural plasticity of the IDL and CC-mediated interactions are pivotal to its roles in the SARS-CoV-2 life cycle.

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Abstract: Pseudomonas aeruginosa employs the Type VI secretion system (T6SS) to outcompete other bacteria in its environment. Among the effectors secreted by the T6SS of P. aeruginosa PAO1, Tse4 is known for its potent antibacterial activity. This study elucidates the molecular function of Tse4, which promotes cell depolarization in competing bacteria. Our results show that Tse4 spontaneously incorporates into lipid monolayers and forms multiionic channels in planar bilayers, with either ohmic conduction or diode-like rectifying currents and a preference for cations over anions. These observations allow us to propose a model of action whereby Tse4 channels couple cell depolarization with K+ efflux. These insights into Tse4’s pore-forming activity enhance our understanding of bacterial competition and exemplify a finely tuned antibacterial strategy, coupling its ability to cause membrane depolarization with potassium efflux that synergises with other T6SS effectors. These results highlight the sophistication of Pseudomonas aeruginosa’s competitive arsenal.

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Abstract: Chirality and non-covalent chemistry are essential features in the construction of complex molecular systems that constitute living organisms. The specific interactions between molecular components with defined geometric orientations enable nature’s dynamism, responsiveness, specificity, and selection. The precision with which nature controls its homochirality and functional structures inspired the design of synthetic supramolecular systems. These systems have aimed at understanding the origins of these properties and mimicking their complexity. Research in the last decades has led to many insights into synthetic and natural supramolecular polymerization processes and principles of amplification of asymmetry. However, the complexity observed in nature remains far from being understood. In particular, detailed studies of assembly mechanisms and amplification of asymmetry principles in water-compatible supramolecular systems are scarce due to the challenging contribution of hydrophobic effects leading to non-equilibrium states of supramolecular assemblies. Valuable contributions to this field have been made in recent years, including the introduction of in situ chemical modifications and dissipative non-equilibrium assembly states, leading to evolutionary features such as self-replication. This chapter introduces the concepts of supramolecular polymerization and shows the analogies between natural and synthetic systems. In particular, the influence of homochirality in natural macroscopic structures is emphasized, showing the importance of this property in water-compatible supramolecular systems. Therefore, key developments in research on the origin of homochirality and challenges for future research to understand this essential feature of life are outlined.