Assignment of Ala, Ile, LeuproS, Met, and ValproS methyl groups of the protruding domain of murine norovirus capsid protein VP1 using methyl–methyl NOEs, site directed mutagenesis, and pseudocontact shifts

Thorben Maass,Robert Creutznacher,Alvaro Mallagaray,Leon Torben Westermann,Charlotte Uetrecht,Thomas Peters,Jasmin Dülfer

doi:10.1007/s12104-022-10066-7

Abstract

The protruding domain (P-domain) of the murine norovirus (MNV) capsid protein VP1 is essential for infection. It mediates receptor binding and attachment of neutralizing antibodies. Protein NMR studies into interactions of the P-domain with ligands will yield insights not easily available from other biophysical techniques and will extend our understanding of MNV attachment to host cells. Such studies require at least partial NMR assignments. Here, we describe the assignment of about 70% of the Ala, Ile, LeuproS, Met, and ValproS methyl groups. An unfavorable distribution of methyl group resonance signals prevents complete assignment based exclusively on 4D HMQC-NOESY-HMQC experiments, yielding assignment of only 55 out of 100 methyl groups. Therefore, we created point mutants and measured pseudo contact shifts, extending and validating assignments based on methyl-methyl NOEs. Of note, the P-domains are present in two different forms caused by an approximate equal distribution of trans- and cis-configured proline residues in position 361.

Highlights

The genus norovirus belongs to the family of Caliciviridae, a family of positive-sense single-stranded RNA viruses
A partial assignment of side chain 13C-methyl groups of murine Norovirus (MNV) P-dimers has been instrumental for that study and will be reported in detail here
We had aimed at a backbone assignment as accomplished for a related human norovirus (HuNoV) P-dimer (Mallagaray et al 2019) but it turned out that MNV P-dimers behave profoundly different

Summary

Introduction

The genus norovirus belongs to the family of Caliciviridae, a family of positive-sense single-stranded RNA viruses. Attempts to obtain uniformly 2H,15N labeled samples of the MNV P-domain employing our published protocol for HuNoV GII.4 Saga P-dimers (Mallagaray et al 2019) failed, yielding unstable protein samples as reflected by corresponding TROSY HSQC spectra (Creutznacher et al 2021a). The monomer–dimer equilibrium leads to spectral crowding and impedes structure-based assignment of methyl-methyl NOEs. 4D HMQCNOESY-HMQC experiments were performed in the presence of saturating amounts of GCDCA, shifting the monomer–dimer equilibrium almost exclusively towards dimers.

Results

Conclusion