The assessment of Pseudomonas aeruginosa lectin LecA binding characteristics of divalent galactosides using multiple techniques

Pseudomonas aeruginosa is a widespread opportunistic pathogen that is capable of colonizing various human tissues and is resistant to many antibiotics. LecA is a galactose binding tetrameric lectin involved in adhesion, infection and biofilm formation. This study reports on the binding characteristics of mono- and divalent (chelating) ligands to LecA using different techniques. These techniques include Affinity Capillary Electrophoresis (ACE), Bio Layer Interferometry (BLI), Native Mass Spectrometry and a Thermal Shift Assay. Aspects of focus include: affinity, selectivity, binding kinetics and residence time. The affinity of a divalent ligand was determined to be in the low nanomolar range for all of the used techniques and with a ligand residence time of approximately 7 hours, while no strong binding was seen to related lectin tetramers. Each of the used techniques provides a unique and complementary insight into the chelation based binding mode of the divalent ligand to the LecA tetramer

The assessment of Pseudomonas aeruginosa lectin LecA binding characteristics of divalent galactosides using multiple techniques

Pseudomonas aeruginosa is a widespread opportunistic pathogen that is capable of colonizing various human tissues and is resistant to many antibiotics. LecA is a galactose binding tetrameric lectin involved in adhesion, infection and biofilm formation. This study reports on the binding characteristics of mono- and divalent (chelating) ligands to LecA using different techniques. These techniques include Affinity Capillary Electrophoresis (ACE), Bio Layer Interferometry (BLI), Native Mass Spectrometry and a Thermal Shift Assay. Aspects of focus include: affinity, selectivity, binding kinetics and residence time. The affinity of a divalent ligand was determined to be in the low nanomolar range for all of the used techniques and with a ligand residence time of approximately 7 hours, while no strong binding was seen to related lectin tetramers. Each of the used techniques provides a unique and complementary insight into the chelation based binding mode of the divalent ligand to the LecA tetramer

The SARS-CoV-2 spike N-terminal domain engages 9-O -acetylated α2-8-linked sialic acids

SARS-CoV-2 viruses engage ACE2 as a functional receptor with their spike protein. The S1 domain of the spike protein contains a C-terminal receptor-binding domain (RBD) and an N-terminal domain (NTD). The NTD of other coronaviruses includes a glycan-binding cleft. However, for the SARS-CoV-2 NTD protein-glycan binding was only observed weakly for sialic acids with highly sensitive methods. Amino acid changes in the NTD of Variants of Concern (VoC) shows antigenic pressure, which can be an indication of NTD-mediated receptor binding. Trimeric NTD proteins of SARS-CoV-2, Alpha, Beta, Delta, and Omicron did not reveal a receptor binding capability. Unexpectedly, the SARS-CoV-2 Beta subvariant strain (501Y.V2-1) NTD binding to Vero E6 cells was sensitive to sialidase pretreatment. Glycan microarray analyses identified a putative 9- O -acetylated sialic acid as a ligand, which was confirmed by catch-and-release ESI-MS, STD-NMR analyses, and a graphene-based electrochemical sensor. The Beta (501Y.V2-1) variant attained an enhanced glycan binding modality in the NTD with specificity towards 9- O -acetylated structures, suggesting a dual-receptor functionality of the SARS-CoV-2 S1 domain, which was quickly selected against. These results indicate that SARS-CoV-2 can probe additional evolutionary space, allowing binding to glycan receptors on the surface of target cells. Graphical abstract: Synopsis: Coronaviruses utilize their N-terminal domain (NTD) for initial reversible low-affinity interaction to (sialylated) glycans. This initial low-affinity/high-avidity engagement enables viral surfing on the target membrane, potentially followed by a stronger secondary receptor interaction. Several coronaviruses, such as HKU1 and OC43, possess a hemagglutinin-esterase for viral release after sialic acid interaction, thus allowing viral dissemination. Other coronaviruses, such as MERS-CoV, do not possess a hemagglutinin-esterase, but interact reversibly to sialic acids allowing for viral surfing and dissemination. The early 501Y.V2-1 subvariant of the Beta SARS-CoV-2 Variant of Concern has attained a receptor-binding functionality towards 9- O -acetylated sialic acid using its NTD. This binding functionality was selected against rapidly, most likely due to poor dissemination. Ablation of sialic acid binding in more recent SARS-CoV-2 Variants of Concern suggests a fine balance of sialic acid interaction of SARS-CoV-2 is required for infection and/or transmission

The SARS-CoV-2 spike N-terminal domain engages 9-O -acetylated α2-8-linked sialic acids

SARS-CoV-2 viruses engage ACE2 as a functional receptor with their spike protein. The S1 domain of the spike protein contains a C-terminal receptor-binding domain (RBD) and an N-terminal domain (NTD). The NTD of other coronaviruses includes a glycan-binding cleft. However, for the SARS-CoV-2 NTD protein-glycan binding was only observed weakly for sialic acids with highly sensitive methods. Amino acid changes in the NTD of Variants of Concern (VoC) shows antigenic pressure, which can be an indication of NTD-mediated receptor binding. Trimeric NTD proteins of SARS-CoV-2, Alpha, Beta, Delta, and Omicron did not reveal a receptor binding capability. Unexpectedly, the SARS-CoV-2 Beta subvariant strain (501Y.V2-1) NTD binding to Vero E6 cells was sensitive to sialidase pretreatment. Glycan microarray analyses identified a putative 9- O -acetylated sialic acid as a ligand, which was confirmed by catch-and-release ESI-MS, STD-NMR analyses, and a graphene-based electrochemical sensor. The Beta (501Y.V2-1) variant attained an enhanced glycan binding modality in the NTD with specificity towards 9- O -acetylated structures, suggesting a dual-receptor functionality of the SARS-CoV-2 S1 domain, which was quickly selected against. These results indicate that SARS-CoV-2 can probe additional evolutionary space, allowing binding to glycan receptors on the surface of target cells. Graphical abstract: Synopsis: Coronaviruses utilize their N-terminal domain (NTD) for initial reversible low-affinity interaction to (sialylated) glycans. This initial low-affinity/high-avidity engagement enables viral surfing on the target membrane, potentially followed by a stronger secondary receptor interaction. Several coronaviruses, such as HKU1 and OC43, possess a hemagglutinin-esterase for viral release after sialic acid interaction, thus allowing viral dissemination. Other coronaviruses, such as MERS-CoV, do not possess a hemagglutinin-esterase, but interact reversibly to sialic acids allowing for viral surfing and dissemination. The early 501Y.V2-1 subvariant of the Beta SARS-CoV-2 Variant of Concern has attained a receptor-binding functionality towards 9- O -acetylated sialic acid using its NTD. This binding functionality was selected against rapidly, most likely due to poor dissemination. Ablation of sialic acid binding in more recent SARS-CoV-2 Variants of Concern suggests a fine balance of sialic acid interaction of SARS-CoV-2 is required for infection and/or transmission