Chitin is a major carbohydrate component of the fungal cell wall and a promising target for novel antifungal agents. However, it is technically challenging to characterize the structure of this polymer in native cell walls. Here, we recorded and compared 13C chemical shifts of chitin using isotopically enriched cells of six Aspergillus, Rhizopus, and Candida strains, with data interpretation assisted by principal component analysis (PCA) and linear discriminant analysis (LDA) methods. The structure of chitin is found to be intrinsically heterogeneous, with peak multiplicity detected in each sample and distinct fingerprints observed across fungal species. Fungal chitin exhibits partial similarity to the model structures of α- and γ-allomorphs; therefore, chitin structure is not significantly affected by interactions with other cell wall components. Addition of antifungal drugs and salts did not significantly perturb the chemical shifts, revealing the structural resistance of chitin to external stress. In addition, the structure of the deacetylated form, chitosan, was found to resemble a relaxed two-fold helix conformation. This study provides high-resolution information on the structure of chitin and chitosan in their cellular contexts. The method is applicable to the analysis of other complex carbohydrates and polymer composites.

New approaches to complement vaccination are needed to combat the spread of SARS-CoV-2 and stop COVID-19 related deaths and medical complications. Human beta defensin 2 (hBD-2) is a naturally occurring epithelial cell derived host defense peptide that has antiviral properties. Our comprehensive in-silico studies demonstrate that hBD-2 binds the site on the CoV-2-RBD that docks with the ACE2 receptor. Biophysical measurements confirm that hBD-2 indeed binds to the CoV-2-receptor binding domain (RBD) (K D ∼ 2μM by surface plasmon resonance), preventing it from binding to ACE2 expressing cells. Importantly, hBD-2 shows specificity by blocking CoV-2/spike pseudoviral infection, but not VSVG mediated infection, of ACE2 expressing human cells with an IC 50 of 2.8 + 0.4 μM. These promising findings offer opportunities to develop hBD-2 and/or its derivatives and mimetics to safely and effectively use as agents to prevent SARS-CoV-2 infection.

Human β-defensins (hBD) play central roles in antimicrobial activities against various microorganisms and in immune-regulation. These peptides perturb phospholipid membranes for function, but it is not well understood how defensins approach, insert and finally disrupt membranes on the molecular level. Here we show that hBD-3 analogs interact with lipid bilayers through a conserved surface that is formed by two adjacent loops in the solution structure. By integrating a collection of 13C, 1H and 31P solid-state NMR methods with long-term molecular dynamic simulations, we reveal that membrane-binding rigidifies the peptide, enhances structural polymorphism, and promotes β-strand conformation. The peptide colocalizes with negatively charged lipids, confines the headgroup motion, and deforms membrane into smaller, ellipsoidal vesicles. This study designates the residue-specific, membrane-bound topology of hBD-3 analogs, serves as the basis for further elucidating the function-relevant structure and dynamics of other defensins, and facilitates the development of defensin-mimetic antibiotics, antifungals, and anti-inflammatories.

New approaches to complement vaccination are needed to combat the spread of SARS-CoV-2 and stop COVID-19 related deaths and long-term medical complications. Human beta defensin 2 (hBD-2) is a naturally occurring epithelial cell derived host defense peptide that has antiviral properties. Our comprehensive in-silico studies demonstrate that hBD-2 binds the site on the CoV-2-RBD that docks with the ACE2 receptor. Biophysical and biochemical assays confirm that hBD-2 indeed binds to the CoV-2-receptor binding domain (RBD) (KD ∼ 300 nM), preventing it from binding to ACE2 expressing cells. Importantly, hBD-2 shows specificity by blocking CoV-2/spike pseudoviral infection, but not VSV-G mediated infection, of ACE2 expressing human cells with an IC50 of 2.4± 0.1 μM. These promising findings offer opportunities to develop hBD-2 and/or its derivatives and mimetics to safely and effectively use as novel agents to prevent SARS-CoV-2 infection.

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