Protein secondary structure affects glycan clustering in native mass spectrometry

Abstract

Infection with human noroviruses (hNoV) for the vast majority of strains requires attachment of the viral capsid to histo blood group antigens (HBGA). The HBGA binding pocket is formed by dimers of the protruding domain (P dimers) of the capsid protein VP1. Several studies have focused on HBGA binding to P dimers, reporting binding affinities and stoichiometries. However, nuclear magnetic resonance spectroscopy (NMR) and native mass spectrometry (MS) analyses yielded incongruent dissociation constants (KD) for binding of HBGAs to P dimers and, in some cases, disagreed whether glycans bind at all. We hypothesized that glycan clustering during electrospray ionization in native MS critically depends on the physicochemical properties of the protein studied. It follows that the choice of the reference protein is crucial. We analyzed carbohydrate clustering using various P dimers and eight non-glycan binding proteins serving as possible references. Data from native and ion mobility MS indicate that the mass fraction of β-sheet has a strong influence on the degree of glycan clustering. Therefore, the determination of specific glycan binding affinities from native MS must be interpreted cautiously.

NMR Experiments Provide Insights into Ligand-Binding to the SARS-CoV-2 Spike Protein Receptor-Binding Domain

“…These findings agree with another NMR study but oppose an MS based report . We have recently observed unspecific glycan clustering due to β-sheet rich regions that obstruct native MS-based glycan interaction studies. , This finding may explain the discrepancy, since the RBD contains a twisted five-stranded antiparallel β-sheet …”

“…19 We have recently observed unspecific glycan clustering due to βsheet rich regions that obstruct native MS-based glycan interaction studies. 32,33 This finding may explain the discrepancy, since the RBD contains a twisted five-stranded antiparallel β-sheet. 27 To further explore the role of sialoglycans as cognate ligands, we subjected 2,3-sialyllactose to STD NMR experiments in the presence of full-length S-glycoprotein, recombinantly produced in HEK cells.…”

Studying the interaction of glycans with intact virions and virus‐like particles by ligand‐observed NMR spectroscopy

“…11 Years later, it was revealed that the binding and recognition of HBGAs by noroviruses is much more complex than previously anticipated 13 and various studies have demonstrated some hidden traps affecting the interpretation of the binding process a stepwise interaction. 14 Mallagaray et al shed light on HBGAs-norovirus interactions revealing that a spontaneous deamidation of N373, near the HBGA binding site, leads to loss of HBGA recognition and is partly responsible for the misinterpretation of previous binding data as caused by cooperative effects. 15 Our laboratory has reported the recognition of influenza virus hemagglutinin (HA)-containing VLPs for competitive selection from a mixture of α(2,6)-Sialyllactose and α(2,3)-Sialyllactose using STD NMR spectroscopy.…”