Interactions of human norovirus capsid P domain with histo-blood group antigens with Hydrogen-Deuterium Exchange Mass Spectrometry
Attachment of human noroviruses to histo blood group antigens (HBGAs) is essential for infection, but how this binding event promotes the infection of host cells is unknown. Here, we employed protein NMR experiments supported by mass spectrometry and crystallography to study HBGA binding to the P domain of a prevalent virus strain (GII.4) in more detail . We report a highly selective transformation of N373, 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.
Sample Processing Protocol
GII.4 Saga 2006 P-domains (residues 224 to 530, GenBank accession number AB447457; due to the cloning strategy the sequence contains an extra GPG sequence prior to S224) were synthesized and purified following an improved version of a previously reported protocol. The lysate was clarified by centrifugation and the protein was purified with Ni-NTA resin (Qiagen). Maltose binding protein and the His-tag were cleaved from the P-domain using HRV3C protease (Novagen). Cleaved P-domain protein eluted from Ni-NTA resin and was further purified by size- exclusion chromatography using a Superdex 26/600 76 pg column (GE Healthcare) in 20 mM sodium phosphate buffer (pH 7.3). Protein purity and dimer concentration were monitored by SDS-polyacrylamide electrophoresis and UV absorption (ε280 70,820 M-1 cm-1), respectively. Separation of fully-, partially and non-deamidated P-dimer species was achieved by cation exchange chromatography using a 6 ml Resource S column (GE Healthcare) at 6 °C. Protein samples were prepared in 20 mM sodium acetate buffer (pH 4.9) and eluted using a 78.4 ml linear salt gradient up to 195 mM NaCl with a flowrate of 1 ml min-1. For HDX measurements P-dimer (50 pmol) was mixed with glycans at tenfold of the final concentration, directly diluted 1:9 in 99% deuterated 20 mM Tris buffer (pH 7.4, 150 mM NaCl, 25°C) and quenched after the respective time point by 1:1 addition of ice-cold quench buffer (300 mM Phosphate buffer, pH 2.3, 6 M Urea). The samples were thawed and injected onto a cooled (0 °C) HPLC System (Agilent Infinity 1260, Agilent Technologies) equipped with a home packed pepsin column (IDEX guard column with an internal volume of 60 µL, Porozyme Immobilized Pepsin beads, Thermo Scientific) in a column oven (25 °C), a peptide trap column (OPTI-TRAP for peptides, Optimize Technologies) and a reversed-phase analytical column (PLRP-S for Biomolecules, Agilent Technologies). Pepsin digestion was performed on-line at a flow rate of 75 µL/min (0.23 % formic acid in water) and peptides were trapped in the trap column. Peptides were eluted and separated on the analytical column using a 7 min gradient of 8-40 % solvent B (solvent A: 0.23 % formic acid in water, solvent B: 0.23 % formic acid in acetonitrile) at 150 µL/min. MS was performed using an Orbitrap Fusion Tribrid in positive ESI MS only mode (Orbitrap resolution 120K, 4 microscans). The experiments were performed in triplicate. Peptide and PTM identification was performed on non-deuterated samples using a longer elution gradient (27 min, 8-40 % solvent B) in data- dependent MS/MS acquisition mode (Orbitrap resolution 120K, 1 microscans, HCD 30 with dynamic exclusion of Top 20 N).
Data Processing Protocol
Precursor and fragment ions were searched and matched against a local protein database in MaxQuant with a minimum score of 20 for unmodified and 40 for modified peptides. Enzyme specificity was set to no enzyme and deamidation on asparagine and gluatamine, oxidation on methionine and N-terminal protein acetylation were set as variable modifications.DeutEx software (obtained from peterslab.org) was used to determine the deuterium uptake. Excel (Microsoft) and GraphPad Prism software (GraphPad Software, Inc.) was used to create uptake plots and perform statistical analysis using Student's T-test with alpha=0.01.
Mallagaray A, Creutznacher R, Dülfer J, Mayer PHO, Grimm LL, Orduña JM, Trabjerg E, Stehle T, Rand KD, Blaum BS, Uetrecht C, Peters T. A post-translational modification of human Norovirus capsid protein attenuates glycan binding. Nat Commun. 2019 10(1):1320 PubMed: 30899001