Infection with human norovirus (hNoV) is the leading cause of acute gastroenteritis worldwide. Attempts to provide antivirals or vaccines have not been successful so far. Infection of hNoV requires attachment to histo blood group antigens (HBGAs), but how this binding event promotes the infection of host cells is unknown. We employ protein NMR experiments supported by mass spectrometry and crystallography to study HBGA binding to the P-domain of a prevalent virus strain (GII.4).
We have successfully assigned 86% of the backbone NH signals and 100% of the methyl groups in a U-[2H,15N] stereo-selective MILVA methyl group labeled hNoV P-domain (72 KDa). Unexpectedly, our NMR analysis revealed a highly selective transformation of asparagine 373, located in an antigenic loop adjoining the HBGA binding site, into an iso-aspartate residue. This spontaneous post-translational modification (PTM) proceeds with an estimated half-life of a few days at physiological temperatures, independent of the presence of HBGAs but dramatically affecting HBGA recognition. Sequence conservation and the surface-exposed position of this PTM suggest an important role in infection and immune recognition for many norovirus strains.
Binding of HBGAs to hNoV P-domains cause long-range chemical shift perturbations (CSPs) of amino acid residues more than 30 Å away from the binding site, indicating the presence of a subtle allosteric cross-talk. To understand the architecture of the allosteric network we generated a library of 17 naturally occurring soft mutants, which yielded distinct CSP patterns in Methyl-TROSY NMR spectra. A CHESCA-like covariance analysis uncovered functional clusters of coupled residues, as well as a residue-specific dissection of the contribution of each amino acid to allostery. A 2nd order Markov analysis revealed the complete structure of the network, reflecting the flow of the allosteric signalling. Critical junctions in the network can be correlated to biologically significant allosteric sites and pathways. We aim to use this information to identify protein “hot-spots”, which could be targeted as allosteric modulators on hNoV infection.