Glycan-Induced Protein Dynamics in Human Norovirus P Dimers Depend on Virus Strain and Deamidation Status

Abstract

Noroviruses are the major cause of viral gastroenteritis and re-emerge worldwide every year, with GII.4 currently being the most frequent human genotype. The norovirus capsid protein VP1 is essential for host immune response. The P domain mediates cell attachment via histo blood-group antigens (HBGAs) in a strain-dependent manner but how these glycan-interactions actually relate to cell entry remains unclear. Here, hydrogen/deuterium exchange mass spectrometry (HDX-MS) is used to investigate glycan-induced protein dynamics in P dimers of different strains, which exhibit high structural similarity but different prevalence in humans. While the almost identical strains GII.4 Saga and GII.4 MI001 share glycan-induced dynamics, the dynamics differ in the emerging GII.17 Kawasaki 308 and rare GII.10 Vietnam 026 strain. The structural aspects of glycan binding to fully deamidated GII.4 P dimers have been investigated before. However, considering the high specificity and half-life of N373D under physiological conditions, large fractions of partially deamidated virions with potentially altered dynamics in their P domains are likely to occur. Therefore, we also examined glycan binding to partially deamidated GII.4 Saga and GII.4 MI001 P dimers. Such mixed species exhibit increased exposure to solvent in the P dimer upon glycan binding as opposed to pure wildtype. Furthermore, deamidated P dimers display increased flexibility and a monomeric subpopulation. Our results indicate that glycan binding induces strain-dependent structural dynamics, which are further altered by N373 deamidation, and hence hint at a complex role of deamidation in modulating glycan-mediated cell attachment in GII.4 strains.

Norovirus–glycan interactions — how strong are they really?

“…For this specific strain, glycan interaction induced folding in a loop proximal to the binding site. This long-range effect is supported by MD simulations [ 47 ].…”

“…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 ].…”

“…for antibodies and half antibodies [ 57 ]. Interestingly, the ratio of P-monomer to P-dimer seems highly dependent on several different factors such as strain and deamidation [ 47 ]. Deamidation appears to increase the monomer fraction.…”

Protein Secondary Structure Affects Glycan Clustering in Native Mass Spectrometry

“…Others used a small-sized monoclonal antibody single-chain fragment (scFv 26 kDa, [5,9,11]), which mostly consisted of β-sheets. While the latter was much better suited, our data show that protein dynamics, as in the deamidated P dimer, further influence clustering [4,27].…”

Protein secondary structure affects glycan clustering in native mass spectrometry

“…Others used a small sized monoclonal antibody single chain fragment (scFv 26 kDa, [5,9,11]), which is mostly consiting of β-sheets. While the latter is much better suited, our data shows that protein dynamics as in the deamidated P dimer further influence the clustering [4,32].…”