Protein Secondary Structure Affects Glycan Clustering in Native Mass Spectrometry

Abstract

Infection by the human noroviruses (hNoV), for the vast majority of strains, requires attachment of the viral capsid to histo blood group antigens (HBGAs). 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 the binding of HBGAs to P dimers and, in some cases, disagreed on 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 a reference protein is crucial. We analysed 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-sheets 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.

Norovirus–glycan interactions — how strong are they really?

“…Inspection of the data published revealed discrepancies that were addressed in follow up studies in our laboratories [ 43–45 ]. It turned out that the experimental approaches employed by us, and others contain hidden traps that are easy to fall into.…”

“…First, it turned out that the data leading to our hypothesis of cooperative binding were only seemingly consistent due to two main causes. For one, it turned out that native MS results were biased by secondary structure-induced glycan clustering [ 43 ]. On the other hand, spontaneous deamidation of a highly conserved asparagine residue (Asn373) and its quantitative conversion into an iso-aspartate residue over time led to mixtures of different protein species with different HBGA affinities.…”

“…Second, we showed that sialic acid containing glycans such as ganglioside head groups do not bind to human or murine norovirus P-dimers [ 44 ]. The reason for this again lies in the secondary structure-induced glycan clustering [ 43 ]. A third point concerns the dissociation constants reported so far by us, and others.…”

“…Therefore, glycan clustering was examined again with different reference proteins. Small proteins such as myoglobin, ubiquitin, and cytochrome c did indeed exhibit less glycan clustering when compared with alcohol dehydrogenase (ADH) [ 43 ] ( Figure 2 ). Interestingly, the K D from STD-NMR could be reproduced using ADH as reference protein.…”

“…The highly flexible but low affinity deamidated P-dimer showed similar or increased glycan interaction compared with the wild-type protein. So even a reference protein with the same number of β-sheets could prove difficult for low-affinity glycan binding and should thus be carefully selected to avoid errors [ 43 , 47 ]. Therefore, protein–glycan interactions are best compared with a proper glycan negative control, which has proven impossible to find except for glycan mimetics [ 48 ].…”